U.S. patent application number 11/235577 was filed with the patent office on 2006-03-30 for methods of diagnosing and treating complications of pregnancy.
Invention is credited to S. Ananth Karumanchi, Vikas Sukhatme.
Application Number | 20060067937 11/235577 |
Document ID | / |
Family ID | 36090722 |
Filed Date | 2006-03-30 |
United States Patent
Application |
20060067937 |
Kind Code |
A1 |
Karumanchi; S. Ananth ; et
al. |
March 30, 2006 |
Methods of diagnosing and treating complications of pregnancy
Abstract
Disclosed herein are methods for diagnosing a pregnancy related
hypertensive disorder or a predisposition to a pregnancy related
hypertensive disorder by measuring the level or biological activity
of soluble endoglin. Also disclosed herein are methods for treating
a a pregnancy related hypertensive disorder, such as pre-eclampsia
and eclampsia, using compounds that alter soluble endoglin levels
or biological activity.
Inventors: |
Karumanchi; S. Ananth;
(Chestnut Hill, MA) ; Sukhatme; Vikas; (Newton,
MA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
36090722 |
Appl. No.: |
11/235577 |
Filed: |
September 26, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60613170 |
Sep 24, 2004 |
|
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Current U.S.
Class: |
424/145.1 ;
424/193.1; 424/697; 514/15.7; 514/20.4; 514/20.5; 514/263.31;
514/269; 514/343; 514/355; 514/47; 514/8.1; 514/8.8; 514/8.9 |
Current CPC
Class: |
A61K 45/06 20130101;
A61P 9/00 20180101; A61K 31/4439 20130101; C07K 16/2896 20130101;
A61P 15/00 20180101; G01N 2800/368 20130101; A61K 38/1841 20130101;
G01N 33/689 20130101; A61K 31/522 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 31/455 20130101; G01N 2333/71
20130101; A61K 38/22 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; C07K 16/2863 20130101; A61P 9/12
20180101; A61K 31/513 20130101; A61K 38/1866 20130101; Y10T
436/143333 20150115; A61K 38/1866 20130101; A61K 31/4439 20130101;
A61K 38/1875 20130101; A61K 31/522 20130101; A61K 38/1891 20130101;
G01N 2500/00 20130101; C07K 14/495 20130101; G01N 33/74 20130101;
A61K 38/1866 20130101; A61K 38/1841 20130101; A61K 38/1875
20130101; A61K 38/1875 20130101; A61K 38/1891 20130101; A61K 31/455
20130101; A61K 39/3955 20130101; A61K 31/513 20130101; A61P 43/00
20180101; A61K 38/1891 20130101; G01N 2800/60 20130101 |
Class at
Publication: |
424/145.1 ;
424/193.1; 514/012; 514/047; 514/263.31; 514/355; 514/343; 514/269;
424/697 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/385 20060101 A61K039/385; A61K 31/522 20060101
A61K031/522; A61K 38/18 20060101 A61K038/18; A61K 31/455 20060101
A61K031/455; A61K 31/4439 20060101 A61K031/4439; A61K 31/513
20060101 A61K031/513 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made in part with support from the
Government through NIH Grant Nos. DK 064255 and HL 079594. The
Government has certain rights in the invention.
Claims
1. A method of treating or preventing a pregnancy related
hypertensive disorder in a subject, said method comprising the step
of administering to said subject a compound capable of specifically
binding to soluble endoglin, wherein said administering is for a
time and in an amount sufficient to treat or prevent said pregnancy
related hypertensive disorder in said subject.
2. The method of claim 1, wherein said pregnancy related
hypertensive disorder is selected from the group consisting of
pre-eclampsia, eclampsia, gestational hypertension, chronic
hypertension, HELLP syndrome, and pregnancy with a small for
gestational age (SGA) infant.
3. The method of claim 2, wherein said pregnancy related
hypertensive disorder is pre-eclampsia or eclampsia.
4. The method claim 1, wherein said compound is a purified soluble
endoglin antibody or a soluble endoglin antigen-binding
fragment.
5. The method of claim 1, wherein said compound is a growth
factor.
6. The method of claim 5, wherein said growth factor is selected
from the group consisting of: transforming growth factor
(TGF)-.beta.1 or TGF-.beta.3, activin A, Bone Morphogenic Protein
(BMP)-2 or BMP-7.
7. The method of claim 1, further comprising administering a
compound selected from the group consisting of a purified sFlt-1
antibody, an sFlt antigen-binding fragment, nicotine, theophylline,
adenosine, nifedipine, minoxidil, magnesium sulfate, vascular
endothelial growth factor (VEGF), and placental growth factor
(PlGF).
8. The method of claim 7, wherein said VEGF is VEGF121 or VEGF
165.
9. The method of claim 1, further comprising the step of
administering to a subject an anti-hypertensive compound.
10. The method of claim 1, wherein said subject is a pregnant
human.
11. The method of claim 1, wherein said subject is a post-partum
human.
12. The method of claim 1, wherein said subject is a non-human.
13. The method of claim 12, wherein said subject is selected from
the group consisting of a cow, a horse, a sheep, a pig, a goat, a
dog, and a cat.
14. A method of treating or preventing a pregnancy related
hypertensive disorder in a subject, said method comprising the step
of administering to said subject a compound that increases the
level of a growth factor capable of binding to soluble endoglin,
wherein said administering is in an amount and for a time
sufficient to treat or prevent said pregnancy related hypertensive
disorder in said subject.
15. The method of claim 14, wherein said growth factor is selected
from the group consisting of TGF-.beta.1, TGF-3.beta., activin A,
BMP-2, and BMP-7.
16. The method of claim 14, wherein said compound is selected from
the group consisting of cyclosporine, alpha tocopherol,
methysergide, bromocriptine, and aldomet.
17. A method of treating or preventing a pregnancy related
hypertensive disorder in a subject, said method comprising
administering to said subject a compound that inhibits growth
factor binding to a soluble endoglin polypeptide, wherein said
administering is sufficient to treat or prevent said pregnancy
related hypertensive disorder in said subject.
18. The method of claim 17, wherein said compound binds to soluble
endoglin and blocks growth factor binding.
19. A method of treating or preventing a pregnancy related
hypertensive disorder in a subject, said method comprising
administering to said subject a compound capable of reducing
soluble endoglin expression or biological activity, wherein said
administering is sufficient to treat or prevent said pregnancy
related hypertensive disorder in said subject.
20. The method of claim 19, wherein said compound is a compound
that inhibits a proteolytic enzyme selected from the group
consisting of a matrix metalloproteinase (MMP), cathepsin, and
elastase.
21. The method of claim 20, wherein said MMP is MMP9.
22. The method of claim 20, wherein MMP is membrane type matrix
metalloproteinase-1.
23. The method of claim 19, wherein said compound comprises an
antisense nucleobase oligomer that is at least 95% complementary to
at least a portion of an endoglin nucleic acid sequence, wherein
said administering is sufficient to treat or prevent said pregnancy
related hypertensive disorder in said subject.
24. The method of claim 23, wherein said antisense nucleobase
oligomer is 8 to 30 nucleotides in length.
25. The method of claim 19, wherein said compound comprises a
double stranded RNA having one strand that is at least 95%
complementary to at least a portion of an endoglin nucleic acid
sequence, wherein said administering is sufficient to treat or
prevent said pregnancy related hypertensive disorder in said
subject.
26. The method of claim 25, wherein said double stranded RNA is a
small interfering RNA (siRNAs) 19 to 25 nucleotides in length.
27. The method of claim 19, said compound comprising a purified
antibody or antigen-binding fragment that specifically binds
soluble endoglin.
28. The method of claim 14, 17, or 19, wherein said pregnancy
related hypertensive disorder is selected from the group consisting
of pre-eclampsia, eclampsia, gestational hypertension, chronic
hypertension, HELLP syndrome, and pregnancy with a small for
gestational age (SGA) infant.
29. The method of claim 28, wherein said pregnancy related
hypertensive disorder is pre-eclampsia or eclampsia.
30. The method of claim 1 or 19, wherein said method further
comprises monitoring said pregnancy related hypertensive disorder
in said subject, wherein said monitoring comprises measuring the
level of soluble endoglin polypeptide in a sample from said
subject.
31. The method of claim 30, wherein said sample is serum or plasma
and a level of soluble endoglin polypeptide less than 25 ng/ml
indicates an improvement in said pregnancy related hypertensive
disorder.
32. The method of claim 30, wherein said measuring of levels is
done on two or more occasions and a decrease in said levels between
measurements is an indicator of an improvement in said pregnancy
related hypertensive disorder.
33. The method of claim 30, wherein said measuring of levels is
compared to a positive reference sample and a decrease in the level
of soluble endoglin relative to said positive reference sample
indicates an improvement in said pregnancy related hypertensive
disorder in said subject.
34. The method of claim 30, wherein said monitoring is used to
determine the therapeutic dosage of the compound.
35. The method of claim 30, wherein said measuring is done using an
immunological assay.
36. The method of claim 30, wherein the level of soluble endoglin
is the level of free, bound, or total soluble endoglin.
37. The method of claim 30, wherein the level of soluble endoglin
is the level of an endoglin polypeptide resulting from degradation
or enzymatic cleavage.
38. The method of claim 30, wherein said monitoring comprises
measuring the level of at least one of sFlt-1, VEGF, or PlGF
polypeptide in a sample from said subject.
39. The method of claim 38, further comprising calculating the
relationship between said levels of soluble endoglin, sFlt-1, VEGF,
or PlGF using a metric, wherein an alteration in the relationship
between said levels in the subject sample relative to a reference
sample, is a diagnostic indicator of a pregnancy related
hypertensive disorder in said subject.
40. The method of claim 39, wherein said metric is a pre-eclampsia
anti-angiogenic index (PAAI): [sFlt-1/VEGF+PlGF].
41. The method of claim 40, wherein a PAAI value less than 20
indicates an improvement in said pre-eclampsia or eclampsia.
42. The method of claim 39, wherein said metric is a soluble
endoglin anti-angiogenic index: [(sFlt-1+0.25 soluble
endoglin)/PlGF] or [(soluble endoglin+sFlt-1)/PlGF].
43. The method of claim 39, wherein said metric is used to
determine the dosage of the therapeutic compound.
44. The method of claim 43, wherein the therapeutic compound is
administered in a dose such that the PAAI is less than 20.
45. The method of claim 1 or 19, wherein said method further
comprises monitoring the pregnancy related hypertensive disorder in
said subject, wherein said monitoring comprises measuring the level
of an endoglin nucleic acid molecule in a sample from said
subject.
46. The method of claim 45, wherein said levels of an endoglin
nucleic acidis compared to a reference sample and an alteration in
said levels in the subject sample compared to said reference is a
diagnostic indicator of a pregnancy related hypertensive disorder
in said subject.
47. An antibody or antigen-binding fragment thereof that
specifically binds soluble endoglin.
48. The antibody or antigen-binding fragment thereof of claim 47,
wherein said antibody or antigen-binding fragment prevents binding
of a growth factor to soluble endoglin.
49. The antibody or antigen-binding fragment thereof of claim 48,
wherein said growth factor is selected from the group consisting of
TGF-.beta.1, TGF-3.beta., activin A, BMP-2, and BMP-7.
50. The antibody or antigen-binding fragment thereof of claim 47,
wherein said antibody is a monoclonal antibody.
51. The antibody or antigen-binding fragment thereof of claim 47,
wherein said antibody or antigen-binding fragment thereof is a
human or humanized antibody.
52. The antibody or antigen-binding fragment thereof of claim 47,
wherein said antibody lacks an Fc portion.
53. The antibody or antigen-binding fragment thereof of claim 47,
wherein said antibody is an F(ab').sub.2, an Fab, or an Fv
structure.
54. The antibody or antigen-binding fragment thereof of claim 47,
wherein said antibody or antigen-binding fragment thereof is
present in a pharmaceutically acceptable carrier.
55. A method of diagnosing a subject as having, or having a
predisposition to, a pregnancy related hypertensive disorder, said
method comprising measuring the level of a soluble endoglin
polypeptide in a sample from said subject.
56. The method of claim 55, wherein said pregnancy related
hypertensive disorder is pre-eclampsia, eclampsia, gestational
hypertension, chronic hypertension, HELLP syndrome, and pregnancy
with a SGA infant.
57. The method of claim 56, wherein said pregnancy related
hypertensive disorder is pre-eclampsia or eclampsia.
58. The method of claim 55, wherein the level of soluble endoglin
is the level of free, bound, or total soluble endoglin.
59. The method of claim 55, wherein a level of soluble endoglin
greater than 20 ng/ml is a diagnostic indicator of a pregnancy
related hypertensive disorder or a predisposition to a pregnancy
related hypertensive disorder.
60. The method of claims 55, wherein said measuring is done using
an immunological assay.
61. The method of claim 60, wherein said immunological assay is an
ELISA.
62. The method of claim 55, wherein an increase in the level of
said soluble endoglin polypeptide relative to a normal reference is
a diagnostic indicator of pregnancy related hypertensive disorder
or a predisposition to a pregnancy related hypertensive
disorder.
63. The method of claim 55 wherein said measuring of levels is done
on two or more occasions and a change in said levels between
measurements is a diagnostic indicator of said pregnancy related
hypertensive disorder.
64. The method of claim 55, wherein said monitoring further
comprises measuring the level of at least one of sFlt-1, VEGF, or
PlGF polypeptide in a sample from said subject.
65. The method of claim 64, further comprising calculating the
relationship between said levels of soluble endoglin, sFlt-1, VEGF,
or PlGF using a metric, wherein an alteration in the relationship
between said levels in the subject sample relative to a reference
sample, is a diagnostic indicator of a pregnancy related
hypertensive disorder in said subject.
66. The method of claim 65, wherein said metric is a pre-eclampsia
anti-angiogenic index (PAAI): [sFlt-1VEGF+PlGF].
67. The method of claim 66, wherein a PAAI value greater than 10 is
a diagnostic indicator of a pregnancy related hypertensive disorder
in said subject.
68. The method of claim 65, wherein said metric is a soluble
endoglin anti-angiogenic index: [(sFlt-1+0.25 soluble
endoglin)/PlGF].
69. The method of claim 68, wherein a soluble endoglin
anti-angiogenic index value greater than 100 is a diagnostic
indicator of a pregnancy related hypertensive disorder in said
subject.
70. The method of claim 65, wherein said metric is [(soluble
endoglin+sFlt-1)/PlGF].
71. The method of claim 66, 68, or 70, wherein said metric further
comprises the body mass index of the mother or the gestational age
of the fetus.
72. A method of diagnosing a subject as having, or having a
predisposition to, a pregnancy related hypertensive disorder, said
method comprising measuring the level of an endoglin nucleic acid
molecule in a sample from said subject and comparing it to a
reference, wherein an alteration in said levels compared to a
reference diagnoses a pregnancy related hypertensive disorder or a
predisposition to a pregnancy related hypertensive disorder.
73. The method of claim 72, wherein said pregnancy related
hypertensive disorder is pre-eclampsia, eclampsia, gestational
hypertension, HELLP syndrome, chronic hypertension, or pregnancy
with a small for gestation age infant.
74. The method of claim 73, wherein said pregnancy related
hypertensive disorder is pre-eclampsia or eclampsia.
75. The method of claim 72, wherein said alteration in said levels
is an increase.
76. The method of claim 72, wherein said endoglin nucleic acid is a
soluble endoglin nucleic acid.
77. The method of claim 72, further comprising measuring the level
of at least one of sFlt-1, VEGF, or PlGF nucleic acid molecule in a
sample from said subject and comparing it to a reference sample,
wherein an alteration in said level diagnoses a pregnancy related
hypertensive disorder or a predisposition to a pregnancy related
hypertensive disorder.
78. The method of claim 72, wherein said measuring of levels is
done on two or more occasions and a change in said levels between
measurements is a diagnostic indicator of said pregnancy related
hypertensive disorder.
79. A method of diagnosing a subject as having, or having a
predisposition to a pregnancy related hypertensive disorder, said
method comprising determining the nucleic acid sequence of an
endoglin gene in a sample from a subject and comparing it to a
reference sequence, wherein an alteration in the subject's nucleic
acid sequence that is an alteration that changes the expression
level of the gene product in said subject diagnoses the subject
with a pregnancy related hypertensive disorder or a predisposition
to a pregnancy related hypertensive disorder.
80. The method of claim 55, 72, or 79, wherein said sample is a
bodily fluid of said subject in which said soluble endoglin is
normally detectable.
81. The method of claim 80, wherein said bodily fluid is selected
from the group consisting of urine, amniotic fluid, blood, serum,
plasma, and cerebrospinal fluid.
82. The method of claim 55, 72, or 79, wherein said sample is a
cell or a tissue.
83. The method of claim 82, wherein said cell is selected from the
group consisting of an endothelial cell, a leukocyte, a monocyte,
or a cell derived from the placenta.
84. The method of claim 82, wherein said tissue is a placental
tissue.
85. The method of claim 55, 72, or 79, wherein said subject is a
non-pregnant human, a pregnant human, a post-partum human, or a
non-human and said method diagnoses a predisposition to
pre-eclampsia or eclampsia.
86. The method of claim 85, wherein said non-human is selected from
the group consisting of a cow, a horse, a sheep, a pig, a goat, a
dog, or a cat.
87. The method of claim 55, 72, or 79, wherein said method is used
to diagnose a pregnancy related hypertensive disorder, or a
predisposition to a pregnancy related hypertensive disorder, at
least 4 weeks prior to the onset of symptoms.
88. A kit for the diagnosis of a pregnancy related hypertensive
disorder, or a predisposition to a pregnancy related hypertensive
disorder, in a subject comprising a nucleic acid molecule having an
endoglin nucleic acid sequence or a sequence complementary thereto,
or any combination thereof, and instructions for using said nucleic
acid molecule to diagnose a pregnancy related hypertensive disorder
or a predisposition to a pregnancy related hypertensive
disorder.
89. The kit of claim 88, wherein said kit further comprises a
nucleic acid sequence of VEGF, sFlt-1 or PlGF or a sequence
complementary thereto, or any combination thereof.
90. The kit of claim 88, wherein said endoglin nucleic acid is a
soluble endoglin nucleic acid.
91. A kit for the diagnosis of a pregnancy related hypertensive
disorder, or a predisposition to a pregnancy related hypertensive
disorder, in a subject comprising a soluble endoglin binding
molecule and instructions for the use of said soluble endoglin
binding molecule for the diagnosis of said pregnancy related
hypertensive disorder, or a predisposition to a pregnancy related
hypertensive disorder.
92. The kit of claim 91, wherein said soluble endoglin binding
molecule is an antibody, or antigen-binding fragment thereof, that
specifically binds soluble endoglin.
93. The kit of claim 91, wherein said kit further comprises a VEGF,
sFlt-1 or PlGF binding molecule.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date of
U.S. provisional application No. 60/613,170, filed Sep. 24, 2004,
herein incorporated by reference.
FIELD OF THE INVENTION
[0003] In general, this invention relates to the detection and
treatment of subjects having a pregnancy related hypertensive
disorder.
BACKGROUND OF THE INVENTION
[0004] Pre-eclampsia is a syndrome of hypertension, edema, and
proteinuria that affects 5 to 10% of pregnancies and results in
substantial maternal and fetal morbidity and mortality.
Pre-eclampsia accounts for at least 200,000 maternal deaths
worldwide per year. The symptoms of pre-eclampsia typically appear
after the 20.sup.th week of pregnancy and are usually detected by
routine measuring of the woman's blood pressure and urine. However,
these monitoring methods are ineffective for diagnosis of the
syndrome at an early stage, which could reduce the risk to the
subject or developing fetus, if an effective treatment were
available.
[0005] Currently there are no known cures for pre-eclampsia.
Pre-eclampsia can vary in severity from mild to life threatening. A
mild form of pre-eclampsia can be treated with bed rest and
frequent monitoring. For moderate to severe cases, hospitalization
is recommended and blood pressure medication or anticonvulsant
medications to prevent seizures are prescribed. If the condition
becomes life threatening to the mother or the baby the pregnancy is
terminated and the baby is delivered pre-term.
[0006] The proper development of the fetus and the placenta is
mediated by several growth factors or angiogenic factors. One of
these angiogenic factors is endoglin, also known as CD105. Endoglin
is a homodimeric cell membrane glycoprotein that is predominantly
expressed on endothelial cells such as syncytiotrophoblasts, human
unbilical vein endothelial cells (HUVEC), and on vascular
endothelial cells. Endoglin shares sequence identity with
betaglycan, a transforming growth factor (TGF)-.beta. receptor type
III. Endoglin has been shown to be a regulatory component of the
TGF-.beta. receptor complex, which modulates angiogenesis,
proliferation, differentiation, and apoptosis. Endoglin also binds
several other members of the TGF-.beta. superfamily including
activin-A, bone morphogenic protein (BMP)-2 and BMP-7. In
particular, endoglin binds TGF-.beta.1 and TGF-.beta.3 with high
affinity and forms heterotrimeric associations with the TGF-.beta.
signaling receptors types I and II. Mutations in the coding region
of the endoglin gene are responsible for haemorrhagic
telangiectasia type 1 (HHT1), a dominantly inherited vascular
disorder characterized by multisystemic vascular dysplasia and
recurrent hemorrhage. A soluble form of endoglin has also been
identified and found to be present at increased levels in patients
with metastatic breast and colorectal cancer; however, the exact
functional role of the soluble endoglin in the pathogenesis of
cancer is unclear. Soluble endoglin production has not been
reported to be associated with pre-eclampsia or normal
pregnancy.
[0007] Several factors have been reported to have an association
with fetal and placental development and, more specifically, with
pre-eclampsia. They include vascular endothelial growth factor
(VEGF), soluble Flt-1 receptor (sFlt-1), and placental growth
factor (PlGF). VEGF is an endothelial cell-specific mitogen, an
angiogenic inducer, and a mediator of vascular permeability. VEGF
has also been shown to be important for glomerular capillary
repair. VEGF binds as a homodimer to one of two homologous
membrane-spanning tyrosine kinase receptors, the fms-like tyrosine
kinase (Flt-1) and the kinase domain receptor (KDR), which are
differentially expressed in endothelial cells obtained from many
different tissues. Flt-1, but not KDR, is highly expressed by
trophoblast cells which contribute to placental formation. PlGF is
a VEGF family member that is also involved in placental
development. PlGF is expressed by cytotrophoblasts and
syncytiotrophoblasts and is capable of inducing proliferation,
migration, and activation of endothelial cells. PlGF binds as a
homodimer to the Flt-1 receptor, but not the KDR receptor. Both
PlGF and VEGF contribute to the mitogenic activity and angiogenesis
that are critical for the developing placenta.
[0008] sFlt-1, which lacks the transmembrane and cytoplasmic
domains of the receptor, was recently identified in a cultured
medium of human umbilical vein endothelial cells and in vivo
expression was subsequently demonstrated in placental tissue.
sFlt-1 binds to VEGF with a high affinity but does not stimulate
mitogenesis of endothelial cells.
[0009] Careful regulation of angiogenic and mitogenic signaling
pathways is critical for maintaining appropriate proliferation,
migration, and angiogenesis by trophoblast cells in the developing
placenta.
[0010] There is a need for methods of accurately diagnosing
subjects at risk for or having pre-eclampsia or eclampsia,
particularly before the onset of the most severe symptoms. A
treatment is also needed.
SUMMARY OF THE INVENTION
[0011] We have discovered methods for diagnosing and treating
pregnancy related hypertensive disorders, including pre-eclampsia
and eclampsia.
[0012] Using gene expression analysis, we have discovered that
levels of soluble endoglin (sE) are markedly elevated in placental
tissue samples from pregnant women suffering from pregnancy
complications associated with hypertension, including
pre-eclampsia. Endoglin is a part of the TGF-.beta. receptor
complex that acts to regulate angiogenesis. Endoglin can bind with
high affinity to TGF-.beta. family members that are ligands for
TGF-.beta. receptors. In affected individuals, excess soluble
endoglin may be depleting the placenta of necessary amounts of
essential angiogenic and mitogenic factors. In the present
invention, compounds that bind to or neutralize soluble endoglin
are used to reduce the elevated levels of soluble endoglin. In
addition, antibodies directed to soluble endoglin as well as RNA
interference and antisense nucleobase oligomers directed to
lowering the levels of biologically active soluble endoglin are
also provided. Finally, the present invention provides for the
measuring of soluble endoglin levels as a detection tool for early
diagnosis and management of pregnancy related hypertensive
disorders, including pre-eclampsia and eclampsia, or a
predisposition thereto.
[0013] Accordingly, in one aspect, the invention provides a method
of treating or preventing a pregnancy related hypertensive
disorder, such as pre-eclampsia or eclampsia, in a subject by
administering to the subject a compound capable of binding to
soluble endoglin, where the administering is for a time and in an
amount sufficient to treat or prevent at least one symptom of the
pregnancy related hypertensive disorder in the subject.
Non-limiting examples of pregnancy related hypertensive disorders
include pre-eclampsia, eclampsia, gestational hypertension, chronic
hypertension, HELLP syndrome, and pregnancy with a small for
gestational age infant (SGA). In a preferred embodiment, the
compound is a purified soluble endoglin antibody or antigen-binding
fragment thereof that specifically binds to soluble endoglin. In
another preferred embodiment, the compound is a growth factor, such
as a TGF-.beta. family member (e.g., TGF-.beta.1, TGF-.beta.3,
activin-A, BMP-2, and BMP-7) or a fragment thereof capable of
specifically binding soluble endoglin.
[0014] In another preferred embodiment, the method also includes
administering a compound, such as a purified sFlt-1 antibody, a
sFlt-1 antigen-binding fragment, nicotine, theophylline, adenosine,
nifedipine, minoxidil, magnesium sulfate, vascular endothelial
growth factor (VEGF), including all isoforms such as VEGF189,
VEGF121, VEGF165, or fragments thereof or placental growth factor
(PlGF), including all isoforms and fragments thereof, where the
administering is for a time and in an amount sufficient to treat or
prevent the pregnancy related hypertensive disorder in a
subject.
[0015] In another aspect, the invention features a method of
treating or preventing a pregnancy related hypertensive disorder,
such as pre-eclampsia or eclampsia, in a subject by administering
to the subject a compound (e.g., chemical compound, polypeptide,
peptide, antibody, or a fragment thereof) that increases the level
of a growth factor capable of binding to soluble endoglin. The
compound is administered for a time and in an amount sufficient to
treat or prevent the pregnancy related hypertensive disorder. In
preferred embodiments, the compound increases the level of a
TGF-.beta. family member (e.g., TGF-.beta.1, TGF-.beta.3,
activin-A, BMP-2, and BMP-7) and fragments thereof. Non-limiting
examples of such compounds include cyclosporine, alpha tocopherol,
methysergide, bromocriptine, and aldomet.
[0016] In another related aspect, the invention features a method
of treating or preventing a pregnancy related hypertensive disorder
in a subject by administering to the subject a compound (e.g.,
chemical compound, polypeptide, peptide, antibody, or a fragment
thereof) that inhibits growth factor binding to a soluble endoglin
polypeptide. The compound is administered for a time and in an
amount sufficient to treat or prevent pregnancy related
hypertensive disorder. In preferred embodiments, the compound binds
to soluble endoglin and prevents growth factor binding.
Non-limiting examples of such compounds include antibodies and
small-molecule compounds obtained through screening.
[0017] In another aspect, the invention provides a method of
treating or preventing a pregnancy related hypertensive disorder,
such as pre-eclampsia or eclampsia, in a subject by administering
to the subject a compound capable of reducing soluble endoglin
expression or biological activity, where the administering is
sufficient to treat or prevent the pregnancy related hypertensive
disorder in the subject. In preferred embodiments, the compound is
a purified antibody or antigen binding fragment thereof or a
compound that inhibits the enzymatic activity of a proteolytic
enzyme selected from the group consisting of: a matrix
metalloproteinae (MMP), a cathepsin, or an elastase. MMPs include
any one of MMP 1-26, preferably MMP9 or membrane-type MMP1.
[0018] Desirably, the compound capable of inhibiting the biological
activity of soluble endoglin is identified by its ability to
inhibit the angiogenic activity of endoglin as measured by an
angiogenesis assay. In one example of such an assay, serum from a
pre-eclamptic patient is used in a matrigel tube formation assay to
induce an anti-angiogenic state. The compound is then added and a
reduction in the anti-angiogenic state by 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90% or more is indicative of a therapeutically
effective compound.
[0019] The compound capable of inhibiting the biological activity
of soluble endoglin can be an antisense nucleobase oligomer having
at least one strand that is at least 80%, preferably 85%, 90%, 95%,
99%, or 100% complementary to at least a portion of the sequence of
soluble endoglin. In one embodiment, the antisense nucleobase
oligomer is complementary to at least 8, 10, preferably 20, 30, 40,
50, 60, 70, 80, 90, 100, or more consecutive nucleotides of soluble
endoglin and can reduce or inhibit the expression or biological
activity of soluble endoglin. Desirably, the antisense nucleobase
oligomer is 8 to 30 nucleotides in length.
[0020] The compound capable of inhibiting the biological activity
of soluble endoglin can also be a double stranded RNA (dsRNA)
molecule having at least one strand that is at least 80%,
preferably 85%, 90%, 95%, 99%, or 100% complementary to at least a
portion of the sequence of a soluble endoglin nucleic acid
molecule. In one embodiment, the double stranded RNA is a small
interfering RNA (siRNA) that is 19 to 25 nucleotides in length and
can reduce or inhibit the expression or biological activity of
soluble endoglin. In additional preferred embodiments, the dsRNA
has 100% nucleic acid identity to at least 18, preferably 19, 20,
21, 22, 23, 24, 25, 35, 45, 50 or more consecutive nucleotides of
the nucleic acid sequence of a soluble endoglin molecule.
Desirably, the dsRNA is an siRNA.
[0021] In various embodiments of any of the above aspects, the
method further involves the step of administering to a subject an
anti-hypertensive compound (e.g., adenosine, nifedipine, minoxidil,
and magnesium sulfate). In other embodiments of the above aspects,
the subject is a pregnant human, a post-partum human, a
non-pregnant human, or a non-human (e.g., a cow, a horse, a sheep,
a pig, a goat, a dog, or a cat). The therapeutic methods of the
invention can be used to treat or prevent a pregnancy related
hypertensive disorder that includes pre-eclampsia, eclampsia,
gestational hypertension, chronic hypertension, HELLP syndrome, and
pregnancy with an SGA infant. Preferred disorders are pre-eclampsia
and eclampsia. In various embodiments of the above aspects, the
method can be combined with the diagnostic methods of the
invention, described below, to monitor the subject during therapy
or to determine effective therapeutic dosages.
[0022] Any of the therapeutic aspects of the invention can also
include administering one ore more additional compounds, such as a
purified sFlt-1 antibody, a sFlt-1 antigen-binding fragment,
nicotine, theophylline, adenosine, nifedipine, minoxidil, magnesium
sulfate, vascular endothelial growth factor (VEGF), including all
isoforms such as VEGF189, VEGF121, or VEGF165, or fragments
thereof; placental growth factor (PlGF), including all isoforms and
fragments thereof, where the administering is for a time and in an
amount sufficient to treat or prevent pre-eclampsia or eclampsia in
a subject. Preferred examples of such compounds are described in
U.S. patent application Publication Nos. 20040126828 and
20050025762 and PCT Publication Number WO 2004/008946. Desirably,
the compound will be a compound capable of binding to sFlt-1 or
decreasing sFlt-1 expression.
[0023] Any of the therapeutic aspects of the invention can be used
alone or in combination with one or more additional methods of the
invention. In one example, an MMP inhibitor and an antibody can be
used in combination to neutralize the soluble endoglin that is
present and to block the further production of soluble endoglin by
cleavage of the membrane bound form.
[0024] In another aspect, the invention features a purified
antibody or antigen-binding fragment thereof that specifically
binds soluble endoglin. In one preferred embodiment, the antibody
prevents binding of a growth factor (e.g., TGF-.beta.1,
TGF-.beta.3, activin-A, BMP-2, and BMP-7) to soluble endoglin. In
another embodiment, the antibody is a monoclonal antibody. In other
preferred embodiments, the antibody or antigen-binding fragment
thereof is a human or humanized antibody. In other embodiments, the
antibody lacks an Fc portion. In still other embodiments, the
antibody is an F(ab').sub.2, an Fab, or an Fv structure. In other
embodiments, the antibody or antigen-binding fragment thereof is
present in a pharmaceutically acceptable carrier.
[0025] In another aspect, the invention provides a method of
diagnosing a subject as having, or having a predisposition to, a
pregnancy related hypertensive disorder, such as pre-eclampsia or
eclampsia, involving measuring the level of soluble endoglin
polypeptide in a sample from the subject. In preferred embodiments
the level of soluble endoglin is the level of free, bound, or total
soluble endoglin. In some enbodiments, the level of soluble
endoglin is the level of a soluble endoglin polypeptide resulting
from degradation or enzymatic cleavage. The diagnosis of a
pregnancy related hypertensive disorder or a predisposition to a
pregnancy related hypertensive disorder can result from an
alteration (e.g., an increase) in the relative level of soluble
endoglin as compared to a normal reference sample or from the
detection of an absolute level of soluble endoglin that is above a
normal reference value. For example, normally, circulating serum or
plasma concentrations of soluble endoglin range from 2-7 ng/ml
during the non-pregnant state and from 10-20 ng/ml during normal
pregnancy. For embodiments that include the measurement of the
absolute levels of soluble endoglin, a level greater than 15 ng/ml,
20 ng/ml, or preferably greater than 25 ng/ml, is considered a
diagnostic indicator of a pregnancy related hypertensive disorder,
such as pre-eclampsia or eclampsia. In additional preferred
embodiments, the method further includes measuring the level of at
least one of sFlt-1, VEGF, or PlGF polypeptide in a sample from a
subject as described in U.S. patent application Publication Nos.
20040126828, 20050025762, and 2005017044 and PCT Publication
Numbers WO 2004/008946 and WO 2005/077007. The method can also
include measuring the level of at least two of sFlt-1, VEGF, or
PlGF polypeptide in a sample from a subject and calculating the
relationship between the levels of sFlt-1, VEGF, or PlGF using a
metric, where an alteration in the subject sample relative to a
reference sample diagnoses a pregnancy related hypertensive
disorder or a predisposition to a pregnancy related hypertensive
disorder. In preferred embodiments, the method also includes
determining the body mass index (BMI), the gestational age (GA) of
the fetus, or both and including the BMI or GA or both in the
metric. In one embodiment, the metric is a pre-eclampsia
anti-angiogenic index (PAAI): [sFlt-1/VEGF+PlGF], where the PAAI is
used as an indicator of anti-angiogenic activity. In one
embodiment, a PAAI greater than 10, more preferably greater than
20, is indicative of pre-eclampsia or eclampsia. In another
embodiment the metric is the following soluble endoglin
anti-angiogenic index: (sFlt-1+0.25(soluble endoglin
polypeptide))/PlGF. An increase in the value of the soluble
endoglin anti-angiogenic index is a diagnostic indicator of a
pregnancy related hypertensive disorder, such as pre-eclampsia or
eclampsia. For example a value above 75 during weeks 21-32 weeks or
a value above 100 after weeks>32 weeks diagnoses a pregnancy
related hypertensive disorder, such as pre-eclampsia or eclampsia.
Another metric useful in the diagnostic methods of the invention
is: (soluble endoglin+sFlt-1)/PlGF. Any of the methods can also
include determining the body mass index (BMI), the gestational age
(GA) of the fetus, or both and including the BMI or GA or both in
the metric.
[0026] In various embodiments of the above aspects, the sample is a
bodily fluid, such as urine, amniotic fluid, blood, serum, plasma,
and cerebrospinal fluid. Desirably, the level of soluble endoglin,
sFlt-1, VEGF, or PlGF polypeptide is determined by an immunological
assay, such as an ELISA. In another example, a level of soluble
endoglin greater than 20, preferably greater than 25 ng/ml, alone
or in combination with increased sFlt-1 and decreased free PlGF or
VEGF is used for the diagnosis of a pregnancy related hypertensive
disorder, such as pre-eclampsia or eclampsia.
[0027] In one embodiment, an increase in the level of soluble
endoglin is indicative of pre-eclampsia or eclampsia or the
propensity to develop pre-eclampsia or eclampsia. In preferred
embodiments of the above aspects, the level of sFlt-1 polypeptide
measured is the level of free, bound, or total sFlt-1 polypeptide.
In additional embodiments, the sFlt-1 polypeptide can also include
sFlt-1 fragments, degradation products, or enzymatic cleavage
products. In other preferred embodiments of the above aspects, the
level of VEGF or PlGF is the level of free VEGF or PlGF.
[0028] In another aspect, the invention provides a method of
diagnosing a subject as having, or having a predisposition to,
pregnancy related hypertensive disorder, such as pre-eclampsia or
eclampsia that includes measuring the level of an endoglin nucleic
acid molecule (e.g., mRNA), preferably a soluble endoglin nucleic
acid, in a sample from the subject and comparing it to a reference
sample, where an alteration (e.g., an increase) in the levels
relative to a reference sample (e.g., a normal reference) diagnoses
a pregnancy related hypertensive disorder, such as pre-eclampsia or
eclampsia, in the subject, or diagnoses a predisposition to
pregnancy related hypertensive disorder, such as pre-eclampsia or
eclampsia. In additional embodiments, the method can further
include measuring the level of a sFlt-1, VEGF, or PlGF nucleic acid
molecule (e.g., mRNA) in a sample from the subject and comparing it
to a reference sample, where an alteration (e.g., a decrease in the
level of VEGF or PlGF or an increase in the level of sFlt-1) in the
levels relative to a normal reference sample diagnoses a pregnancy
related hypertensive disorder, such as pre-eclampsia or eclampsia
in the subject, or diagnoses a predisposition to a pregnancy
related hypertensive disorder, such as pre-eclampsia or
eclampsia.
[0029] In preferred embodiments of the above diagnostic aspects,
the levels are measured on two or more occasions and a change in
the levels between measurements is a diagnostic indicator of
pregnancy related hypertensive disorder, such as pre-eclampsia or
eclampsia. In one preferred embodiment, an increase (e.g., 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or
greater) in the level of soluble endoglin from the first
measurement to the next measurement is used to diagnose pregnancy
related hypertensive disorder, such as pre-eclampsia or eclampsia.
In another embodiment of the above diagnostic aspects, the levels
of soluble endoglin are compared to a normal reference sample and
an increase (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, 96%, 97%, 98%, 99% or greater) in the level of soluble
endoglin as compared to a normal reference sample is indicative of
pre-eclampsia or eclampsia.
[0030] In another aspect, the invention provides a method of
diagnosing a subject as having, or having a predisposition to, a
pregnancy related hypertensive disorder, such as pre-eclampsia or
eclampsia, that includes determining the nucleic acid sequence of
an endoglin gene in a subject and comparing it to a reference
sequence, where an alteration in the subject's nucleic acid
sequence that changes the level or the biological activity of the
gene product in the subject diagnoses the subject with a pregnancy
related hypertensive disorder, or a predisposition to a pregnancy
related hypertensive disorder. In one embodiment, the alteration is
a polymorphism in the nucleic acid sequence. In another embodiment,
the nucleic acid sequence of sFlt-1, VEGF, or PlGF, or any
combination thereof, gene is also determined and compared to a
reference sequence. An alteration in any one or more of these
sequences that changes the level or the biological activity of the
gene product in the subject diagnoses the subject with a pregnancy
related hypertensive disorder.
[0031] In various embodiments of the above aspects, the sample is a
bodily fluid (e.g., urine, amniotic fluid, blood, serum, plasma, or
cerebrospinal fluid) of the subject in which the soluble endoglin
and the sFlt-1, VEGF, or PlGF is normally detectable. In additional
embodiments, the sample is a tissue or a cell (e.g., placental
tissue or placental cells, endothelial cells, leukocytes, and
monocytes). In other embodiments of the above aspects, the subject
is a non-pregnant human, a pregnant human, or a post-partum human.
In other embodiments of the above aspects, the subject is a
non-human (e.g., a cow, a horse, a sheep, a pig, a goat, a dog, or
a cat). In one embodiment, the subject is a non-pregnant or
pregnant human and the method is used to diagnose a predisposition
to pre-eclampsia or eclampsia. In additional embodiments, the BMI
or GA or both is also measured. In various embodiments of the above
aspects, an increase in the level of soluble endoglin nucleic acid
or polypeptide relative to a reference is a diagnostic indicator of
pre-eclampsia or eclampsia.
[0032] In another aspect, the invention provides a method of
diagnosing a subject as having, or having a predisposition to,
pregnancy related hypertensive disorder, such as pre-eclampsia or
eclampsia that includes measuring the level of a soluble endoglin
ligand, such as TGF-.beta.1, TGF-.beta.3, activin-A, BMP-2, and
BMP-7 in a sample from a subject.
[0033] In various embodiments of any of the above diagnostic
aspects, the pregnancy related hypertensive disorder is
pre-eclampsia, eclampsia, gestational hypertension, chrnoic
hypertension, HELLP syndrome, or pregnancy with an SGA infant. In
any of the diagnostic aspects, the measuring of levels is done on
two or more occasions and an increase in the levels between
measurements us a diagnostic indicator of the pregnancy related
hypertensive disorder. The diagnostic methods are desirable used to
diagnose a pregnancy related hypertensive disorder prior to the
onset of symptoms (e.g., at least 4, 5, 6, 7, 8, 9, or 10 weeks
prior).
[0034] In another aspect, the invention provides a kit for the
diagnosis of a pregnancy related hypertensive disorder, such as
pre-eclampsia or eclampsia, in a subject comprising a nucleic acid
sequence useful for detecting an endoglin nucleic acid, or fragment
thereof, or a sequence complementary thereto. In a preferred
embodiment, the nucleic acid sequence hybridizes, preferably at
high stringency, to a nucleic acid encoding soluble endoglin, or a
fragment thereof. In a preferred embodiment, the kit further
comprises a nucleic acid sequence for detecting sFlt-1, VEGF, or
PlGF.
[0035] In a related aspect, the invention provides a kit for the
diagnosis of a pregnancy related hypertensive disorder, such as
pre-eclampsia or eclampsia, in a subject that includes a soluble
endoglin binding molecule (e.g., an antibody or antigen binding
fragment thereof that specifically binds soluble endoglin). In one
embodiment, the component is an immunological assay, an enzymatic
assay, or a colorimetric assay. In other embodiments of the above
aspects, the kit diagnoses a predisposition to pre-eclampsia or
eclampsia in a pregnant or a non-pregnant subject. In preferred
embodiments of the above aspects, the kit also includes a component
for detecting sFlt-1, VEGF, or PlGF polypeptide. In additional
preferred embodiments, the kit is used to detect soluble endoglin
and to further detect VEGF, sFlt-1 and PlGF and determine a
diagnostic ratio for the sample (e.g., PAAI or soluble endoglin
anti-angiogenic index).
[0036] In preferred embodiments, the diagnostic kits include a
label or instructions for the intended use of the kit components.
In one embodiment, the diagnostic kit is labeled or includes
instructions for use in the diagnosis of a pregnancy related
hypertensive disorder, such as pre-eclampsia or eclampsia, or a
predisposition to a pregnancy related hypertensive disorder, such
as pre-eclampsia or eclampsia in a subject. In a preferred
embodiment, the diagnostic kit includes a label or instructions for
the use of the kit to determine the levels of soluble endoglin of
the subject sample and to compare the soluble endoglin levels to a
reference value. It will be understood that the reference values
will depend on the intended use of the kit. For example, the sample
can be compared to a normal soluble endoglin reference value,
wherein an increase in the soluble endoglin levels is indicative of
a pregnancy related hypertensive disorder, such as pre-eclampsia or
eclampsia. The sample can also be compared to a reference that is a
value or a sample from a subject known to have pre-eclampsia,
wherein a decrease in the soluble endoglin levels is indicative of
a pregnancy related hypertensive disorder, such as pre-eclampsia or
eclampsia.
[0037] In a related aspect, the invention features a device for
diagnosing a subject as having or having a predisposition to a
pregnancy related hypertensive disorder, such as pre-eclampsia or
eclampsia.by providing components for measuring and/or comparing
the levels of soluble endoglin polypeptide or nucleic acid to a
reference sample, wherein an alteration in the levels of soluble
endoglin compared to a normal reference value diagnoses a pregnancy
related hypertensive disorder, such as pre-eclampsia or eclampsia
in the subject. In preferred embodiments, the device includes a
membrane in a lateral flow or dipstick format used to measure and
compare polypeptide levels in urine sample.
[0038] The device can also include components for comparing the
levels of soluble endoglin and at least one of sFlt-1, VEGF, and
PlGF nucleic acid molecules or polypeptides in a sample from a
subject relative to a reference sample, wherein an alteration in
the levels of soluble endoglin, and at least one of sFlt-1, VEGF,
and PlGF nucleic acid molecules or polypeptides diagnoses
pre-eclampsia or eclampsia or a predisposition to pre-eclampsia or
eclampsia in the subject. In a preferred embodiment the device
includes components for a metric to compare the levels of soluble
endoglin, and at least one of sFlt-1, VEGF, and PlGF
polypeptides.
[0039] Any of the diagnostic methods and kits described herein can
also be used to monitor a subject already diagnosed as having or
being at risk for having pre-eclampsia or eclampsia in order to
monitor the subject during therapy or to determine effective
therapeutic dosages. In one example, a kit used for therapeutic
monitoring can have a reference soluble endoglin value that is
indicative of pre-eclampsia or eclampsia, wherein a decrease in the
soluble endoglin value of the subject sample relative to the
reference sample can be used to indicate therapeutic efficacy or
effective dosages of therapeutic compounds. In preferred
embodiments, the kit is labeled or includes instructions for use in
therapeutic monitoring or therapeutic dosage determination and the
therapeutic compound can be included in the kit. The level of
soluble endoglin protein or nucleic acid is measured alone or in
combination with the levels of sFlt-1, VEGF, or PlGF protein or
nucleic acids, or any combination thereof. In additional preferred
embodiments, the level of soluble endoglin is compared to a
reference sample that is indicative of a pregnancy related
hypertensive disorder, such as pre-eclampsia or eclampsia, and an
alteration (e.g., a decrease) in the levels of soluble endoglin
relative to the reference sample is indicative of therapeutic
efficacy or an effective dosage of a therapeutic compound. In one
example, a decrease (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90% or more) in the level of soluble endoglin polypeptide or
nucleic acid measured during or after administering therapy
relative to the value before therapy is admininistered indicates an
improvement in the pregnancy related hypertensive disorder. In
another example, the absolute levels of soluble endoglin in the
serum or plasma are measured and used for monitoring the
therapeutic efficacy of that compound. For example, a therapeutic
compound is preferably administered in a dose such that the level
of soluble endoglin is less than 25 ng/ml, preferably less than 20
ng/ml.
[0040] In embodiments of the above therapeutic monitoring aspects
that include the measurement of sFlt-1, a decrease in the level of
sFlt-1 polypeptide or nucleic acid indicates an improvement in the
pre-eclampsia or eclampsia. In another embodiment, a therapeutic
compound is administered in a dose such that the level of sFlt-1
polypeptide is less than 2 ng/ml. In embodiments that include the
measurement of VEGF or PlGF, an increase in the level of VEGF or
PlGF polypeptide or nucleic acid measured during or after
administering therapy relative to the value before therapy
indicates an improvement in the pre-eclampsia or eclampsia. In
embodiments that include the measurement of sFlt-1, VEGF, or PlGF
in addition to the measurement of soluble endoglin, the method can
include calculating the relationship between the levels of sFlt-1,
VEGF, or PlGF using a metric, wherein an alteration in the
relationship between said levels in the subject sample relative to
a reference sample, is a diagnostic indicator of pre-eclampsia or
eclampsia. On example of such a metric is the PAAI. In this
example, a decrease in the PAAI value of a subject (e.g., less than
20, preferably less than 10) indicates an improvement in the
pre-eclampsia or eclampsia. A decrease in the PAAI (e.g., less than
20, preferably less than 10) can also indicate an effective dosage
of a therapeutic compound. Another example is the following soluble
endoglin anti-angiogenic index, wherein an increase in the value is
a diagnostic indicator of a pregnancy related hypertensive
disorder, such as pre-eclampsia or eclampsia.
[0041] In preferred embodiments of the aspects relating to
diagnosis or monitoring of therapeutic treatments, polypeptides are
measured using an immunological assay such as ELISA or western
blot. The level of soluble endoglin can be the level of free, bound
(i.e., bound to a ligand), or total (i.e., free+bound) soluble
endoglin, as well as the level of soluble endoglin resulting from
degradation or enzymatic cleavage. For any of the monitoring
methods, the measuring of levels can be done on two or more
occasions and a change in the levels between measurements is a
diagnostic indicator or pre-eclampsia or eclampsia.
[0042] In another aspect, the invention provides a method of
identifying a compound that ameliorates a pregnancy related
hypertensive disorder, such as pre-eclampsia or eclampsia, that
involves contacting a cell that expresses an endoglin nucleic acid
molecule with a candidate compound, and comparing the level of
expression of the nucleic acid molecule in the cell contacted by
the candidate compound with the level of expression in a control
cell not contacted by the candidate compound, where an alteration
in expression of the endoglin nucleic acid molecule identifies the
candidate compound as a compound that may be useful to ameliorate a
pregnancy related hypertensive disorder, such as pre-eclampsia or
eclampsia.
[0043] In another aspect, the invention provides a method of
identifying a compound that ameliorates a pregnancy related
hypertensive disorder, such as pre-eclampsia or eclampsia, that
involves contacting a cell that expresses a soluble endoglin
polypeptide with a candidate compound, and comparing the level of
expression of the polypeptide in the cell contacted by the
candidate compound with the level of polypeptide expression in a
control cell not contacted by the candidate compound, where an
alteration in the expression of the soluble endoglin polypeptide
identifies the candidate compound as a compound that may be useful
to ameliorate the pregnancy related hypertensive disorder. In one
embodiment, the alteration in expression is assayed using an
immunological assay, an enzymatic assay, or an immunoassay. In one
embodiment, the alteration in expression is a decrease in the level
of soluble endoglin. The alteration in expression can result from
an alteration in transcription or an alteration in translation.
[0044] In another aspect, the invention provides a method of
identifying a compound that ameliorates a pregnancy related
hypertensive disorder, such as pre-eclampsia or eclampsia, that
involves contacting a cell that expresses a soluble endoglin
polypeptide with a candidate compound, and comparing the biological
activity of the soluble endoglin polypeptide in the cell contacted
by the candidate compound with the level of biological activity in
a control cell not contacted by the candidate compound, where an
alteration in the biological activity of the soluble endoglin
polypeptide identifies the candidate compound as a compound that
ameliorates the pregnancy related hypertensive disorder. In one
embodiment, the alteration is a decrease in the biological activity
of soluble endoglin as assayed using an angiogenesis assay, a
growth factor binding assay, or any of the assays described
herein.
[0045] In another aspect, the invention provides a method of
identifying a compound that ameliorates a pregnancy related
hypertensive disorder, such as pre-eclampsia or eclampsia,
comprising detecting binding of a soluble endoglin polypeptide and
a candidate compound, where a compound that binds the soluble
endoglin polypeptide may be useful to ameliorate a pregnancy
related hypertensive disorder.
[0046] In another aspect, the invention provides a method of
identifying a compound that ameliorates a pregnancy related
hypertensive disorder, such as pre-eclampsia or eclampsia, that
involves detecting binding between a soluble endoglin polypeptide
and a growth factor in the presence of a candidate compound, where
a decrease in the binding, relative to binding between the soluble
endoglin polypeptide and the growth factor in the absence of the
candidate compound identifies the candidate compound as a compound
that may be useful to ameliorate the pregnancy related hypertensive
disorder. In one embodiment, the growth factor is a TGF-.beta.
family member.
[0047] In another aspect, the invention provides a method of
identifying a polypeptide that prevents binding between a soluble
endoglin polypeptide and a growth factor. The method involves
detecting binding between a soluble endoglin polypeptide and a
growth factor in the presence of the candidate polypeptide, where a
decrease in the binding, relative to binding between the soluble
endoglin polypeptide and the growth factor in the absence of the
candidate polypeptide identifies the candidate polypeptide as a
polypeptide that prevents binding between a soluble endoglin
polypeptide and a growth factor. In one embodiment, the growth
factor is a TGF-.beta. family member.
[0048] In a related aspect, the invention provides a compound
identified according to the previous aspect, where the compound is
a polypeptide that specifically binds a soluble endoglin
polypeptide and prevents the soluble endoglin polypeptide from
binding a TGF-.beta. family member. In one preferred embodiment,
the polypeptide is an antibody that binds soluble endoglin,
preferably an antibody that specifically binds soluble
endoglin.
[0049] While the methods described herein refer to pre-eclampsia
and eclampsia specifically, it should be understood that the
diagnostic and monitoring methods of the invention also apply to
general complications of pregnancy associated with hypertension
including but not limited to gestational hypertension, HELLP
syndrome, and pregnancy with a small for gestational age (SGA)
infant.
[0050] For the purpose of the present invention, the following
abbreviations and terms are defined below.
[0051] By "alteration" is meant a change (increase or decrease) in
the expression levels of a gene or polypeptide as detected by
standard art known methods such as those described below. As used
herein, an alteration includes a 10% change in expression levels,
preferably a 25% change, more preferably a 40%, 50%, 60%, 70%, 80%,
90% or greater change in expression levels. "Alteration" can also
indicate a change (increase or decrease) in the biological activity
of any of the polypeptides of the invention (e.g., soluble
endoglin, sFlt-1, VEGF, or PlGF). As used herein, an alteration
includes a 10% change in biological activity, preferably a 25%
change, more preferably a 40%, 50%, 60%, 70%, 80%, 90% or greater
change in biological activity. Examples of biological activity for
soluble endoglin are angiogenesis and binding assays using known
ligands such as activin-A, BMP 2, BMP-7, TGF-.beta.1 and
TGF-.beta.3. The biological activity of soluble endoglin can be
measured by ligand binding assays, immunoassays, and angiogenesis
assays that are standard in the art or are described herein. An
example of such an assay is the in vitro matrigel endothelial tube
formation assay in which antagonism of endoglin signaling led to
massive loss of capillary formation (Li et al., Faseb Journal
14:55-64 (2000)). Other examples of biological activity for PlGF or
VEGF include binding to receptors as measured by immunoassays,
ligand binding assays or Scatchard plot analysis, and induction of
cell proliferation or migration as measured by BrdU labeling, cell
counting experiments, or quantitative assays for DNA synthesis such
as .sup.3H-thymidine incorporation. Examples of biological activity
for sFlt-1 include binding to PlGF and VEGF as measured by
immunoassays, ligand binding assays, or Scatchard plot analysis.
Additional examples of assays for biological activity for each of
the polypeptides are described herein.
[0052] By "antisense nucleobase oligomer" is meant a nucleobase
oligomer, regardless of length, that is complementary to the coding
strand or mRNA of an endoglin gene. By a "nucleobase oligomer" is
meant a compound that includes a chain of at least eight
nucleobases, preferably at least twelve, and most preferably at
least sixteen bases, joined together by linkage groups. Included in
this definition are natural and non-natural oligonucleotides, both
modified and unmodified, as well as oligonucleotide mimetics such
as Protein Nucleic Acids, locked nucleic acids, and arabinonucleic
acids. Numerous nucleobases and linkage groups may be employed in
the nucleobase oligomers of the invention, including those
described in U.S. patent Publication Nos. 20030114412 (see for
example paragraphs 27-45 of the publication) and 20030114407 (see
for example paragraphs 35-52 of the publication), incorporated
herein by reference. The nucleobase oligomer can also be targeted
to the translational start and stop sites. Preferably the antisense
nucleobase oligomer comprises from about 8 to 30 nucleotides. The
antisense nucleobase oligomer can also contain at least 40, 60, 85,
120, or more consecutive nucleotides that are complementary to
endoglin mRNA or DNA, and may be as long as the full-length mRNA or
gene.
[0053] By "body mass index" is meant a number, derived by using
height and weight measurements, that gives a general indication of
whether or not weight falls within a healthy range. The formula
generally used to determine the body mass index is a person's
weight in kilograms divided by a person's height in meters squared
or weight (kg)/(height (m)).sup.2.
[0054] By "compound" is meant any small molecule chemical compound,
antibody, nucleic acid molecule, or polypeptide, or fragments
thereof. Compounds particularly useful for the therapeutic methods
of the invention can alter, preferably decrease, the levels or
biological activity of soluble endoglin by at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90% or more.
[0055] By "chimeric antibody" is meant a polypeptide comprising at
least the antigen-binding portion of an antibody molecule linked to
at least part of another protein (typically an immunoglobulin
constant domain).
[0056] By "double-stranded RNA (dsRNA)" is meant a ribonucleic acid
molecule comprised of both a sense and an anti-sense strand. dsRNAs
are typically used to mediate RNA interference.
[0057] By "endoglin," also known as CD105, is meant a mammalian
growth factor that has endoglin biological activity (see Fonsatti
et al., Oncogene 22:6557-6563, 2003; Fonsatti et al., Curr. Cancer
Drug Targets 3:427-432, 2003) and is homologous to the protein
defined by any of the following GenBank accession numbers: AAH29080
and NP.sub.--031958 (mouse); AAS67893 (rat); NP.sub.--000109,
P17813, VSP.sub.--004233, and CAA80673 (human); and A49722 (pig),
or described in U.S. Pat. No. 6,562,957. Endoglin is a homodimeric
cell membrane glycoprotein which is expressed at high levels in
proliferating vascular cells and syncytiotrophoblasts from
placentas. There are two distinct isoforms of endoglin, L and S,
which differ in their cytoplasmic tails by 47 amino acids. Both
isoforms are included in the term endoglin as used herein. Endoglin
binds to TGF-.beta. family members and, in the presence of
TGF-.beta., endoglin can associate with the TGF-.beta. signaling
receptors RI and RII, and potentiate the response to the growth
factors. Endoglin biological activities include binding to
TGF-.beta. family members such as activin-A, BMP 2, BMP-7,
TGF-.beta.1 and TGF-.beta.3; induction of angiogenesis, regulation
of cell proliferation, attachment, migration, invasion; and
activation of endothelial cells. Assays for endoglin biological
activities are known in the art and include ligand binding assays
or Scatchard plot analysis; BrdU labeling, cell counting
experiments, or quantitative assays for DNA synthesis such as
.sup.3H-thymidine incorporation used to measure cell proliferation;
and angiogenesis assays such as those described herein or in
McCarty et al., Intl. J. Oncol. 21:5- 10, 2002; Akhtar et al. Clin.
Chem. 49:32-40, 2003; and Yamashita et al, J. Biol. Chem.
269:1995-2001, 1994). By "soluble endoglin" is meant any
circulating, non-membrane bound form of endoglin which includes at
least a part of the extracellular portion of the protein (see FIG.
1). Soluble endoglin can result from the cleavage of the membrane
bound form of endoglin by a proteolytic enzyme. One potential
cleavage site is at amino acid 437 producing a soluble endoglin
polypeptide that includes amino acids 1-437 of the endoglin
polypeptide (see FIGS. 2A and 2B). Although not wishing to be bound
by theory, it is likely that the extracellular ligand binding
domain retained by soluble endoglin would allow it to bind ligands
such as TGF-.beta.1 and TGF-.beta.3, thereby creating a resulting
deficiency in TGF-.beta.. Furthermore, since endoglin is an
endothelial-specific molecule, it is likely that the TGF-.beta.
deficiencies would be maximal in endothelial cells. Soluble
endoglin can also include circulating degradation products or
fragments that result from enzymatic cleavage of endoglin and that
maintain endoglin biological activity. The biological function of
soluble endoglin is unknown at the present time, but is predicted
to create a deficiency of TGF-.beta. and other known ligands.
Soluble endoglin biological activity can be assayed by measuring
the levels of free Activin A or free TGF-.beta.3 or by using an
angiogenesis assay known in the art or described herein.
[0058] By "endoglin nucleic acid" is meant a nucleic acid that
encodes any of the endoglin proteins described above. For example,
the gene for human endoglin consists of 14 exons, where exon 1
encodes the signal peptide sequence, exons 2-12 encode the
extracellular domain, exon 13 encodes the transmembrane domain, and
exon 14 encodes C-terminal cytoplasmic domain (see FIGS. 1, 2A, and
2B). Desirable, the endoglin nucleic acid encodes soluble endoglin
(see FIG. 2A).
[0059] By "expression" is meant the detection of a gene or
polypeptide by standard art known methods. For example, polypeptide
expression is often detected by western blotting, DNA expression is
often detected by Southern blotting or polymerase chain reaction
(PCR), and RNA expression is often detected by northern blotting,
PCR, or RNAse protection assays.
[0060] By "fragment" is meant a portion of a polypeptide or nucleic
acid molecule. This portion contains, preferably, at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of
the reference nucleic acid molecule or polypeptide. Fragments may
contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400,
500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
Preferably, fragments of soluble endoglin include from 4 to 437
amino acids and from 10 to 1311 nucleotides.
[0061] By "gestational age" is meant a reference to the age of the
fetus, counting from the first day of the mother's last menstrual
period usually referred to in weeks.
[0062] By "gestational hypertension" is meant the development of
high blood pressure without proteinuria after 20 weeks of
pregnancy.
[0063] By a "history of pre-eclampsia or eclampsia" is meant a
previous diagnosis of pre-eclampsia or eclampsia or pregnancy
induced hypertension in the subject themselves or in a related
family member.
[0064] By "homologous" is meant any gene or protein sequence that
bears at least 30% homology, more preferably 40%, 50%, 60%, 70%,
80%, and most preferably 90% or more homology to a known gene or
protein sequence over the length of the comparison sequence. A
"homologous" protein can also have at least one biological activity
of the comparison protein. In general, for proteins, the length of
comparison sequences will be at least 10 amino acids, preferably
20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200,
250, 300, 350, 400, or at least 437 amino acids or more. For
nucleic acids, the length of comparison sequences will generally be
at least 25, 50, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500,
550, 600, 650, 700, 800, 900, 1000, 1100, 1200, or at least 1311
nucleotides or more. "Homology" can also refer to a substantial
similarity between an epitope used to generate antibodies and the
protein or fragment thereof to which the antibodies are directed.
In this case, homology refers to a similarity sufficient to elicit
the production of antibodies that can specifically recognize the
protein at issue.
[0065] By "humanized antibody" is meant an immunoglobulin amino
acid sequence variant or fragment thereof that is capable of
binding to a predetermined antigen. Ordinarily, the antibody will
contain both the light chain as well as at least the variable
domain of a heavy chain. The antibody also may include the CH1,
hinge, CH2, CH3, or CH4 regions of the heavy chain. The humanized
antibody comprises a framework region (FR) having substantially the
amino acid sequence of a human immunoglobulin and a complementarity
determining region (CDR) having substantially the amino acid
sequence of a non-human immunoglobulin (the "import"
sequences).
[0066] Generally, a humanized antibody has one or more amino acid
residues introduced into it from a source that is non-human. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains (Fab, Fab',
F(ab').sub.2, Fabc, Fv) in which all or substantially all of the
CDR regions correspond to those of a non-human immunoglobulin and
all or substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. By
"complementarity determining region (CDR)" is meant the three
hypervariable sequences in the variable regions within each of the
immunoglobulin light and heavy chains. By "framework region (FR)"
is meant the sequences of amino acids located on either side of the
three hypervariable sequences (CDR) of the immunoglobulin light and
heavy chains.
[0067] The FR and CDR regions of the humanized antibody need not
correspond precisely to the parental sequences, e.g., the import
CDR or the consensus FR may be mutagenized by substitution,
insertion or deletion of at least one residue so that the CDR or FR
residue at that site does not correspond to either the consensus or
the import antibody. Such mutations, however, will not be
extensive. Usually, at least 75%, preferably 90%, and most
preferably at least 95% of the humanized antibody residues will
correspond to those of the parental FR and CDR sequences.
[0068] By "hybridize" is meant pair to form a double-stranded
molecule between complementary polynucleotide sequences, or
portions thereof, under various conditions of stringency. (See,
e.g., Wahl and Berger Methods Enzymol. 152:399, 1987; Kimmel,
Methods Enzymol. 152:507, 1987.) For example, stringent salt
concentration will ordinarily be less than about 750 mM NaCl and 75
mM trisodium citrate, preferably less than about 500 mM NaCl and 50
mM trisodium citrate, and most preferably less than about 250 mM
NaCl and 25 mM trisodium citrate. Low stringency hybridization can
be obtained in the absence of organic solvent, e.g., formamide,
while high stringency hybridization can be obtained in the presence
of at least about 35% formamide, and most preferably at least about
50% formamide. Stringent temperature conditions will ordinarily
include temperatures of at least about 30.degree. C., more
preferably of at least about 37.degree. C., and most preferably of
at least about 42.degree. C. Varying additional parameters, such as
hybridization time, the concentration of detergent, e.g., sodium
dodecyl sulfate (SDS), and the inclusion or exclusion of carrier
DNA, are well known to those skilled in the art. Various levels of
stringency are accomplished by combining these various conditions
as needed. In a preferred embodiment, hybridization will occur at
30.degree. C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
In a more preferred embodiment, hybridization will occur at
37.degree. C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35%
formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In
a most preferred embodiment, hybridization will occur at 42.degree.
C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide,
and 200 .mu.g/ml ssDNA. Useful variations on these conditions will
be readily apparent to those skilled in the art.
[0069] For most applications, washing steps that follow
hybridization will also vary in stringency. Wash stringency
conditions can be defined by salt concentration and by temperature.
As above, wash stringency can be increased by decreasing salt
concentration or by increasing temperature. For example, stringent
salt concentration for the wash steps will preferably be less than
about 30 mM NaCl and 3 mM trisodium citrate, and most preferably
less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent
temperature conditions for the wash steps will ordinarily include a
temperature of at least about 25.degree. C., more preferably of at
least about 42.degree. C., and most preferably of at least about
68.degree. C. In a preferred embodiment, wash steps will occur at
25.degree. C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS.
In a more preferred embodiment, wash steps will occur at 42.degree.
C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a most
preferred embodiment, wash steps will occur at 68.degree. C. in 15
mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional
variations on these conditions will be readily apparent to those
skilled in the art. Hybridization techniques are well known to
those skilled in the art and are described, for example, in Benton
and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc.
Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current
Protocols in Molecular Biology, Wiley Interscience, New York,
2001); Berger and Kimmel (Guide to Molecular Cloning Techniques,
1987, Academic Press, New York); and Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
New York.
[0070] By "intrauterine growth retardation (IUGR)" is meant a
syndrome resulting in a birth weight which is less that 10 percent
of the predicted fetal weight for the gestational age of the fetus.
The current World Health Organization criterion for low birth
weight is a weight less than 2,500 gm (5 lbs. 8 oz.) or below the
10.sup.th percentile for gestational age according to U.S. tables
of birth weight for gestational age by race, parity, and infant sex
(Zhang and Bowes, Obstet. Gynecol. 86:200-208, 1995). These low
birth weight babies are also referred to as "small for gestational
age (SGA)". Pre-eclampsia is a condition known to be associated
with IUGR or SGA.
[0071] By "metric" is meant a measure. A metric may be used, for
example, to compare the levels of a polypeptide or nucleic acid
molecule of interest. Exemplary metrics include, but are not
limited to, mathematical formulas or algorithms, such as ratios.
The metric to be used is that which best discriminates between
levels of soluble endoglin, sFlt-1, VEGF, PlGF, or any combination
thereof, in a subject having pregnancy related hypertensive
disorder, such as pre-eclampsia or eclampsia, and a normal control
subject. Depending on the metric that is used the diagnostic
indicator of pregnancy related hypertensive disorder may be
significantly above or below a reference value (e.g., from a
control subject not having a pregnancy related hypertensive
disorder). Soluble endoglin level is determined by measuring the
amount of free, bound (i.e., bound to growth factor), or total
(free+bound) soluble endoglin. sFlt-1 level is measured by
measuring the amount of free, bound (i.e., bound to growth factor),
or total sFlt-1 (bound+free). VEGF or PlGF levels are determined by
measuring the amount of free PlGF or free VEGF (i.e., not bound to
sFlt-1). One exemplary metric is [sFlt-1/(VEGF+PlGF)], also
referred to as the pre-eclampsia anti-angiogenic index (PAAI).
Another example is the following soluble endoglin anti-angiogenic
index: (sFlt-1+0.25(soluble endoglin polypeptide))/PlGF. An
increase in the value of the soluble endoglin anti-angiogenic index
is a diagnostic indicator of pre-eclampsia or eclampsia. Yet
another exemplary metric is the following: (soluble
enodglin+sFlt-1)/PlGF. Any of the metrics of the invention can
further include the BMI of the mother or GA of the infant.
[0072] By "pre-eclampsia anti-angiogenesis index (PAAI)" is meant
the ratio of sFlt-1/VEGF+PlGF used as an indicator of
anti-angiogenic activity. A PAAI greater than 10, more preferably
greater than 20, is indicative of a pregnancy related hypertensive
disorder, such as pre-eclampsia or risk of pre-eclampsia.
[0073] By "soluble endoglin anti-angiogenic index" is meant the
ratio of (sFlt-1+0.25 soluble endoglin)/PlGF. For example, a value
of 75, or higher, preferably 100 or higher, or more preferably 200
or higher is indicative of a pregnancy complication associated with
hypertension, such as pre-eclampsia or eclampsia.
[0074] By "operably linked" is meant that a gene and a regulatory
sequence(s) are connected in such a way as to permit gene
expression when the appropriate molecules (e.g., transcriptional
activator proteins) are bound to the regulatory sequence(s).
[0075] By "pharmaceutically acceptable carrier" is meant a carrier
that is physiologically acceptable to the treated mammal while
retaining the therapeutic properties of the compound with which it
is administered. One exemplary pharmaceutically acceptable carrier
substance is physiological saline. Other physiologically acceptable
carriers and their formulations are known to one skilled in the art
and described, for example, in Remington's Pharmaceutical Sciences,
(20.sup.th edition), ed. A. Gennaro, 2000, Lippincott, Williams
& Wilkins, Philadelphia, Pa.
[0076] By "placental growth factor (PlGF)" is meant a mammalian
growth factor that is homologous to the protein defined by GenBank
accession number P49763 and that has PlGF biological activity. PlGF
is a glycosylated homodimer belonging to the VEGF family and can be
found in two distinct isoforms through alternative splicing
mechanisms. PlGF is expressed by cyto- and syncytiotrophoblasts in
the placenta and PlGF biological activities include induction of
proliferation, migration, and activation of endothelial cells,
particularly trophoblast cells.
[0077] By "polymorphism" is meant a genetic variation, mutation,
deletion or addition in a soluble endoglin, sFlt-1, PlGF, or VEGF
nucleic acid molecule that is indicative of a predisposition to
develop pre-eclampsia or eclampsia. Such polymorphisms are known to
the skilled artisan and are described, for example, by Raab et al.
(Biochem. J. 339:579-588, 1999) and Parry et al. (Eur. J
Immunogenet. 26:321-323, 1999). A polymorphism may be present in
the promoter sequence, an open reading frame, intronic sequence, or
untranslated 3' region of a gene. Known examples of such
polymorphisms in the endoglin gene include a 6 base insertion of
GGGGGA in intron 7 at 26 bases beyond the 3' end of exon 7 (Ann.
Neurol. 41:683-6, 1997).
[0078] By "pregnancy related hypertensive disorder" is meant any
condition or disease or pregnancy that is associated with or
characterized by an increase in blood pressure. Included among
these conditions are pre-eclampsia (including premature
pre-eclampsia, severe pre-eclampsia), eclampsia, gestational
hypertension, HELLP syndrome, (hemolysis, elevated liver enzymes,
low platelets), abruption placenta, chronic hypertension, pregnancy
with intra uterine growth restriction, and pregnancy with a small
for gestational age (SGA) infant. It should be noted that although
pregnancy with a SGA infant is not often associated with
hypertension, it is included in this definition.
[0079] By "pre-eclampsia" is meant the multi-system disorder that
is characterized by hypertension with proteinuria or edema, or
both, glomerular dysfunction, brain edema, liver edema, or
coagulation abnormalities due to pregnancy or the influence of a
recent pregnancy. All forms of pre-eclampsia, such as premature,
mild, moderate, and severe pre-eclampsia are included in this
definition. Pre-eclampsia generally occurs after the 20.sup.th week
of gestation. Pre-eclampsia is generally defined as some
combination of the following symptoms: (1) a systolic blood
pressure (BP)>140 mmHg and a diastolic BP>90 mmHg after 20
weeks gestation (generally measured on two occasions, 4-168 hours
apart), (2) new onset proteinuria (1+by dipstik on urinanaysis,
>300 mg of protein in a 24-hour urine collection, or a single
random urine sample having a protein/creatinine ratio >0.3), and
(3) resolution of hypertension and proteinuria by 12 weeks
postpartum. Severe pre-eclampsia is generally defined as (1) a
diastolic BP>110 mmHg (generally measured on two occasions,
4-168 hours apart) or (2) proteinuria characterized by a
measurement of 3.5 g or more protein in a 24-hour urine collection
or two random urine specimens with at least 3+protein by dipstick.
In pre-eclampsia, hypertension and proteinuria generally occur
within seven days of each other. In severe pre-eclampsia, severe
hypertension, severe proteinuria and HELLP syndrome (hemolysis,
elevated liver enzymes, low platelets) or eclampsia can occur
simultaneously or only one symptom at a time. HELLP syndrome is
characterized by evidence of thrombocytopenia (<100000
cells/.mu.l), increased LDH (>600 IU/L) and increased AST
(>70 IU/L). Occasionally, severe pre-eclampsia can lead to the
development of seizures. This severe form of the syndrome is
referred to as "eclampsia." Eclampsia can also include dysfunction
or damage to several organs or tissues such as the liver (e.g.,
hepatocellular damage, periportal necrosis) and the central nervous
system (e.g., cerebral edema and cerebral hemorrhage). The etiology
of the seizures is thought to be secondary to the development of
cerebral edema and focal spasm of small blood vessels in the
kidney.
[0080] By "premature pre-eclampsia" is meant pre-eclampsia with
onset of symptoms <37 weeks or <34 weeks.
[0081] By "protein" or "polypeptide" or "polypeptide fragment" is
meant any chain of more than two amino acids, regardless of
post-translational modification (e.g., glycosylation or
phosphorylation), constituting all or part of a naturally occurring
polypeptide or peptide, or constituting a non-naturally occurring
polypeptide or peptide.
[0082] By "reference sample" is meant any sample, standard, or
level that is used for comparison purposes. A "normal reference
sample" can be a prior sample taken from the same subject, a sample
from a pregnant subject not having any pregnancy related
hypertensive disorder, such as pre-eclampsia or eclampsia, a sample
from a pregnant subject not having a pregnancy related hypertensive
disorder, such as pre-eclampsia or eclampsia, a subject that is
pregnant but the sample was taken early in pregnancy (e.g., in the
first or second trimester or before the detection of a pregnancy
related hypertensive disorder, such as pre-eclampsia or eclampsia),
a subject that is pregnant and has no history of a pregnancy
related hypertensive disorder, such as pre-eclampsia or eclampsia,
a subject that is not pregnant, a sample of a purified reference
polypeptide at a known normal concentration (i.e., not indicative
of a pregnancy related hypertensive disorder, such as pre-eclampsia
or eclampsia). By "reference standard or level" is meant a value or
number derived from a reference sample. A normal reference standard
or level can be a value or number derived from a normal subject
that is matched to the sample subject by at least one of the
following criteria: gestational age of the fetus, maternal age,
maternal blood pressure prior to pregnancy, maternal blood pressure
during pregnancy, BMI of the mother, weight of the fetus, prior
diagnosis of pre-eclampsia or eclampsia, and a family history of
pre-eclampsia or eclampsia. A "positive reference" sample, standard
or value is a sample or value or number derived from a subject that
is known to have a pregnancy related hypertensive disorder, such as
pre-eclampsia or eclampsia, that is matched to the sample subject
by at least one of the following criteria: gestational age of the
fetus, maternal age, maternal blood pressure prior to pregnancy,
maternal blood pressure during pregnancy, BMI of the mother, weight
of the fetus, prior diagnosis of a pregnancy related hypertensive
disorder, and a family history of a pregnancy related hypertensive
disorder
[0083] By "reduce or inhibit" is meant the ability to cause an
overall decrease preferably of 20% or greater, more preferably of
40%, 50%, 60%, 70%, 80%, 90% or greater change in the level of
protein or nucleic acid, detected by the aforementioned assays (see
"expression"), as compared to an untreated sample
[0084] By "sample" is meant a tissue biopsy, cell, bodily fluid
(e.g., blood, serum, plasma, urine, saliva, amniotic fluid, or
cerebrospinal fluid) or other specimen obtained from a subject.
Desirably, the biological sample includes soluble endoglin nucleic
acid molecules or polypeptides or both.
[0085] By "small interfering RNAs (siRNAs)" is meant an isolated
dsRNA molecule, preferably greater than 10 nucleotides (nt) in
length, more preferably greater than 15 nucleotides in length, and
most preferably greater than 19 nucleotides in length that is used
to identify the target gene or mRNA to be degraded. A range of
19-25 nucleotides is the most preferred size for siRNAs. siRNAs can
also include short hairpin RNAs in which both strands of an siRNA
duplex are included within a single RNA molecule. siRNA includes
any form of dsRNA (proteolytically cleaved products of larger
dsRNA, partially purified RNA, essentially pure RNA, synthetic RNA,
recombinantly produced RNA) as well as altered RNA that differs
from naturally occurring RNA by the addition, deletion,
substitution, and/or alteration of one or more nucleotides. Such
alterations can include the addition of non-nucleotide material,
such as to the end(s) of the 19, 20, 21, 22, 23, 24, or 25 nt RNA
or internally (at one or more nucleotides of the RNA). In a
preferred embodiment, the RNA molecules contain a 3' hydroxyl
group. Nucleotides in the RNA molecules of the present invention
can also comprise non-standard nucleotides, including non-naturally
occurring nucleotides or deoxyribonucleotides. Collectively, all
such altered RNAs are referred to as analogs of RNA. siRNAs of the
present invention need only be sufficiently similar to natural RNA
that it has the ability to mediate RNA interference (RNAi). As used
herein, RNAi refers to the ATP-dependent targeted cleavage and
degradation of a specific mRNA molecule through the introduction of
small interfering RNAs or dsRNAs into a cell or an organism. As
used herein "mediate RNAi" refers to the ability to distinguish or
identify which RNAs are to be degraded.
[0086] By "soluble endoglin binding molecule" is meant a protein or
small molecule compound that specifically binds a soluble endoglin
polypeptide. A soluble endoglin binding molecule may be, for
example, an antibody, antibody-related peptide, one or more CDR
regions of a soluble endoglin binding antibody, or soluble endoglin
interacting protein.
[0087] By "soluble Flt-1 (sFlt-1)" (also known as sVEGF-R1) is
meant the soluble form of the Flt-1 receptor, that is homologous to
the protein defined by GenBank accession number U01134, and that
has sFlt-1 biological activity. The biological activity of an
sFlt-1 polypeptide may be assayed using any standard method, for
example, by assaying sFlt-1 binding to VEGF. sFlt-1 lacks the
transmembrane domain and the cytoplasmic tyrosine kinase domain of
the Flt-1 receptor. sFlt-1 can bind to VEGF and PlGF with high
affinity, but it cannot induce proliferation or angiogenesis and is
therefore functionally different from the Flt-1 and KDR receptors.
sFlt-1 was initially purified from human umbilical endothelial
cells and later shown to be produced by trophoblast cells in vivo.
As used herein, sFlt-1 includes any sFlt-1 family member or
isoform. sFlt-1 can also mean degradation products or fragments
that result from enzymatic cleavage of the Flt-1 receptor and that
maintain sFlt-1 biological activity. In one example, specific
metalloproteinases released from the placenta may cleave the
extracellular domain of Flt-1 receptor to release the N-terminal
portion of Flt-1 into circulation.
[0088] By "specifically binds" is meant a compound or antibody
which recognizes and binds a polypeptide of the invention but that
does not substantially recognize and bind other molecules in a
sample, for example, a biological sample, which naturally includes
a polypeptide of the invention. In one example, an antibody that
specifically binds soluble endoglin does not bind membrane bound
endoglin. In another example, an antibody that specifically binds
to soluble endoglin recognizes a region within amino acids 1 to 437
of endoglin that is unique to soluble endoglin but not the
full-length endoglin.
[0089] By "subject" is meant a mammal, including, but not limited
to, a human or non-human mammal, such as a cow, a horse, a sheep, a
pig, a goat, a dog, or a cat. Included in this definition are
pregnant, post-partum, and non-pregnant mammals.
[0090] By "substantially identical" is meant a nucleic acid or
amino acid sequence that, when optimally aligned, for example using
the methods described below, share at least 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100sequence identity
with a second nucleic acid or amino acid sequence, e.g., an
endoglin or soluble endoglin sequence. "Substantial identity" may
be used to refer to various types and lengths of sequence, such as
full-length sequence, epitopes or immunogenic peptides, functional
domains, coding and/or regulatory sequences, exons, introns,
promoters, and genomic sequences. Percent identity between two
polypeptides or nucleic acid sequences is determined in various
ways that are within the skill in the art, for instance, using
publicly available computer software such as Smith Waterman
Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol
147:195-7); "BestFit" (Smith and Waterman, Advances in Applied
Mathematics, 482-489 (1981)) as incorporated into GeneMatcher
Plus.TM., Schwarz and Dayhof (1979) Atlas of Protein Sequence and
Structure, Dayhof, M. O., Ed pp 353-358; BLAST program (Basic Local
Alignment Search Tool; (Altschul, S. F., W. Gish, et al. (1990) J
Mol Biol 215: 403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X,
WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR)
software. In addition, those skilled in the art can determine
appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over the length of
the sequences being compared. In general, for proteins, the length
of comparison sequences will be at least 10 amino acids, preferably
20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200,
250, 300, 350, 400, or at least 437 amino acids or more. For
nucleic acids, the length of comparison sequences will generally be
at least 25, 50, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500,
550, 600, 650, 700, 800, 900, 1000, 1100, 1200, or at least 1311
nucleotides or more. It is understood that for the purposes of
determining sequence identity when comparing a DNA sequence to an
RNA sequence, a thymine nucleotide is equivalent to a uracil
nucleotide. Conservative substitutions typically include
substitutions within the following groups: glycine, alanine;
valine, isoleucine, leucine; aspartic acid, glutamic acid,
asparagine, glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine.
[0091] By "symptoms of pre-eclampsia" is meant any of the
following: (1) a systolic blood pressure (BP)>140 mmHg and a
diastolic BP>90 mmHg after 20 weeks gestation, (2) new onset
proteinuria (1+by dipstik on urinanaysis, >300 mg of protein in
a 24 hour urine collection, or random urine protein/creatinine
ratio >0.3), and (3) resolution of hypertension and proteinuria
by 12 weeks postpartum. The symptoms of pre-eclampsia can also
include renal dysfunction and glomerular endotheliosis or
hypertrophy. By "symptoms of eclampsia" is meant the development of
any of the following symptoms due to pregnancy or the influence of
a recent pregnancy: seizures, coma, thrombocytopenia, liver edema,
pulmonary edema, and cerebral edema.
[0092] By "transforming growth factor .beta. (TGF-.beta.)" is meant
a mammalian growth factor that has TGF-.beta. biological activity
and is a member of a family of structurally related paracrine
polypeptides found ubiquitously in vertebrates, and prototypic of a
large family of metazoan growth, differentiation, and morphogenesis
factors (see, for review, Massaque et al. Ann Rev Cell Biol
6:597-641 (1990); Massaque et al. Trends Cell Biol 4:172-178
(1994); Kingsley Gene Dev. 8:133-146 (1994); and Sporn et al. J
Cell Biol 119:1017-1021 (1992). As described in Kingsley, supra,
the TGF-.beta. superfamily has at least 25 members, and can be
grouped into distinct sub-families with highly related sequences.
The most obvious sub-families include the following: the TGF-.beta.
sub-family, which comprises at least four genes that are much more
similar to TGF-.beta.1 than to other members of the TGF-.beta.
superfamily; the activin sub-family, comprising homo- or
hetero-dimers or two sub-units, inhibin.beta.-A and
inhibin.beta.-B. The decapentaplegic sub-family, which includes the
mammalian factors BMP2 and BMP4, which can induce the formation of
ectopic bone and cartilage when implanted under the skin or into
muscles. The 60A sub-family, which includes a number of mammalian
homologs, with osteoinductive activity, including BMP5-8. Other
members of the TGF-.beta. superfamily include the gross
differentiation factor 1 (GDF-1), GDF-3/VGR-2, dorsalin, nodal,
mullerian-inhibiting substance (MIS), and glial-derived
neurotrophic growth factor (GDNF). It is noted that the DPP and 60A
sub-families are related more closely to one another than to other
members of the TGF-.beta. superfamily, and have often been grouped
together as part of a larger collection of molecules called DVR
(dpp and vg1 related). Unless evidenced from the context in which
it is used, the term TGF-.beta. as used throughout this
specification will be understood to generally refer to members of
the TGF-.beta. superfamily as appropriate. (Massague et al, Annu.
Rev. Biochem. 67:753-91, 1998; Josso et al, Curr. Op. Gen. Dev.,
7:371-377, 1997). TGF-.beta. functions to regulate growth,
differentiation, motility, tissue remodeling, neurogenesis, would
repair, apoptosis, and angiogenesis in many cell types. TGF-.beta.
also inhibits cell proliferation in many cell types and can
stimulate the synthesis of matrix proteins.
[0093] By "therapeutic amount" is meant an amount that when
administered to a patient suffering from pre-eclampsia or eclampsia
is sufficient to cause a qualitative or quantitative reduction in
the symptoms of pre-eclampsia or eclampsia as described herein. A
"therapeutic amount" can also mean an amount that when administered
to a patient suffering from pre-eclampsia or eclampsia is
sufficient to cause a reduction in the expression levels of
endoglin or sFlt-1 or an increase in the expression levels of VEGF
or PlGF as measured by the assays described herein.
[0094] By "treating" is meant administering a compound or a
pharmaceutical composition for therapeutic purposes. To "treat
disease" or use for "therapeutic treatment" refers to administering
treatment to a subject already suffering from a disease to improve
the subject's condition. Preferably, the subject is diagnosed as
suffering from a pregnancy complication associated with
hypertension, such as pre-eclampsia or eclampsia, based on
identification of any of the characteristic symptoms described
below or the use of the diagnostic methods described herein. To
"prevent disease" refers to prophylactic treatment of a subject who
is not yet ill, but who is susceptible to, or otherwise at risk of,
developing a particular disease. Preferably a subject is determined
to be at risk of developing pre-eclampsia or eclampsia using the
diagnostic methods described herein. Thus, in the claims and
embodiments, treating is the administration to a mammal either for
therapeutic or prophylactic purposes.
[0095] By "trophoblast" is meant the mesectodermal cell layer
covering the blastocyst that erodes the uterine mucosa and through
which the embryo receives nourishment from the mother; the cells
contribute to the formation of the placenta.
[0096] By "vascular endothelial growth factor (VEGF)" is meant a
mammalian growth factor that is homologous to the growth factor
defined in U.S. Pat. Nos. 5,332,671; 5,240,848; 5,194,596; and
Charnock-Jones et al. (Biol. Reproduction, 48: 1120-1128, 1993),
and has VEGF biological activity. VEGF exists as a glycosylated
homodimer and includes at least four different alternatively
spliced isoforms. The biological activity of native VEGF includes
the promotion of selective growth of vascular endothelial cells or
umbilical vein endothelial cells and induction of angiogenesis. As
used herein, VEGF includes any VEGF family member or isoform (e.g.,
VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF189, VEGF165, or VEGF
121). Preferably, VEGF is the VEGF121 or VEGF165 isoform (Tischer
et al., J. Biol. Chem. 266, 11947-11954, 1991; Neufed et al. Cancer
Metastasis 15:153-158, 1996), which is described in U.S. Pat. Nos.
6,447,768; 5,219,739; and 5,194,596, hereby incorporated by
reference. Also included are mutant forms of VEGF such as the
KDR-selective VEGF and Flt-selective VEGF described in Gille et al.
(J. Biol. Chem. 276:3222-3230, 2001). As used herein VEGF also
includes any modified forms of VEGF such as those described in
LeCouter et al. (Science 299:890-893, 2003). Although human VEGF is
preferred, the invention is not limited to human forms and can
include other animal forms of VEGF (e.g. mouse, rat, dog, or
chicken).
[0097] By "vector" is meant a DNA molecule, usually derived from a
plasmid or bacteriophage, into which fragments of DNA may be
inserted or cloned. A recombinant vector will contain one or more
unique restriction sites, and may be capable of autonomous
replication in a defined host or vehicle organism such that the
cloned sequence is reproducible. A vector contains a promoter
operably linked to a gene or coding region such that, upon
transfection into a recipient cell, an RNA is expressed.
[0098] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0099] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0100] FIG. 1 is a schematic showing the endoglin protein. SP:
signal peptide, ZP: zona pellucida domain, CL: potential cleavage
site (amino acid 437) for the release of soluble endoglin, TM:
transmembrane domain, Cyto: cytoplasmic domain.
[0101] FIG. 2A shows the predicted cDNA sequence (SEQ ID NO: 1) of
soluble endoglin. FIG. 2B shows the predicted amino acid sequence
(SEQ ID NO: 2) of soluble endoglin.
[0102] FIG. 3 is a Northern blot showing endoglin mRNA levels in
placentas from normal pregnancies (N), placentas from mild
pre-eclamptic pregnancies (p) and placentas from severe
pre-eclamptic pregnancies (P).
[0103] FIG. 4 is a western blot showing endoglin protein levels in
the placenta. Samples are from two pre-eclamptic patients, p32 and
p36, that presented to the Beth Israel Deaconess Medical Center in
2003 and maternal serum from a pregnant woman. The Western blot was
probed using a N-terminal antibody obtained from Santa Cruz
Biotechnology, Inc., (Santa Cruz, Calif.) that shows both the 110
kD band in the placenta and a smaller 63 kD band that is present in
the placenta and the serum samples.
[0104] FIG. 5 is a graph that shows the circulating concentrations
of soluble endoglin in women with normal pregnancy, mild
pre-eclampsia, severe pre-eclampsia and non-pre-eclamptic
pregnancies complicated by pre-term delivery. All blood specimens
were obtained within 24 hours prior to delivery. Soluble endoglin
was measured using an ELISA kit from R & D Systems, MN (Cat #
DNDG00). These data show that soluble endoglin levels are
significantly elevated in pre-eclamptic patients at the time of
clinical disease.
[0105] FIG. 6 is a graph showing the mean soluble endoglin
concentration for the five different study groups of pregnant women
throughout pregnancy during the various gestational age group
windows.
[0106] FIG. 7 is a graph showing the mean sFlt1 concentrations for
the five different study groups of pregnant women throughout
pregnancy during the various gestational age group windows.
[0107] FIG. 8 is a graph showing the mean PlGF concentrations for
the five different study groups of pregnant women throughout
pregnancy during the various gestational age group windows.
[0108] FIG. 9 is a graph showing the values for the soluble
endoglin anti-angiogenic index for pre-eclampsia anti-angiogenesis
for samples taken prior to clinical symptoms.
[0109] FIG. 10 is a graph showing the mean concentrations of
soluble endoglin according to the number of weeks before clinical
premature pre-eclampsia (PE<37 weeks).
[0110] FIG. 11 is a graph showing the soluble endoglin
anti-angiogenic index values according to the number of weeks
before clinical premature pre-eclampsia (PE<37 weeks).
[0111] FIG. 12 is a graph showing the alteration in soluble
endoglin levels throughout pregnancy for term pre-eclampsia
(PE>37 weeks) before and after symptoms.
[0112] FIG. 13 is a graph showing the alteration in the soluble
endoglin anti-angiogenic index levels throughout pregnancy for term
pre-eclampsia (PE>37 weeks) before and after symptoms.
[0113] FIG. 14 is a graph showing the soluble endoglin levels
detected in women during gestational hypertension and before
gestational hypertension (1-5 weeks preceding gestational
hypertension (during 33-36 week of pregnancy)) and normotensive
controls.
[0114] FIG. 15 is a graph showing the soluble endoglin
anti-angiogenic index levels in women during gestational
hypertension and before gestational hypertension (1-5 weeks
preceding gestational hypertension (during 33-36 week of
pregnancy)) and normotensive controls.
[0115] FIG. 16 is a graph showing the soluble endoglin levels
detected during the 33-36 week gestational windows in women with
severe SGA, mild SGA, and normotensive controls.
[0116] FIG. 17 is a graph showing the soluble endoglin
anti-angiogenic index levels detected during the 33-36 week
gestational windows in women with severe SGA, mild SGA, and
normotensive controls.
[0117] FIG. 18 is a graph showing the concentration of sFlt1 and
soluble endoglin in the same pregnant patients plotted against each
other.
[0118] FIG. 19 shows photomicrographs of double immunofluorescence
staining of endoglin (red) and smooth muscle actin (green) for
pre-eclamptic placentas taken at 25.2 weeks. The antibody used to
detect endoglin stains both full-length endoglin and the soluble
endoglin. Control placentas for the appropriate gestational windows
were derived from patients with pre-term labor.
[0119] FIG. 20 shows photomicrographs of double immunofluorescence
staining of endoglin (red) and smooth muscle actin (green) for
pre-eclamptic placentas taken at 41.3 weeks. The antibody used to
detect endoglin stains both full-length endoglin and the soluble
endoglin. Control placentas for the appropriate gestational windows
were derived from patients with pre-term labor.
[0120] FIG. 21 A is a photograph of the autoradiogram from
immunoprecipitation and western blots experiments for endoglin
using both pre-eclamptic placentas and serum. FIG. 21 B is a
photograph of the autoradiogram from immunoprecipitation and
western blots experiments for endoglin using pre-eclamptic
placentas. The three different N and P samples represent individual
patients. For both figures commercially available monoclonal
antibodies were used for immunoprecipitations and polyclonal
antibodies were used for the western blots. Both these antibodies
were raised against the N-terminal region of the endoglin protein
and detect both the full length and the truncated soluble endoglin
protein.
[0121] FIG. 22 is a graph showing the results of angiogenesis
assays using HUVECs in growth factor reduced matrigels.
Angiogenesis assays were performed in the presence of soluble
endoglin or sFlt1 or both and the endothelial tube lengths
quantitated. C--represents control, E--represents 1 .mu.g/ml of
soluble endoglin and S represents 1 .mu.g/ml of sFlt1. E+S
represent the combination of 1 .mu.g/ml of E+1 .mu.g/ml of sFlt1.
Data represents a mean of three independent experiments.
[0122] FIG. 23 is a graph showing the microvascular permeability in
several organ beds assessed using Evans blue leakage in mice as
described in the materials and methods. C-control (GFP), E-soluble
endoglin, S-sFlt1 and S+E-sFlt1+soluble endoglin. Data represents a
mean of 4 independent experiments.
[0123] FIG. 24 is a graph showing the percent change in rat renal
microvessel diameter were subjected to microvascular reactivity
experiments in the presence of TGF-.beta.1 (B1) and TGF-.beta.3
(B3) from doses ranging from 200 pg/ml-200 ng/ml. These same
experiments were repeated in the presence of soluble endoglin (E)
at 1 .mu.g/ml. These data presented are a mean of 4 independent
experiments.
[0124] FIG. 25 is a graph showing the percent change in the
vascular diameter of renal microvessels in the presence of 1 ng/ml
of VEGF (V), TGF-.beta.1 (B1) and the combination (V+B1). Also
shown is the effect of this combination in the presence of 1
.mu.g/ml each of sFlt1 (S) and soluble endoglin (E) (V+B1+S+E). The
data represents a mean of 4 independent experiments.
[0125] FIG. 26A is a photograph of a peripheral smear of blood
samples taken at the time of sacrifice from pregnant rats injected
with the combination of sFlt1 and a control adenoviruses (CMV).
FIG. 26B is a photograph of a peripheral smear of blood samples
taken at the time of sacrifice from pregnant rats injected with the
combination of sFlt and adenoviruses expressing soluble endoglin
and demonstrates active hemolysis as evidenced by schistocyes and
increased reticulocyte count. Arrowheads represent schistocyte.
[0126] FIGS. 27A-D are a series of photomicrographs showing the
renal histology (H&E stain) of the various animal groups
described in Table 8. FIG. 27A shows the renal histology for the
control group with no evidence of glomerular endotheliosis. FIG.
27B shows the renal histology for the soluble endoglin injected
group with no evidence of glomerular endotheliosis. FIG. 27C shows
the renal histology for sFlt1 injected rats showing moderate
endotheliosis (shown by arrow head). FIG. 27D shows the renal
histology for the soluble endoglin and sFlt1 injected rats showing
extremely swollen glomeruli and severe glomerular endotheliosis
with protein resorption droplets in the podocytes. All light
micrographs were taken at 60.times. (original magnification).
DETAILED DESCRIPTION
[0127] We have discovered that soluble endoglin levels are elevated
in blood serum samples taken from women with a pregnancy related
hypertensive disorder, such as pre-eclampsia or eclampsia. Soluble
endoglin may be formed by cleavage of the extracellular portion of
the membrane bounds form by proteolytic enzymes. Excess soluble
endoglin may be depleting the placenta of necessary amounts of
these essential angiogenic and mitogenic factors. Thus, soluble
endoglin is an excellent diagnostic marker pregnancy related
hypertensive disorders, including pre-eclampsia and eclampsia.
Furthermore, we have discovered therapeutic agents that interfere
with soluble endoglin binding to growth factors, agents that reduce
soluble endoglin expression or biological activity, or agents that
increase levels of growth factors, can be used to treat or prevent
pregnancy related hypertensive disorders, such as pre-eclampsia or
eclampsia in a subject. Such agents include, but are not limited
to, antibodies to soluble endoglin, oligonucleotides for antisense
or RNAi that reduce levels of soluble endoglin, compounds that
increase the levels of growth factors, compounds that prevent the
proteolytic cleavage of the membrane bound form of endoglin thereby
preventing the release of soluble endoglin, and small molecules
that bind soluble endoglin and block the growth factor binding
site. The invention also features methods for measuring levels of
soluble endoglin as a detection tool for early diagnosis and
management of a pregnancy related hypertensive disorder, including
pre-eclampsia and eclampsia.
[0128] While the detailed description presented herein refers
specifically to soluble endoglin, sFlt-1, VEGF, or PlGF, it will be
clear to one skilled in the art that the detailed description can
also apply to family members, isoforms, and/or variants of soluble
endoglin, sFlt-1, VEGF, or PlGF.
Diagnostics
[0129] The present invention features assays based on the detection
of soluble endoglin to pregnancy related hypertensive disorders,
such as pre-eclampsia, eclampsia, or the propensity to develop such
conditions. While the methods described herein refer to
pre-eclampsia and eclampsia specifically, it should be understood
that the diagnostic and monitoring methods of the invention apply
to any pregnancy related hypertensive disorder including, but not
limited to, gestational hypertension, pregnancy with a small for
gestational age (SGA) infant, HELLP, chronic hypertension,
pre-eclampsia (mild, moderate, and severe), and eclampsia.
[0130] Levels of endoglin, either free, bound, or total levels, are
measured in a subject sample and used as an indicator of
pre-eclampsia, eclampsia, or the propensity to develop such
conditions.
[0131] A subject having pre-eclampsia, eclampsia, or a
predisposition to such conditions will show an increase in the
expression of a soluble endoglin polypeptide. The soluble endoglin
polypeptide can include full-length soluble endoglin, degradation
products, alternatively spliced isoforms of soluble endoglin,
enzymatic cleavage products of soluble endoglin, and the like. An
antibody that specifically binds a soluble endoglin polypeptide may
be used for the diagnosis of pre-eclampsia or eclampsia or to
identify a subject at risk of developing such conditions. One
example of an antibody useful in the methods of the invention is a
monoclonal antibody against the N-terminal region of endoglin that
is commercially available from Santa Cruz Biotechnology, Inc. (cat
# sc-20072). A variety of protocols for measuring an alteration in
the expression of such polypeptides are known, including
immunological methods (such as ELISAs and RIAs), and provide a
basis for diagnosing pre-eclampsia or eclampsia or a risk of
developing such conditions. Again, an increase in the level of the
soluble endoglin polypeptide is diagnostic of a subject having
pre-eclampsia, eclampsia, or a predisposition to such
conditions.
[0132] Elevated levels of soluble endoglin are a positive indicator
of pre-eclampsia or eclampsia. For example, if the level of soluble
endoglin is increased relative to a reference (e.g., 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90% or more), this is considered a
positive indicator of pre-eclampsia or eclampsia. Additionally, any
detectable alteration in levels of soluble endoglin, sFlt-1, VEGF,
or PlGF relative to normal levels is indicative of eclampsia,
pre-eclampsia, or the propensity to develop such conditions.
Normally, circulating serum concentrations of soluble endoglin
range from 2-7 ng/ml during the non-pregnant state and from 10-20
ng/ml during normal pregnancy. Elevated serum levels, greater than
15 ng/ml, preferably greater than 20 ng/ml, and most preferably
greater than 25 ng/ml or more, of soluble endoglin is considered a
positive indicator of pre-eclampsia.
[0133] In one embodiment, the level of soluble endoglin is measured
in combination with the level of sFlt-1, VEGF, or PlGF polypeptide
or nucleic acid, or any combination thereof. Methods for the
measurement of sFlt-1, VEGF, and PlGF are described in PCT
Publication Number WO 2004/008946 and U.S. Publication No.
20040126828, hereby incorporated by reference in their entirety. In
additional preferred embodiments, the body mass index (BMI) and
gestational age of the fetus is also measured and included the
diagnostic metric.
[0134] In one embodiment, a metric incorporating soluble endoglin,
sFlt-1, VEGF, or PlGF, or any combination therein, is used to
determine whether a relationship between levels of at least two of
the proteins is indicative of pre-eclampsia or eclampsia. In one
example, the metric is a PAAI (sFlt-1/VEGF+PlGF), which is used, in
combination with soluble endoglin measurement, as an
anti-angiogenic index that is diagnostic of pre-eclampsia,
eclampsia, or the propensity to develop such conditions. If the
level of soluble endoglin is increased relative to a reference
sample (e.g., 1.5-fold, 2-fold, 3-fold, 4-fold, or even by as much
as 10-fold or more), and the PAAI is greater than 10, more
preferably greater than 20, then the subject is considered to have
pre-eclampsia, eclampsia, or to be in imminent risk of developing
the same. The PAAI (sFlt-1/VEGF+PlGF) ratio is merely one example
of a useful metric that may be used as a diagnostic indicator. It
is not intended to limit the invention. Virtually any metric that
detects an alteration in the levels of soluble endoglin, sFlt-1,
PlGF, or VEGF, or any combination thereof, in a subject relative to
a normal control may be used as a diagnostic indicator. Another
example is the following soluble endoglin anti-angiogenic index:
(sFlt-1+0.25(soluble endoglin polypeptide))/PlGF. An increase in
the value of the soluble endoglin metric is a diagnostic indicator
of pre-eclampsia or eclampsia. A soluble endoglin index above 100,
preferably above 200 is a diganostic indicator of pre-eclampsia or
eclampsia. Another example is the following index: (soluble
endoglin+sFlt-1)/PlGF. The indexes can further include the BMI of
the mother or the GA of the infant.
[0135] Standard methods may be used to measure levels of soluble
endoglin, VEGF, PlGF, or sFlt-1 polypeptide in any bodily fluid,
including, but not limited to, urine, serum, plasma, saliva,
amniotic fluid, or cerebrospinal fluid. Such methods include
immunoassay, ELISA, western blotting using antibodies directed to
soluble endoglin, VEGF, PlGF or sFlt-1, and quantitative enzyme
immunoassay techniques such as those described in Ong et al.
(Obstet. Gynecol. 98:608-611, 2001) and Su et al. (Obstet.
Gynecol., 97:898-904, 2001). ELISA assays are the preferred method
for measuring levels of soluble endoglin, VEGF, PlGF, or sFlt-1.
Preferably, soluble endoglin is measured.
[0136] Oligonucleotides or longer fragments derived from an
endoglin, sFlt-1, PlGF, or VEGF nucleic acid sequence may be used
as a probe not only to monitor expression, but also to identify
subjects having a genetic variation, mutation, or polymorphism in
an endoglin, sFlt-1, PlGF, or VEGF nucleic acid molecule that are
indicative of a predisposition to develop the pre-eclampsia or
eclampsia. These polymorphisms may affect nucleic acid or
polypeptide expression levels or biological activity. Such
polymorphisms are known to the skilled artisan and are described,
for example, by Raab et al., supra, and Parry et al. (Eur. J
Immunogenet. 26:321-3, 1999). For example, polymorphisms in the
endoglin gene have been described and many of these are associated
with the dominant vascular disorder known as hereditary
haemorrhagic telengiectasia type I (HHT1). Many of these mutations
lead to the production of a soluble form of endoglin that is
unstable, resulting in the decreased expression of endoglin found
in endothelial cells and monocytes of HHT patients (Raab et al.,
supra). In another example, a survey of the GenBank database
(www.ncbi.nlm.nih.gov) reveals at least 330 known polymorphisms in
the gene and the promoter region of Flt-1/sFlt-1. Detection of
genetic variation, mutation, or polymorphism relative to a normal,
reference sample can be used as a diagnostic indicator of
pre-eclampsia, eclampsia, or the propensity to develop
pre-eclampsia or eclampsia.
[0137] Such genetic alterations may be present in the promoter
sequence, an open reading frame, intronic sequence, or untranslated
3' region of a gene. Information related to genetic alterations can
be used to diagnose a subject as having pre-eclampsia, eclampsia,
or a predisposition to such conditions. As noted throughout,
specific alterations in the levels or biological activity of
soluble endoglin, sFlt-1, VEGF, or PlGF, or any combination
thereof, can be correlated with the likelihood of pre-eclampsia or
eclampsia, or the predisposition to the same. As a result, one
skilled in the art, having detected a given mutation, can then
assay one or more of the biological activities of the protein to
determine if the mutation causes or increases the likelihood of
pre-eclampsia or eclampsia.
[0138] In one embodiment, a subject having pre-eclampsia,
eclampsia, or a predisposition to such conditions will show an
increase in the expression of a nucleic acid encoding endoglin,
preferably soluble endoglin, or sFlt-1, or an alteration in PlGF or
VEGF levels. Methods for detecting such alterations are standard in
the art and are described in Ausubel et al., supra. In one example
northern blotting or real-time PCR is used to detect endoglin,
preferably soluble endoglin, sFlt-1, PlGF, or VEGF mRNA levels.
[0139] Hybridization with PCR probes that are capable of detecting
an endoglin or soluble endoglin nucleic acid molecule, including
genomic sequences, or closely related molecules, may be used to
hybridize to a nucleic acid sequence derived from a subject having
pre-eclampsia or eclampsia or at risk of developing such
conditions. The specificity of the probe, whether it is made from a
highly specific region, e.g., the 5' regulatory region, or from a
less specific region, e.g., a conserved motif, and the stringency
of the hybridization or amplification (maximal, high, intermediate,
or low), determine whether the probe hybridizes to a naturally
occurring sequence, allelic variants, or other related sequences.
Hybridization techniques may be used to identify mutations
indicative of a pre-eclampsia or eclampsia in a soluble endoglin
nucleic acid molecule, or may be used to monitor expression levels
of a gene encoding a soluble endoglin polypeptide (for example, by
Northern analysis, Ausubel et al., supra).
[0140] In yet another embodiment, subjects may be diagnosed for a
predisposition to pre-eclampsia or eclampsia by direct analysis of
the sequence of an endoglin or soluble endoglin nucleic acid
molecule.
[0141] The measurement of any of the nucleic acids or polypeptides
described herein can occur on at least two different occasions and
an alteration in the levels as compared to normal reference levels
over time is used as an indicator of pre-eclampsia, eclampsia, or
the propensity to develop such conditions.
[0142] The level of any of the soluble endoglin polypeptide or
nucleic acid present in the bodily fluids of a subject having
pre-eclampsia, eclampsia, or the propensity to develop such
conditions may be increased by as little as 10%, 20%, 30%, or 40%,
or by as much as 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%
or more relative to levels in a normal control subject or relative
to a previous sampling obtained from the same bodily fluids of the
same subject. The level of a soluble endoglin polypeptide or
nucleic acid in the bodily fluids of a subject having
pre-eclampsia, eclampsia, or the propensity to develop such
conditions may be altered by as little as 10%, 20%, 30%, or 40%, or
by as much as 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over
time from one measurement to the next.
[0143] The level of sFlt-1, VEGF, or PlGF measured in combination
with the level of soluble endoglin in the bodily fluids of a
subject having pre-eclampsia, eclampsia, or the propensity to
develop such conditions may be altered by as little as 10%, 20%,
30%, or 40%, or by as much as 50%, 60%, 70%, 80%, 90%, 95%, 96%,
97%, 98%, 99% or more relative to the level of sFlt-1, VEGF, or
PlGF in a normal control. The level of sFlt-1, VEGF, or PlGF
measured in combination with the level of soluble endoglin in the
bodily fluids of a subject having pre-eclampsia, eclampsia, or the
propensity to develop such conditions may be altered by as little
as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, 90%,
95%, 96%, 97%, 98%, 99% over time from one measurement to the
next.
[0144] In one embodiment, a subject sample of a bodily fluid (e.g.,
urine, plasma, serum, amniotic fluid, or cerebrospinal fluid) is
collected early in pregnancy prior to the onset of pre-eclampsia
symptoms. In another example, the sample can be a tissue or cell
collected early in pregnancy prior to the onset of pre-eclampsia
symptoms. Non-limiting examples of tissues and cells include
placental tissue, placental cells, endothelial cells, and
leukocytes such as monocytes. In humans, for example, maternal
blood serum samples are collected from the antecubital vein of
pregnant women during the first, second, or third trimesters of the
pregnancy. Preferably, the assay is carried out during the first
trimester, for example, at 4, 6, 8, 10, or 12 weeks, or any
interval therein, or during the second trimester, for example at
14, 16, 18, 20, 22, or 24 weeks, or any interval therein. Such
assays may also be conducted at the end of the second trimester or
the third trimester, for example at 26, 28, 30, 32, 34, 36, or 38
weeks, or any interval therein. It is preferable that levels of
soluble endoglin be measured twice during this period of time. For
the diagnosis of post-partum pre-eclampsia or eclampsia, assays for
soluble endoglin may be carried out postpartum. For the diagnosis
of a predisposition to pre-eclampsia or eclampsia, the assay is
carried out prior to the onset of pregnancy. In one example, for
the monitoring and management of therapy, the assay is carried out
during the pregnancy after the diagnosis of pre-eclampsia.
[0145] In one particular example, serial blood samples can be
collected during pregnancy and the levels of soluble endoglin
polypeptide determined by ELISA. In another example, a sample is
collected during the second trimester and early in the third
trimester and in increase in the level of soluble endoglin from the
first sampling to the next is indicative of pre-eclampsia or
eclampsia, or the propensity to develop either.
[0146] The invention also include the measurment of any ligands of
soluble endoglin (e.g., TGF-.beta.1, TGF-.beta.3, activin-A, BMP-2,
and BMP-7) ligand in a bodily fluid from a subject, preferably
urine, and an altelration (e.g., increase or decrease) in the level
of the soluble endoglin ligand is indicative of pre-eclampsia or
eclampsia.
[0147] In veterinary practice, assays may be carried out at any
time during the pregnancy, but are, preferably, carried out early
in pregnancy, prior to the onset of pre-eclampsia symptoms. Given
that the term of pregnancies varies widely between species, the
timing of the assay will be determined by a veterinarian, but will
generally correspond to the timing of assays during a human
pregnancy.
[0148] The diagnostic methods described herein can be used
individually or in combination with any other diagnostic method
described herein for a more accurate diagnosis of the presence of,
severity of, or estimated time of onset of pre-eclampsia or
eclampsia. In addition, the diagnostic methods described herein can
be used in combination with any other diagnostic methods determined
to be useful for the accurate diagnosis of the presence of,
severity of, or estimated time of onset of pre-eclampsia or
eclampsia.
[0149] The diagnostic methods described herein can also be used to
monitor and manage pre-eclampsia or eclampsia in a subject. In one
example, a therapy is administed until the blood, plasma, or serum
soluble endoglin level is less than 25 ng/ml. In another example,
if a subject is determined to have an increased level of soluble
endoglin relative to a normal control then the therapy can be
administered until the serum PlGF level rises to approximately 400
pg/mL. In this embodiment, the levels of soluble endoglin, sFlt-1,
PlGF, and VEGF, or any and all of these, are measured repeatedly as
a method of not only diagnosing disease but monitoring the
treatment and management of the pre-eclampsia and eclampsia.
Diagnostic Kits
[0150] The invention also provides for a diagnostic test kit. For
example, a diagnostic test kit can include antibodies to soluble
endoglin and means for detecting, and more preferably evaluating,
binding between the antibodies and the soluble endoglin
polypeptide. For detection, either the antibody or the soluble
endoglin polypeptide is labeled, and either the antibody or the
soluble endoglin polypeptide is substrate-bound, such that soluble
endoglin polypeptide-antibody interaction can be established by
determining the amount of label attached to the substrate following
binding between the antibody and the soluble endoglin polypeptide.
A conventional ELISA is a common, art-known method for detecting
antibody-substrate interaction and can be provided with the kit of
the invention. Soluble endoglin polypeptides can be detected in
virtually any bodily fluid including, but not limited to urine,
serum, plasma, saliva, amniotic fluid, or cerebrospinal fluid. The
invention also provides for a diagnostic test kit that includes a
soluble endoglin nucleic acid that can be used to detect and
determine levels of soluble endoglin nucleic acids. A kit that
determines an alteration in the level of soluble endoglin
polypeptide relative to a reference, such as the level present in a
normal control, is useful as a diagnostic kit in the methods of the
invention.
[0151] The diagnostic kits of the invention can include antibodies
or nucleic acids for the detection of sFlt-1, VEGF, or PlGF
polypeptides or nucleic acids as described in U.S. patent
application Publication Nos. 20040126828, 20050025762, and
2005017044 and PCT Publication Numbers WO 2004/008946 and WO
2005/077007.
[0152] Desirably, the kit includes any of the components needed to
perform any of the diagnostic methods described above. For example,
the kit desirably includes a membrane, where the soluble endoglin
binding agent or the agent that binds the soluble endoglin binding
agent is immobilized on the membrane. The membrane can be supported
on a dipstick structure where the sample is deposited on the
membrane by placing the dipstick structure into the sample or the
membrane can be supported in a lateral flow cassette where the
sample is deposited on the membrane through an opening in the
cassette.
[0153] The diagnostic kits also generally include a label or
instructions for the intended use of the kit components and a
reference sample or purified proteins to be used to establish a
standard curve. In one example, the kit contains instructions for
the use of the kit for the diagnosis of pre-eclampsia, eclampsia,
or the propensity to develop pre-eclampsia or eclampsia. In yet
another example, the kit contains instructions for the use of the
kit to monitor therapeutic treatment or dosage regimens for the
treatment of pre-eclampsia or eclampsia. The diagnostic kit may
also include a label or instructions for the use of the kit to
determine the PAAI or soluble endoglin anti-angiogenesis index of
the subject sample and to compare the PAAI or soluble endoglin
anti-angiogenesis index to a reference sample value. It will be
understood that the reference sample values will depend on the
intended use of the kit. For example, the sample can be compared to
a normal reference value, wherein an increase in the PAAI or
soluble endoglin anti-angiogenesis index or in the soluble endoglin
value is indicative of pre-eclampsia or eclampsia, or a
predisposition to pre-eclampsia or eclampsia. In another example, a
kit used for therapeutic monitoring can have a reference PAAI or
soluble endoglin anti-angiogenesis index value or soluble endoglin
value that is indicative of pre-eclampsia or eclampsia, wherein a
decrease in the PAAI or soluble endoglin anti-angiogenesis index
value or a decrease in the soluble endoglin value of the subject
sample relative to the reference sample can be used to indicate
therapeutic efficacy or effective dosages of therapeutic
compounds.
[0154] Screening Assays
[0155] As discussed above, the level of a soluble endoglin nucleic
acid or polypeptide is increased in a subject having pre-eclampsia,
eclampsia, or a predisposition to such conditions. Based on these
discoveries, compositions of the invention are useful for the
high-throughput low-cost screening of candidate compounds to
identify those that modulate the expression of a soluble endoglin
polypeptide or nucleic acid molecule whose expression is altered in
a subject having a pre-eclampsia or eclampsia.
[0156] Any number of methods are available for carrying out
screening assays to identify new candidate compounds that alter the
expression of a soluble endoglin nucleic acid molecule. In one
working example, candidate compounds are added at varying
concentrations to the culture medium of cultured cells expressing a
soluble endoglin nucleic acid sequence. Exemplary cell cultures
include trophoblasts (e.g., BEWO, JAR, and JEG cells) and HUVECs.
Cells that express high levels of the membrane bound form of
endoglin can be treated with a proteinase (e.g., a matrix
metalloproteinase) that cleaves the extracellular domain of
endoglin to form soluble endoglin. These cells can then be used to
screen for new candidate compounds. Gene expression is then
measured, for example, by microarray analysis, Northern blot
analysis (Ausubel et al., supra), or RT-PCR, using any appropriate
fragment prepared from the nucleic acid molecule as a hybridization
probe. The level of gene expression in the presence of the
candidate compound is compared to the level measured in a control
culture medium lacking the candidate compound. A compound that
promotes a decrease in the expression of a soluble endoglin gene,
nucleic acid molecule, or polypeptide, or a functional equivalent
thereof, is considered useful in the invention; such a molecule may
be used, for example, as a therapeutic to treat pre-eclampsia or
eclampsia in a subject.
[0157] In another working example, the effect of candidate
compounds may be measured at the level of polypeptide production
using the same general approach and standard immunological
techniques, such as western blotting or immunoprecipitation with an
antibody specific for a soluble endoglin polypeptide. For example,
immunoassays may be used to detect or monitor the expression of at
least one of the polypeptides of the invention in an organism.
Polyclonal or monoclonal antibodies (produced as described above)
that are capable of binding to such a polypeptide may be used in
any standard immunoassay format (e.g., ELISA, western blot, or RIA
assay) to measure the level of the polypeptide. In some
embodiments, a compound that promotes a decrease in the expression
or biological activity of a soluble endoglin polypeptide is
considered particularly useful. Again, such a molecule may be used,
for example, as a therapeutic to delay, ameliorate, or treat a
pre-eclampsia or eclampsia, or the symptoms of a pre-eclampsia or
eclampsia, in a subject.
[0158] In yet another working example, candidate compounds may be
screened for those that specifically bind to a soluble endoglin
polypeptide. The efficacy of such a candidate compound is dependent
upon its ability to interact with such a polypeptide or a
functional equivalent thereof. Such an interaction can be readily
assayed using any number of standard binding techniques and
functional assays (e.g., those described in Ausubel et al., supra).
In one embodiment, a candidate compound may be tested in vitro for
its ability to specifically bind a polypeptide of the invention. In
another embodiment, a candidate compound is tested for its ability
to decrease the biological activity of a soluble endoglin
polypeptide by decreasing binding of a soluble endoglin polypeptide
and a growth factor, such as TGF-.beta.1, TGF-.beta.3, activin-A,
BMP-2 and BMP-7.
[0159] In another working example, a soluble endoglin nucleic acid
is expressed as a transcriptional or translational fusion with a
detectable reporter, and expressed in an isolated cell (e.g.,
mammalian or insect cell) under the control of a heterologous
promoter, such as an inducible promoter. The cell expressing the
fusion protein is then contacted with a candidate compound, and the
expression of the detectable reporter in that cell is compared to
the expression of the detectable reporter in an untreated control
cell. A candidate compound that decreases the expression of a
soluble endoglin detectable reporter is a compound that is useful
for the treatment of pre-eclampsia or eclampsia. In preferred
embodiments, the candidate compound alters the expression of a
reporter gene fused to a nucleic acid or nucleic acid.
[0160] In one particular working example, a candidate compound that
binds to a soluble endoglin polypeptide may be identified using a
chromatography-based technique. For example, a recombinant
polypeptide of the invention may be purified by standard techniques
from cells engineered to express the polypeptide (e.g., those
described above) and may be immobilized on a column. A solution of
candidate compounds is then passed through the column, and a
compound specific for the soluble endoglin polypeptide is
identified on the basis of its ability to bind to the polypeptide
and be immobilized on the column. To isolate the compound, the
column is washed to remove non-specifically bound molecules, and
the compound of interest is then released from the column and
collected. Similar methods may be used to isolate a compound bound
to a polypeptide microarray. Compounds isolated by this method (or
any other appropriate method) may, if desired, be further purified
(e.g., by high performance liquid chromatography). In addition,
these candidate compounds may be tested for their ability to
decrease the activity of a soluble endoglin polypeptide. Compounds
isolated by this approach may also be used, for example, as
therapeutics to treat pre-eclampsia or eclampsia in a human
subject. Compounds that are identified as binding to a polypeptide
of the invention with an affinity constant less than or equal to 10
mM are considered particularly useful in the invention.
Alternatively, any in vivo protein interaction detection system,
for example, any two-hybrid assay may be utilized to identify
compounds or proteins that bind to a polypeptide of the
invention.
[0161] Potential antagonists include organic molecules, peptides,
peptide mimetics, polypeptides, nucleic acids, and antibodies that
bind to a soluble endoglin nucleic acid sequence or a soluble
endoglin polypeptide.
[0162] Soluble endoglin DNA sequences may also be used in the
discovery and development of a therapeutic compound for the
treatment of pre-eclampsia or eclampsia. The encoded protein, upon
expression, can be used as a target for the screening of drugs.
Additionally, the DNA sequences encoding the amino terminal regions
of the encoded protein or Shine-Delgarno or other translation
facilitating sequences may be isolated by standard techniques
(Ausubel et al., supra).
[0163] Optionally, compounds identified in any of the
above-described assays may be confirmed as useful in an assay for
compounds that decrease the biological activity of soluble
endoglin.
[0164] Small molecules of the invention preferably have a molecular
weight below 2,000 daltons, more preferably between 300 and 1,000
daltons, and most preferably between 400 and 700 daltons. It is
preferred that these small molecules are organic molecules.
[0165] Test Compounds and Extracts
[0166] In general, compounds capable of decreasing the activity of
a soluble endoglin polypeptide are identified from large libraries
of both natural product or synthetic (or semi-synthetic) extracts
or chemical libraries or from polypeptide or nucleic acid
libraries, according to methods known in the art. Those skilled in
the field of drug discovery and development will understand that
the precise source of test extracts or compounds is not critical to
the screening procedure(s) of the invention. Compounds used in
screens may include known compounds (for example, known
therapeutics used for other diseases or disorders). Alternatively,
virtually any number of unknown chemical extracts or compounds can
be screened using the methods described herein. Examples of such
extracts or compounds include, but are not limited to, plant-,
fungal-, prokaryotic- or animal-based extracts, fermentation
broths, and synthetic compounds, as well as modification of
existing compounds. Numerous methods are also available for
generating random or directed synthesis (e.g., semi-synthesis or
total synthesis) of any number of chemical compounds, including,
but not limited to, saccharide-, lipid-, peptide-, and nucleic
acid-based compounds. Synthetic compound libraries are commercially
available from Brandon Associates (Merrimack, N.H.) and Aldrich
Chemical (Milwaukee, Wis.). Alternatively, libraries of natural
compounds in the form of bacterial, fungal, plant, and animal
extracts are commercially available from a number of sources,
including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch
Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A.
(Cambridge, Mass.). In addition, natural and synthetically produced
libraries are produced, if desired, according to methods known in
the art, e.g., by standard extraction and fractionation methods.
Furthermore, if desired, any library or compound is readily
modified using standard chemical, physical, or biochemical
methods.
[0167] In addition, those skilled in the art of drug discovery and
development readily understand that methods for dereplication
(e.g., taxonomic dereplication, biological dereplication, and
chemical dereplication, or any combination thereof) or the
elimination of replicates or repeats of materials already known for
their molt-disrupting activity should be employed whenever
possible.
[0168] When a crude extract is found to decrease the activity of a
soluble endoglin polypeptide, or to bind to a soluble endoglin
polypeptide, further fractionation of the positive lead extract is
necessary to isolate chemical constituents responsible for the
observed effect. Thus, the goal of the extraction, fractionation,
and purification process is the careful characterization and
identification of a chemical entity within the crude extract that
decreases the activity of a soluble endoglin polypeptide. Methods
of fractionation and purification of such heterogeneous extracts
are known in the art. If desired, compounds shown to be useful as
therapeutics for the treatment of a human pre-eclampsia or
eclampsia are chemically modified according to methods known in the
art.
Therapeutics
[0169] The present invention features methods and compositions for
treating or preventing pre-eclampsia or eclampsia in a subject.
Given that levels of soluble endoglin are increased in subjects
having pre-eclampsia, eclampsia, or having a predisposition to such
conditions, any agent that decreases the expression levels and/or
biological activity of a soluble endoglin polypeptide or nucleic
acid molecule is useful in the methods of the invention. Such
agents include compounds such as TGF-.beta.1, TGF-.beta.3,
activin-A, BMP2, or BMP7, that can disrupt soluble endoglin binding
to ligands; a purified antibody or antigen-binding fragment that
specifically binds soluble endoglin; antisense nucleobase
oligomers; and dsRNAs used to mediate RNA interference. Additional
useful compounds inlcude any compounds that can alter the
biological activity of soluble endoglin, for example, as measured
by an angiogenesis assay. Exemplary treatment methods are described
in detail below. These methods can also be combined with methods to
decrease sFlt-1 levels or to increase VEGF or PlGF levels or
decrease sFlt-1 levels as described in PCT Publication Number WO
2004/008946 and U.S. patent Publication No. 20040126828.
Therapeutics Targeting the TGF-.beta. Signaling Pathway
[0170] TGF-.beta. is the prototype of a family of at least 25
growth factors which regulate growth, differentiation, motility,
tissue remodeling, neurogenesis, wound repair, apoptosis, and
angiogenesis in many cell types. TGF-.beta. also inhibits cell
proliferation in many cell types and can stimulate the synthesis of
matrix proteins. Unless evidenced from the context in which it is
used, the term TGF-.beta. as used throughout this specification
will be understood to generally refer to any and all members of the
TGF-.beta. superfamily as appropriate. Soluble endoglin binds
several specific members of the TGF-.beta. family including
TGF-.beta.1, TGF-.beta.3, activin, BMP-2 and BMP-7, and may serve
to deplete the developing fetus or placenta of these necessary
mitogenic and angiogenic factor. The present invention features
methods of increasing the levels of these ligands to bind to
soluble endoglin and to neutralize the effects of soluble
endoglin.
Purified Proteins
[0171] In a preferred embodiment of the present invention, purified
forms of any soluble endoglin ligand such as TGF-.beta. family
proteins, including but not limited to TGF-.beta.1, TGF-.beta.3,
activin-A, BMP2, and BMP7, are administered to the subject in order
to treat or prevent pre-eclampsia or eclampsia.
[0172] Purified TGF-.beta. family proteins include any protein with
an amino acid sequence that is homologous, more desirably,
substantially identical to the amino acid sequence of TGF-.beta.1
or TGF-.beta.3, or any known TGF-.beta. family member, that can
induce angiogenesis. Non-limiting examples include human
TGF-.beta.1 (Cat #240-B-002) and human TGF-.beta.3 (Cat
#243-B3-002) from R & D Systems, MN.
Therapeutic Compounds that Inhibit Proteolytic Cleavage of
Endgolin
[0173] We have identified a potential cleavage site in the
extracellular domain of endoglin where a proteolytic enzyme could
cleave the membrane bound form of endoglin, releasing the
extracellular domain as a soluble form. Our sequence alignments of
the cleavage site suggest that a matrix metalloproteinase (MMP) may
be responsible for the cleavage and release of soluble endoglin.
Alternatively, a cathepsin or an elastase may also be involved in
the cleavage event. MMPs are also known as collagenases,
gelatinases, and stromelysins and there are currently 26 family
members known (for a review see Whittaker and Ayscough, Cell
Transmissions 17:1 (2001)). A preferred MMP is MMP9, which is known
to be up-regulated in placentas from pre-eclamptic patients (Lim et
al., Am. J. Pathol. 151:1809-1818, 1997). The activity of MMPs is
controlled through activation of pro-enzymes and inhibition by
endogenous inhibitors such as the tissue inhibitors of
metalloproteinases (TIMPS). Inhibitors of MMPs are zinc binding
proteins. There are 4 known endogenous inhibitors (TIMP 1-4), which
are reviewed in Whittaker et al., supra. One preferred MMP
inhibitor is the inhibitor of membrane type-MMP1 that has been
shown to cleave betaglycan, a molecule that shares similarity to
enodglin (Velasco-Loyden et al., J. Biol. Chem. 279:7721-7733
(2004)). In addition, a variety of naturally-occurring and
synthetic MMP inhibitors have been identified and are also reviewed
in Whittaker et al., supra. Examples include antibodies directed to
MMPs, and various compounds including marimastat, batimastat,
CT1746, BAY 12-9566, Prinomastat, CGS-27023A, D9120, BMS275291
(Bristol Myers Squibb), and trocade, some of which are currently in
clinical trials. Given the potential role of MMPs, cathepsins, or
elastases in the release and up-regulation of soluble endoglin
levels, the present invention also provides for the use of any
compound, such as those described above, known to inhibit the
activity of any MMP, cathepsin, or elastase involved in the
cleavage and release of soluble endoglin, for the treatment or
prevention of pre-eclampsia or eclampsia in a subject.
Therapeutic Compounds that Increase Soluble Endoglin Binding
Proteins
[0174] The present invention provides for the use of any compound
known to stimulate or increase blood serum levels of soluble
endoglin binding proteins, including but not limited to
TGF-.beta.1, TGF-.beta.3, activin-A, BMP2, and BMP7, for the
treatment or prevention of pre-eclampsia in a subject. These
compounds can be used alone or in combination with the purified
proteins described above or any of the other methods used to
increase TGF-.beta. family proteins protein levels described
herein. In one example, cyclosporine is used to stimulate
TGF-.beta.1 production at a dosage of 100-200 mg twice a day.
[0175] In addition to the use of compounds that can increase serum
levels of soluble endoglin binding proteins, the invention provides
for the use of any chronic hypertension medications used in
combination with any of the therapeutic methods described herein.
Medications used for the treatment of hypertension during pregnancy
include methyldopa, hydralazine hydrochloride, or labetalol. For
each of these medications, modes of administration and dosages are
determined by the physician and by the manufacturer's
instructions.
Therapeutic Compounds that Alter the Anti-Angiogenic Activity of
Soluble Endoglin
[0176] Additional therapeutic compounds can be identified using
angiogenesis assays. For example, pre-eclamptic serum having
elevated levels of soluble endoglin are added to a matrigel tube
formation assay will induce an anti-angiogenic state. Test
compounds can then be added to the assay and a reversion in the
anti-angiogenic state by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90% or more indicates that the compound can reduce the biological
activity of soluble endoglin and is useful as a therapeutic
compound.
Therapeutic Nucleic Acids
[0177] Recent work has shown that the delivery of nucleic acid (DNA
or RNA) capable of expressing an endothelial cell mitogen such as
VEGF to the site of a blood vessel injury will induce proliferation
and reendothelialization of the injured vessel. While the present
invention does not relate to blood vessel injury, these general
techniques for the delivery of nucleic acid to endothelial cells
can be used in the present invention for the delivery of nucleic
acids encoding soluble endoglin binding proteins, such as
TGF-.beta.1, TGF-.beta.3, activin-A, BMP2 and BMP7. The techniques
can also be used for the delivery of nucleic acids encoding
proteins, such as those described above, known to inhibit the
activity of any MMP, cathepsin, or elastase involved in the
cleavage and release of soluble endoglin, for the treatment or
prevention of pre-eclampsia or eclampsia in a subject. These
general techniques are described in U.S. Pat. Nos. 5,830,879 and
6,258,787 and are incorporated herein by reference.
[0178] In the present invention the nucleic acid may be any nucleic
acid (DNA or RNA) including genomic DNA, cDNA, and mRNA, encoding a
soluble endoglin binding proteins such as TGF-.beta.1, TGF-.beta.3,
activin-A, BMP2 and BMP7. The nucleic acids encoding the desired
protein may be obtained using routine procedures in the art, e.g.
recombinant DNA, PCR amplification.
Modes for Delivering Nucleic Acids
[0179] For any of the nucleic acid applications described herein,
standard methods for administering nucleic acids can be used. For
example, to simplify the manipulation and handling of the nucleic
acid encoding the soluble endoglin binding protein, the nucleic
acid is preferably inserted into a cassette where it is operably
linked to a promoter. The promoter must be capable of driving
expression of the soluble endoglin binding protein in the desired
target host cell. The selection of appropriate promoters can
readily be accomplished. Preferably, one would use a high
expression promoter. An example of a suitable promoter is the
763-base-pair cytomegalovirus (CMV) promoter. The Rous sarcoma
virus (RSV) (Davis, et al., Hum. Gene Ther. 4:151-159, 1993) and
mouse mammary tumor virus (MMTV) promoters may also be used.
Certain proteins can be expressed using their native promoter.
Other elements that can enhance expression can also be included
(e.g., enhancers or a system that results in high levels of
expression such as a tat gene and tar element). The recombinant
vector can be a plasmid vector such as pUC118, pBR322, or other
known plasmid vectors, that includes, for example, an E. coli
origin of replication (see, Sambrook, et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory press, 1989). The
plasmid vector may also include a selectable marker such as the
.beta. lactamase gene for ampicillin resistance, provided that the
marker polypeptide does not adversely affect the metabolism of the
organism being treated. The cassette can also be bound to a nucleic
acid binding moiety in a synthetic delivery system, such as the
system disclosed in PCT Publication No. W095/22618.
[0180] The nucleic acid can be introduced into the cells by any
means appropriate for the vector employed. Many such methods are
well known in the art (Sambrook et al., supra, and Watson et al.,
"Recombinant DNA", Chapter 12, 2d edition, Scientific American
Books, 1992). Recombinant vectors can be transferred by methods
such as calcium phosphate precipitation, electroporation,
liposome-mediated transfection, gene gun, microinjection, viral
capsid-mediated transfer, polybrene-mediated transfer, or
protoplast fusion. For a review of the procedures for liposome
preparation, targeting and delivery of contents, see Mannino and
Gould-Fogerite, (Bio Techniques, 6:682-690, 1988), Felgner and
Holm, (Bethesda Res. Lab. Focus, 11:21, 1989) and Maurer (Bethesda
Res. Lab. Focus, 11:25, 1989).
[0181] Transfer of the recombinant vector (either plasmid vector or
viral vectors) can be accomplished through direct injection into
the amniotic fluid or intravenous delivery.
[0182] Gene delivery using adenoviral vectors or adeno-associated
vectors (AAV) can also be used. Adenoviruses are present in a large
number of animal species, are not very pathogenic, and can
replicate equally well in dividing and quiescent cells. As a
general rule, adenoviruses used for gene delivery are lacking one
or more genes required for viral replication. Replication-defective
recombinant adenoviral vectors used for the delivery of a soluble
endoglin binding protein, can be produced in accordance with
art-known techniques (see Quantin et al., Proc. Natl. Acad. Sci.
USA, 89:2581-2584, 1992; Stratford-Perricadet et al., J. Clin.
Invest., 90:626-630, 1992; and Rosenfeld et al., Cell, 68:143-155,
1992). For an example of the use of gene therapy in utero see U.S.
Pat. No. 6,399,585.
[0183] Once transferred, the nucleic acid is expressed by the cells
at the site of injury for a period of time sufficient to increase
blood serum levels of a soluble endoglin binding protein. Because
the vectors containing the nucleic acid are not normally
incorporated into the genome of the cells, expression of the
protein of interest takes place for only a limited time. Typically,
the protein is expressed at therapeutic levels for about two days
to several weeks, preferably for about one to two weeks.
Re-application of the DNA can be utilized to provide additional
periods of expression of the therapeutic protein.
Therapeutic Nucleic Acids that Inhibit Soluble Endoglin
Expression
[0184] The present invention also features the use of antisense
nucleobase oligomers to downregulate expression of soluble endoglin
mRNA directly. By binding to the complementary nucleic acid
sequence (the sense or coding strand), antisense nucleobase
oligomers are able to inhibit protein expression presumably through
the enzymatic cleavage of the RNA strand by RNAse H. Preferably the
antisense nucleobase oligomer is capable of reducing soluble
endoglin protein expression in a cell that expresses increased
levels of soluble endoglin. Preferably the decrease in soluble
endoglin protein expression is at least 10% relative to cells
treated with a control oligonucleotide, preferably 20% or greater,
more preferably 40%, 50%, 60%, 70%, 80%, 90% or greater. Methods
for selecting and preparing antisense nucleobase oligomers are well
known in the art. For an example of the use of antisense nucleobase
oligomers to downregulate VEGF expression see U.S. Pat. No.
6,410,322, incorporated herein by reference. Methods for assaying
levels of protein expression are also well known in the art and
include western blotting, immunoprecipitation, and ELISA.
[0185] The present invention also features the use of RNA
interference (RNAi) to inhibit expression of soluble endoglin. RNA
interference (RNAi) is a recently discovered mechanism of
post-transcriptional gene silencing (PTGS) in which double-stranded
RNA (dsRNA) corresponding to a gene or mRNA of interest is
introduced into an organism resulting in the degradation of the
corresponding mRNA. In the RNAi reaction, both the sense and
anti-sense strands of a dsRNA molecule are processed into small RNA
fragments or segments ranging in length from 21 to 23 nucleotides
(nt) and having 2-nucleotide 3' tails. Alternatively, synthetic
dsRNAs, which are 21 to 23 nt in length and have 2-nucleotide 3'
tails, can be synthesized, purified and used in the reaction. These
21 to 23 nt dsRNAs are known as "guide RNAs" or "short interfering
RNAs" (siRNAs).
[0186] The siRNA duplexes then bind to a nuclease complex composed
of proteins that target and destroy endogenous mRNAs having
homology to the siRNA within the complex. Although the identity of
the proteins within the complex remains unclear, the function of
the complex is to target the homologous mRNA molecule through base
pairing interactions between one of the siRNA strands and the
endogenous mRNA. The mRNA is then cleaved approximately 12 nt from
the 3' terminus of the siRNA and degraded. In this manner, specific
genes can be targeted and degraded, thereby resulting in a loss of
protein expression from the targeted gene. siRNAs can also be
chemically synthesized or obtained from a company that chemically
synthesizes siRNAs (e.g., Dharmacon Research Inc., Pharmacia, or
ABI).
[0187] The specific requirements and modifications of dsRNA are
described in PCT Publication No. WO01/75164 (incorporated herein by
reference). While dsRNA molecules can vary in length, it is most
preferable to use siRNA molecules which are 21- to 23-nucleotide
dsRNAs with characteristic 2- to 3-nucleotide 3' overhanging ends
typically either (2'-deoxy)thymidine or uracil. The siRNAs
typically comprise a 3' hydroxyl group. Single stranded siRNA as
well as blunt ended forms of dsRNA can also be used. In order to
further enhance the stability of the RNA, the 3' overhangs can be
stabilized against degradation. In one such embodiment, the RNA is
stabilized by including purine nucleotides, such as adenosine or
guanosine. Alternatively, substitution of pyrimidine nucleotides by
modified analogs, e.g.,substitution of uridine 2-nucleotide
overhangs by (2'-deoxy)thymide is tolerated and does not affect the
efficiency of RNAi. The absence of a 2' hydroxyl group
significantly enhances the nuclease resistance of the overhang in
tissue culture medium.
[0188] Alternatively siRNA can be prepared using any of the methods
set forth in PCT Publication No. WO01/75164 (incorporated herein by
reference) or using standard procedures for in vitro transcription
of RNA and dsRNA annealing procedures as described in Elbashir et
al. (Genes & Dev., 15:188-200, 2001). siRNAs are also obtained
as described in Elbashir et al., supra, by incubation of dsRNA that
corresponds to a sequence of the target gene in a cell-free
Drosophila lysate from syncytial blastoderm Drosophila embryos
under conditions in which the dsRNA is processed to generate siRNAs
of about 21 to about 23 nucleotides, which are then isolated using
techniques known to those of skill in the art. For example, gel
electrophoresis can be used to separate the 21-23 nt RNAs and the
RNAs can then be eluted from the gel slices. In addition,
chromatography (e.g., size exclusion chromatography), glycerol
gradient centrifugation, and affinity purification with antibody
can be used to isolate the 21 to 23 nt RNAs.
[0189] A variety of methods are available for transfection, or
introduction, of dsRNA or oligonucleotides into mammalian cells.
For example, there are several commercially available transfection
reagents including but not limited to: TransIT-TKO.TM. (Mirus, Cat.
# MIR 2150), Transmessenger.TM. (Qiagen, Cat. # 301525), and
Oligofectamine.TM. (Invitrogen, Cat. # MIR 12252-011). Protocols
for each transfection reagent are available from the
manufacturer.
[0190] In the present invention, the dsRNA, or siRNA, is
complementary to the mRNA sequence of soluble endoglin mRNA and can
reduce or inhibit expression of soluble endoglin. Preferably, the
decrease in soluble endoglin protein expression is at least 10%
relative to cells treated with a control dsRNA or siRNA, more
preferably 25%, and most preferably at least 40%, 50%, 60%, 70%,
80%, 90%, or more. Methods for assaying levels of protein
expression are also well known in the art and include western
blotting, immunoprecipitation, and ELISA.
[0191] In the present invention, the nucleic acids used include any
modification that enhances the stability or function of the nucleic
acid in any way. Examples include modifications to the phosphate
backbone, the internucleotide linkage, or to the sugar moiety.
[0192] Soluble Endoglin Based Therapeutic Compounds Useful in Early
Pregnancy
[0193] Inhibition of full-length endoglin signaling has been shown
to enhance trophoblast invasivness in villous explant cultures
(Caniggia I et al, Endocrinology, 1997, 138:4977-88). Soluble
endoglin is therefore likely to enhance trophoblast invasviness
during early pregnancy. Accordingly, compositions that increase
soluble endoglin levels early in pregnancy in a woman who does not
have a pregnancy related hypertensive disorder or a predisposition
to a pregnancy related hypertensive disorder may be beneficial for
enhancing placentation. Examples of compositions that increase
soluble endoglin levels include purified soluble endoglin
polypeptides, soluble endoglin encoding nucleic acid molecules, and
compounds or growth factors that increase the levels or biological
activity of soluble endoglin.
Assays for Gene and Protein Expression
[0194] The following methods can be used to evaluate protein or
gene expression and determine efficacy for any of the
above-mentioned methods for increasing soluble endoglin binding
protein levels, or for decreasing soluble endoglin protein
levels.
[0195] Blood serum from the subject is measured for levels of
soluble endoglin, using methods such as ELISA, western blotting, or
immunoassays using specific antibodies. Blood serum from the
subject can also be measured for levels of TGF-.beta.1,
TGF-.beta.3, activin-A, BMP2, BMP7, or any protein ligand known to
bind to soluble endoglin. Methods used to measure serum levels of
proteins include ELISA, western blotting, or immunoassays using
specific antibodies. In addition, in vitro angiogenesis assays can
be performed to determine if the subject's blood has converted from
an anti-angiogenic state to a pro-angiogenic state. Such assays are
described above in Example 4. A positive result is considered an
increase of at least 10%, 20%, preferably 30%, more preferably at
least 40% or 50%, and most preferably at least 60%, 70%, 80%, 90%
or more in the levels of soluble endoglin, TGF-.beta.1,
TGF-.beta.3, activin-A, BMP2, BMP7, or any protein ligand known to
bind to soluble endoglin. A positive result can also be considered
conversion by at least 10%, preferably 20%, 30%, 40%, 50%, and most
preferably at least 60%, 70%, 80%, 90% or more from an
anti-angiogenic state to a pro-angiogenic state using the in vitro
angiogenesis assay.
[0196] Blood serum or urine samples from the subject can also be
measured for levels of nucleic acids or polypeptides encoding
TGF-.beta.1, TGF-.beta.3, activin-A, BMP2, BMP7, or soluble
endoglin. There are several art-known methods to assay for gene
expression. Some examples include the preparation of RNA from the
blood samples of the subject and the use of the RNA for northern
blotting, PCR based amplification, or RNAse protection assays. A
positive result is considered an increase of at least least 10%,
20%, preferably 30%, more preferably at least 40% or 50%, and most
preferably at least 60%, 70%, 80%, 90% or more in the levels of
soluble endoglin, TGF-.beta.1, TGF-.beta.3, activin-A, BMP2, BMP7
nucleic acids.
Use of Antibodies for Therapeutic Treatment
[0197] The elevated levels of soluble endoglin found in the serum
samples taken from pregnant women suffering from pre-eclampsia
suggests that soluble endoglin is acting as a "physiologic sink" to
bind to and deplete the trophoblast cells and maternal endothelial
cells of functional growth factors required for the proper
development and angiogenesis of the fetus or the placenta. The use
of compounds, such as antibodies, to bind to soluble endoglin and
neutralize the activity of soluble endoglin (e.g., binding to
TGF-.beta.1, TGF-.beta.3, activin-A, BMP2, BMP7), may help prevent
or treat pre-eclampsia or eclampsia, by producing an increase in
free TGF-.beta.1, TGF-.beta.3, activin-A, BMP2, and BMP7. Such an
increase would allow for an increase in trophoblast proliferation,
migration and angiogenesis required for placental development and
fetal nourishment, and for systemic maternal endothelial cell
health.
[0198] The present invention provides antibodies that bind
specifically to the ligand-binding domain of soluble endoglin. The
antibodies are used to neutralize the activity of soluble endoglin
and the most effective mechanism is believed to be through direct
blocking of the binding sites for TGF-.beta.1, TGF-.beta.3,
activin-A, BMP2, or BMP7, however, other mechanisms cannot be ruled
out. Methods for the preparation and use of antibodies for
therapeutic purposes are described in several patents including
U.S. Pat. Nos. 6,054,297; 5,821,337; 6,365,157; and 6,165,464 and
are incorporated herein by reference. Antibodies can be polyclonal
or monoclonal; monoclonal antibodies are preferred.
[0199] Monoclonal antibodies, particularly those derived from
rodents including mice, have been used for the treatment of various
diseases; however, there are limitations to their use including the
induction of a human anti-mouse immunoglobulin response that causes
rapid clearance and a reduction in the efficacy of the treatment.
For example, a major limitation in the clinical use of rodent
monoclonal antibodies is an anti-globulin response during therapy
(Miller et al., Blood, 62:988-995 1983; Schroff et al., Cancer
Res., 45:879-885, 1985).
[0200] The art has attempted to overcome this problem by
constructing "chimeric" antibodies in which an animal
antigen-binding variable domain is coupled to a human constant
domain (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad.
Sci. USA, 81:6851-6855, 1984; Boulianne et al., Nature,
312:643-646, 1984; Neuberger et al., Nature, 314:268-270, 1985).
The production and use of such chimeric antibodies are described
below.
[0201] Competitive inhibition of ligand binding to soluble endoglin
is useful for the prevention or treatment of pre-eclampsia or
eclampsia. Such an increase can result in a rescue of endothelial
dysfunction and a shift in the balance of
pro-angiogenic/anti-angiogenic factors towards angiogenesis.
[0202] A cocktail of the monoclonal antibodies of the present
invention can be used as an effective treatment for pre-eclampsia
or eclampsia. The cocktail may include as few as two, three, or
four different antibodies or as many as six, eight, or ten
different antibodies. In addition, the antibodies of the present
invention can be combined with an anti-hypertensive drug (e.g.,
methyldopa, hydralazine hydrochloride, or labetalol) or any other
medication used to treat pre-eclampsia, eclampsia, or the symptoms
associated with pre-eclampsia or eclampsia.
[0203] Preparation of Antibodies
[0204] Monoclonal antibodies that specifically bind to the sFlt-1
receptor may be produced by methods known in the art. These methods
include the immunological method described by Kohler and Milstein
(Nature, 256: 495-497, 1975) and Campbell ("Monoclonal Antibody
Technology, The Production and Characterization of Rodent and Human
Hybridomas" in Burdon et al., Eds., Laboratory Techniques in
Biochemistry and Molecular Biology, Volume 13, Elsevier Science
Publishers, Amsterdam, 1985), as well as by the recombinant DNA
method described by Huse et al. (Science, 246, 1275-1281,
1989).
[0205] Monoclonal antibodies may be prepared from supernatants of
cultured hybridoma cells or from ascites induced by
intra-peritoneal inoculation of hybridoma cells into mice. The
hybridoma technique described originally by Kohler and Milstein
(Eur. J. Immunol, 6, 511-519, 1976) has been widely applied to
produce hybrid cell lines that secrete high levels of monoclonal
antibodies against many specific antigens.
[0206] The route and schedule of immunization of the host animal or
cultured antibody-producing cells therefrom are generally in
keeping with established and conventional techniques for antibody
stimulation and production. Typically, mice are used as the test
model, however, any mammalian subject including human subjects or
antibody producing cells therefrom can be manipulated according to
the processes of this invention to serve as the basis for
production of mammalian, including human, hybrid cell lines.
[0207] After immunization, immune lymphoid cells are fused with
myeloma cells to generate a hybrid cell line that can be cultivated
and subcultivated indefinitely, to produce large quantities of
monoclonal antibodies. For purposes of this invention, the immune
lymphoid cells selected for fusion are lymphocytes and their normal
differentiated progeny, taken either from lymph node tissue or
spleen tissue from immunized animals. The use of spleen cells is
preferred, since they offer a more concentrated and convenient
source of antibody producing cells with respect to the mouse
system. The myeloma cells provide the basis for continuous
propagation of the fused hybrid. Myeloma cells are tumor cells
derived from plasma cells. Murine myeloma cell lines can be
obtained, for example, from the American Type Culture Collection
(ATCC; Manassas, Va.). Human myeloma and mouse-human heteromyeloma
cell lines have also been described (Kozbor et al., J. Immunol.,
133:3001-3005, 1984; Brodeur et al., Monoclonal Antibody Production
Techniques and Applications, Marcel Dekker, Inc., New York, pp.
51-63, 1987).
[0208] The hybrid cell lines can be maintained in vitro in cell
culture media. Once the hybridoma cell line is established, it can
be maintained on a variety of nutritionally adequate media such as
hypoxanthine-aminopterin-thymidine (HAT) medium. Moreover, the
hybrid cell lines can be stored and preserved in any number of
conventional ways, including freezing and storage under liquid
nitrogen. Frozen cell lines can be revived and cultured
indefinitely with resumed synthesis and secretion of monoclonal
antibody. The secreted antibody is recovered from tissue culture
supernatant by conventional methods such as precipitation, ion
exchange chromatography, affinity chromatography, or the like.
[0209] The antibody may be prepared in any mammal, including mice,
rats, rabbits, goats, and humans. The antibody may be a member of
one of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or
IgE, and the subclasses thereof, and preferably is an IgG
antibody.
[0210] While the preferred animal for producing monoclonal
antibodies is mouse, the invention is not so limited; in fact,
human antibodies may be used and may prove to be preferable. Such
antibodies can be obtained by using human hybridomas (Cole et al.,
"Monoclonal Antibodies and Cancer Therapy", Alan R. Liss Inc., p.
77-96, 1985). In the present invention, techniques developed for
the production of chimeric antibodies by splicing the genes from a
mouse antibody molecule of appropriate antigen specificity together
with genes from a human antibody molecule can be used (Morrison et
al., Proc. Natl. Acad. Sci. 81, 6851-6855, 1984; Neuberger et al.,
Nature 312, 604-608, 1984; Takeda et al., Nature 314, 452-454,
1985); such antibodies are within the scope of this invention and
are described below.
[0211] As another alternative to the cell fusion technique,
Epstein-Barr virus (EBV) immortalized B cells are used to produce
the monoclonal antibodies of the present invention (Crawford et
al., J. Gen. Virol., 64:697-700, 1983; Kozbor and Roder, J.
Immunol., 4:1275-1280, 1981; Kozbor et al., Methods Enzymol.,
121:120-140, 1986). In general, the procedure consists of isolating
Epstein-Barr virus from a suitable source, generally an infected
cell line, and exposing the target antibody secreting cells to
supernatants containing the virus. The cells are washed, and
cultured in an appropriate cell culture medium. Subsequently,
virally transformed cells present in the cell culture can be
identified by the presence of the Epstein-Barr viral nuclear
antigen, and transformed antibody secreting cells can be identified
using standard methods known in the art. Other methods for
producing monoclonal antibodies, such as recombinant DNA, are also
included within the scope of the invention.
[0212] Preparation of Soluble Endoglin Immunogens
[0213] Soluble endoglin may be used by itself as an immunogen, or
may be attached to a carrier protein or to other objects, such as
sepharose beads. Soluble endoglin may be purified from cells known
to express the endogenous protein such as human umbilical vein
endothelial cells (trophoblasts or HUVEC; Burrows et al., Clin.
Cancer Res. 1:1623-1634, 1995; Fonsatti et al., Clin. Cancer Res.
6:2037-2043, 2000). Cells that express membrane bound endoglin can
be treated with a proteolytic enzyme (e.g., a matrix
metalloproteinase) to cleave the extracellular domain, thereby
producing the soluble form. Additionally, nucleic acid molecules
that encode soluble endoglin, or portions thereof, can be inserted
into known vectors for expression in host cells using standard
recombinant DNA techniques. Suitable host cells for soluble
endoglin expression include baculovirus cells (e.g., Sf9 cells),
bacterial cells (e.g., E. coli), and mammalian cells (e.g., NIH3T3
cells).
[0214] In addition, peptides can be synthesized and used as
immunogens. The methods for making antibody to peptides are well
known in the art and generally require coupling the peptide to a
suitable carrier molecule, such as serum albumin. Peptides include
any amino acid sequence that is substantially identical to any part
of the soluble endoglin amino acid sequence corresponding to
GenBank accession numbers AAH29080 and NP.sub.--031958 (mouse);
AAS67893 (rat); NP.sub.--000109, P17813, VSP.sub.--004233, and
CAA80673 (human); and A49722 (pig). Peptides can be any length,
preferably 10 amino acids or greater, more preferably 25 amino
acids or greater, and most preferably 40, 50, 60, 70, 80, 100, 200,
300, 400, 437 amino acids or greater. Preferably, the amino acid
sequences are at least 60%, more preferably 85%, and, most
preferably 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the
sequence of any of the human endoglin sequences. The peptides can
be commercially obtained or made using techniques well known in the
art, such as, for example, the Merrifield solid-phase method
(Science, 232:341-347, 1985). The procedure may use commercially
available synthesizers such as a Biosearth 9500 automated peptide
machine, with cleavage of the blocked amino acids being achieved
with hydrogen fluoride, and the peptides purified by preparative
HPLC using a Waters Delta Prep 3000 instrument, on a 15-20 .mu.m
Vydac C4 PrepPAK column.
[0215] Functional Equivalents of Antibodies
[0216] The invention also includes functional equivalents of the
antibodies described in this specification. Functional equivalents
include polypeptides with amino acid sequences substantially
identical to the amino acid sequence of the variable or
hypervariable regions of the antibodies of the invention.
Functional equivalents have binding characteristics comparable to
those of the antibodies, and include, for example, chimerized,
humanized and single chain antibodies as well as fragments thereof.
Methods of producing such functional equivalents are disclosed, for
example, in PCT Publication No. WO93/21319; European Patent
Application No. 239,400; PCT Publication No. WO89/09622; European
Patent Application No. 338,745; European Patent Application No.
332424; and U.S. Pat. No. 4,816,567; each of which is herein
incorporated by reference.
[0217] Chimerized antibodies preferably have constant regions
derived substantially or exclusively from human antibody constant
regions and variable regions derived substantially or exclusively
from the sequence of the variable region from a mammal other than a
human. Such humanized antibodies are chimeric immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab',
F(ab').sub.2 or other antigen-binding subsequences of antibodies)
which contain minimal sequence derived from non-human
immunoglobulin. Methods for humanizing non-human antibodies are
well known in the art (for reviews see Vaswani and Hamilton, Ann.
Allergy Asthma Immunol., 81:105-119, 1998 and Carter, Nature
Reviews Cancer, 1:118-129, 2001). Generally, a humanized antibody
has one or more amino acid residues introduced into it from a
source that is non-human. These non-human amino acid residues are
often referred to as import residues, which are typically taken
from an import variable domain. Humanization can be essentially
performed following the methods known in the art (Jones et al.,
Nature, 321:522-525, 1986; Riechmann et al., Nature, 332:323-329,
1988; and Verhoeyen et al., Science, 239:1534-1536 1988), by
substituting rodent CDRs or other CDR sequences for the
corresponding sequences of a human antibody. Accordingly, such
humanized antibodies are chimeric antibodies wherein substantially
less than an intact human variable domain has been substituted by
the corresponding sequence from a non-human species (see for
example, U.S. Pat. No. 4,816,567). In practice, humanized
antibodies are typically human antibodies in which some CDR
residues and possibly some FR residues are substituted by residues
from analogous sites in rodent antibodies (Presta, Curr. Op.
Struct. Biol., 2:593-596, 1992).
[0218] Additional methods for the preparation of humanized
antibodies can be found in U.S. Pat. Nos. 5,821,337, and 6,054,297,
and Carter, (supra) which are all incorporated herein by reference.
The humanized antibody is selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any
isotype, including IgG.sub.1, IgG.sub.2, IgG.sub.3, and IgG.sub.4.
Where cytotoxic activity is not needed, such as in the present
invention, the constant domain is preferably of the IgG.sub.2
class. The humanized antibody may comprise sequences from more than
one class or isotype, and selecting particular constant domains to
optimize desired effector functions is within the ordinary skill in
the art.
[0219] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
(Marks et al., J. Mol. Biol., 222:581-597, 1991 and Winter et al.
Annu. Rev. Immunol., 12:433-455, 1994). The techniques of Cole et
al. and Boerner et al. are also useful for the preparation of human
monoclonal antibodies (Cole et al., supra; Boerner et al., J.
Immunol., 147: 86-95, 1991).
[0220] Suitable mammals other than a human include any mammal from
which monoclonal antibodies may be made. Examples of mammals other
than a human include, for example a rabbit, rat, mouse, horse,
goat, or primate; a mouse is preferred.
[0221] Functional equivalents of antibodies also include
single-chain antibody fragments, also known as single-chain
antibodies (scFvs). Single-chain antibody fragments are recombinant
polypeptides which typically bind antigens or receptors; these
fragments contain at least one fragment of an antibody variable
heavy-chain amino acid sequence (V.sub.H) tethered to at least one
fragment of an antibody variable light-chain sequence (V.sub.L)
with or without one or more interconnecting linkers. Such a linker
may be a short, flexible peptide selected to assure that the proper
three-dimensional folding of the V.sub.L and V.sub.H domains occurs
once they are linked so as to maintain the target molecule
binding-specificity of the whole antibody from which the
single-chain antibody fragment is derived. Generally, the carboxyl
terminus of the V.sub.L or V.sub.H sequence is covalently linked by
such a peptide linker to the amino acid terminus of a complementary
V.sub.L and V.sub.H sequence. Single-chain antibody fragments can
be generated by molecular cloning, antibody phage display library
or similar techniques. These proteins can be produced either in
eukaryotic cells or prokaryotic cells, including bacteria.
[0222] Single-chain antibody fragments contain amino acid sequences
having at least one of the variable regions or CDRs of the whole
antibodies described in this specification, but are lacking some or
all of the constant domains of those antibodies. These constant
domains are not necessary for antigen binding, but constitute a
major portion of the structure of whole antibodies. Single-chain
antibody fragments may therefore overcome some of the problems
associated with the use of antibodies containing part or all of a
constant domain. For example, single-chain antibody fragments tend
to be free of undesired interactions between biological molecules
and the heavy-chain constant region, or other unwanted biological
activity. Additionally, single-chain antibody fragments are
considerably smaller than whole antibodies and may therefore have
greater capillary permeability than whole antibodies, allowing
single-chain antibody fragments to localize and bind to target
antigen-binding sites more efficiently. Also, antibody fragments
can be produced on a relatively large scale in prokaryotic cells,
thus facilitating their production. Furthermore, the relatively
small size of single-chain antibody fragments makes them less
likely than whole antibodies to provoke an immune response in a
recipient.
[0223] Functional equivalents further include fragments of
antibodies that have the same or comparable binding characteristics
to those of the whole antibody. Such fragments may contain one or
both Fab fragments or the F(ab').sub.2 fragment. Preferably the
antibody fragments contain all six CDRs of the whole antibody,
although fragments containing fewer than all of such regions, such
as three, four or five CDRs, are also functional.
[0224] Further, the functional equivalents may be or may combine
members of any one of the following immunoglobulin classes: IgG,
IgM, IgA, IgD, or IgE, and the subclasses thereof.
[0225] Preparation of Functional Equivalents of Antibodies
[0226] Equivalents of antibodies are prepared by methods known in
the art. For example, fragments of antibodies may be prepared
enzymatically from whole antibodies. Preferably, equivalents of
antibodies are prepared from DNA encoding such equivalents. DNA
encoding fragments of antibodies may be prepared by deleting all
but the desired portion of the DNA that encodes the full-length
antibody.
[0227] DNA encoding chimerized antibodies may be prepared by
recombining DNA substantially or exclusively encoding human
constant regions and DNA encoding variable regions derived
substantially or exclusively from the sequence of the variable
region of a mammal other than a human. DNA encoding humanized
antibodies may be prepared by recombining DNA encoding constant
regions and variable regions other than the CDRs derived
substantially or exclusively from the corresponding human antibody
regions and DNA encoding CDRs derived substantially or exclusively
from a mammal other than a human.
[0228] Suitable sources of DNA molecules that encode fragments of
antibodies include cells, such as hybridomas, that express the
full-length antibody. The fragments may be used by themselves as
antibody equivalents, or may be recombined into equivalents, as
described above.
[0229] The DNA deletions and recombinations described in this
section may be carried out by known methods, such as those
described in the published patent applications listed above.
[0230] Antibody Screening and Selection
[0231] Monoclonal antibodies are isolated and purified using
standard art-known methods. For example, antibodies can be screened
using standard art-known methods such as ELISA against the soluble
endoglin peptide antigen or western blot analysis. Non-limiting
examples of such techniques are described in Examples II and III of
U.S. Pat. No. 6,365,157, herein incorporated by reference.
[0232] Therapeutic Uses of Antibodies
[0233] When used in vivo for the treatment or prevention of
pre-eclampsia or eclampsia, the antibodies of the subject invention
are administered to the subject in therapeutically effective
amounts. Preferably, the antibodies are administered parenterally
or intravenously by continuous infusion. The dose and dosage
regimen depends upon the severity of the disease, and the overall
health of the subject. The amount of antibody administered is
typically in the range of about 0.001 to about 10 mg/kg of subject
weight, preferably 0.01 to about 5 mg/kg of subject weight.
[0234] For parenteral administration, the antibodies are formulated
in a unit dosage injectable form (solution, suspension, emulsion)
in association with a pharmaceutically acceptable parenteral
vehicle. Such vehicles are inherently nontoxic, and
non-therapeutic. Examples of such vehicles are water, saline,
Ringer's solution, dextrose solution, and 5% human serum albumin.
Nonaqueous vehicles such as fixed oils and ethyl oleate may also be
used. Liposomes may be used as carriers. The vehicle may contain
minor amounts of additives such as substances that enhance
isotonicity and chemical stability, e.g., buffers and
preservatives. The antibodies typically are formulated in such
vehicles at concentrations of about 1 mg/ml to 10 mg/ml.
Combination Therapies
[0235] Optionally, a pre-eclampsia or eclampsia therapeutic may be
administered in combination with any other standard pre-eclampsia
or eclampsia therapy; such methods are known to the skilled artisan
and include the methods described in U.S. patent application
Publication Nos. 20040126828, 20050025762, and 2005017044 and PCT
Publication Numbers WO 2004/008946 and WO 2005/077007.
Dosages and Modes of Administration
[0236] Preferably, the therapeutic is administered during pregnancy
for the treatment or prevention of pre-eclampsia or eclampsia or
after pregnancy to treat post-partum pre-eclampsia or eclampsia.
Techniques and dosages for administration vary depending on the
type of compound (e.g., chemical compound, purified protein,
antibody, antisense, RNAi, or nucleic acid vector) and are well
known to those skilled in the art or are readily determined.
[0237] Therapeutic compounds of the present invention may be
administered with a pharmaceutically acceptable diluent, carrier,
or excipient, in unit dosage form. Administration may be
parenteral, intravenous, subcutaneous, oral or local by direct
injection into the amniotic fluid. Intravenous delivery by
continuous infusion is the preferred method for administering the
therapeutic compounds of the present invention.
[0238] The composition can be in the form of a pill, tablet,
capsule, liquid, or sustained release tablet for oral
administration; or a liquid for intravenous, subcutaneous or
parenteral administration; or a polymer or other sustained release
vehicle for local administration.
[0239] Methods well known in the art for making formulations are
found, for example, in "Remington: The Science and Practice of
Pharmacy" (20th ed., ed. A. R. Gennaro AR., 2000, Lippincott
Williams & Wilkins, Philadelphia, Pa.). Formulations for
parenteral administration may, for example, contain excipients,
sterile water, saline, polyalkylene glycols such as polyethylene
glycol, oils of vegetable origin, or hydrogenated napthalenes.
Biocompatible, biodegradable lactide polymer, lactide/glycolide
copolymer, or polyoxyethylene-polyoxypropylene copolymers may be
used to control the release of the compounds. Nanoparticulate
formulations (e.g., biodegradable nanoparticles, solid lipid
nanoparticles, liposomes) may be used to control the
biodistribution of the compounds. Other potentially useful
parenteral delivery systems include ethylene-vinyl acetate
copolymer particles, osmotic pumps, implantable infusion systems,
and liposomes. The concentration of the compound in the formulation
varies depending upon a number of factors, including the dosage of
the drug to be administered, and the route of administration.
[0240] The compound may be optionally administered as a
pharmaceutically acceptable salts, such as non-toxic acid addition
salts or metal complexes that are commonly used in the
pharmaceutical industry. Examples of acid addition salts include
organic acids such as acetic, lactic, pamoic, maleic, citric,
malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic,
tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic
acids or the like; polymeric acids such as tannic acid,
carboxymethyl cellulose, or the like; and inorganic acid such as
hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid,
or the like. Metal complexes include zinc, iron, and the like.
[0241] Formulations for oral use include tablets containing the
active ingredient(s) in a mixture with non-toxic pharmaceutically
acceptable excipients. These excipients may be, for example, inert
diluents or fillers (e.g., sucrose and sorbitol), lubricating
agents, glidants, and anti-adhesives (e.g., magnesium stearate,
zinc stearate, stearic acid, silicas, hydrogenated vegetable oils,
or talc).
[0242] Formulations for oral use may also be provided as chewable
tablets, or as hard gelatin capsules wherein the active ingredient
is mixed with an inert solid diluent, or as soft gelatin capsules
wherein the active ingredient is mixed with water or an oil
medium.
[0243] The dosage and the timing of administering the compound
depends on various clinical factors including the overall health of
the subject and the severity of the symptoms of pre-eclampsia. In
general, once pre-eclampsia or a predisposition to pre-eclampsia is
detected, continuous infusion of the purified protein is used to
treat or prevent further progression of the condition. Treatment
can be continued for a period of time ranging from 1 to 100 days,
more preferably 1 to 60 days, and most preferably 1 to 20 days, or
until the completion of pregnancy. Dosages vary depending on each
compound and the severity of the condition and are titrated to
achieve a steady-state blood serum concentration ranging from 10 to
20 ng/ml soluble endoglin; and/or 1 to 500 pg/mL free VEGF or free
PlGF, or both, preferably 1 to 100 pg/mL, more preferably 5 to 50
pg/mL and most preferably 5 to 10 pg/mL VEGF or PlGF, or 1-5 ng of
sFlt-1.
[0244] The diagnostic methods described herein can be used to
monitor the pre-eclampsia or eclampsia during therapy or to
determine the dosages of therapeutic compounds. In one example, a
therapeutic compound is administered and the PAAI is determined
during the course of therapy. If the PAAI is less than 20,
preferably less than 10, then the therapeutic dosage is considered
to be an effective dosage. In another example, a therapeutic
compound is administered and the soluble endoglin anti-angiogenic
index is determined during the course of therapy. If the soluble
endoglin anti-angiogenic index is less than 200, preferably less
than 100, then the therapeutic dosage is considered to be an
effective dosage.
Subject Monitoring
[0245] The disease state or treatment of a subject having
pre-eclampsia, eclampsia, or a predisposition to such a condition
can be monitored using the methods and compositions of the
invention. For example, the expression of a soluble endoglin
polypeptide present in a bodily fluid, such as urine, plasma,
amniotic fluid, or CSF, is monitored. The soluble endoglin
monitoring can be combined with methods for monitoring the
expression of an sFlt-1, VEGF, or PlGF polypeptide. Such monitoring
may be useful, for example, in assessing the efficacy of a
particular drug in a subject or in assessing disease progression.
Therapeutics that decrease the expression of a soluble endoglin
nucleic acid molecule or polypeptide are taken as particularly
useful in the invention.
EXAMPLES
[0246] The following examples are intended to illustrate the
invention. They are not meant to limit the invention in any
way.
Example 1
Increased Levels of Endoglin mRNA and Protein in Pregnant Women
with Pre-Eclampsia
[0247] In an attempt to identify novel secreted factors playing a
pathologic role in pre-eclampsia, we performed gene expression
profiling of placental tissue from 17 pregnant women with
pre-eclampsia and 13 normal pregnant women using Affymetrix U95A
microarray chips. We found that the gene for endoglin was
upregulated in women with pre-eclampsia.
[0248] In order to confirm the upregulation of endoglin in
pre-eclampsia, we performed Northern blots to analyze the placental
endoglin mRNA levels (FIG. 3) and western blot analysis to measure
serum protein levels of endoglin (FIG. 4) in pre-eclamptic pregnant
women as compared with normotensive pregnant women. Pre-eclampsia
was defined as (1) a systolic blood pressure (BP)>140 mmHg and a
diastolic BP>90 mmHg after 20 weeks gestation, (2) new onset
proteinuria (1+by dipstik on urinanalysis, >300 mg of protein in
a 24 hour urine collection, or random urine protein/creatinine
ratio >0.3, and (3) resolution of hypertension and proteinuria
by 12 weeks postpartum. Patients with underlying hypertension,
proteinuria, or renal disease were excluded. Patients were divided
into mild and severe pre-eclampsia based on the presence or absence
of nephritic range proteinuria (>3 g of protein on a 24 hour
urine collection or urine protein/creatinine ratio greater than
3.0). The mean urine protein/creatinine ratios in the mild
pre-eclampsia group were 0.94.+-.0.2 and in the severe
pre-eclampsia group were 7.8.+-.2.1. The mean gestational ages of
the various groups were as follows: normal 38.8.+-.0.2 weeks, mild
pre-eclampsia 34.+-.1.2 weeks, severe pre-eclampsia 31.3.+-.0.6
weeks, and pre-term 29.5.+-.2.0 weeks. Placental samples were
obtained immediately after delivery. Four random samples were taken
from each placenta, placed in RNAlater stabilization solution
(Ambion, Austin, Tex.) and stored at -70.degree. C. RNA isolation
was performed using Qiagen RNAeasy Maxi Kit (Qiagen, Valencia,
Calif.).
[0249] Northern blots probed with a 400 base pair probe in the
coding region of endoglin (Unigene Hs.76753) and a GAPDH probe as a
normalization control showed an increase in placental endoglin
mRNA. Western blots probed with an antibody to the amino terminus
of endoglin showed an increase in both placental and maternal serum
levels of endoglin protein in pre-eclamptic pregnant women as
compared to normotensive pregnant women.
Example 2
Demonstration of a Soluble Endoglin Polypeptide in the Placentas
and Serum of Pre-Eclamptic Patients
[0250] The western blot analysis used to measure the levels of
endoglin protein in placentas and serum from pre-eclamptic women
suggested the presence of a smaller protein (63 kDa), that was
present in the placenta and serum of pre-eclamptic pregnant women.
We have demonstrated that this smaller fragment is the
extracellular domain of endoglin. This truncated version is likely
to be shed from the placental syncitiotrophoblasts and endothelial
cells and circulated in excess quantities in patients with
pre-eclampsia. This soluble form of endoglin may be acting as an
anti-angiogenic agent by binding to circulating ligands that are
necessary for normal vascular health.
Example 3
Circulating Concentrations of Soluble Endoglin in Women with Normal
Versus Pre-Eclamptic Pregnancies
[0251] In order to compare the levels of circulating, soluble
endoglin from the serum of normal, mildly pre-eclamptic, or
severely pre-eclamptic women, we performed ELISA analysis on blood
samples taken from these women. Patients were divided into mild and
severe pre-eclampsia based on the presence or absence of nephritic
range proteinuria (>3 g of protein on a 24 hour urine collection
or urine protein/creatinine ratio greater than 3.0). The mean urine
protein/creatinine ratios in the mild pre-eclampsia group were
0.94.+-.0.2 and in the severe pre-eclampsia group were 7.8.+-.2.1
(FIG. 5). ELISA was performed using a commercially available ELISA
kit from R & D Systems, MN (Cat # DNDG00) as previously
described (Maynard et al, J. Clin. Invest. 111:649-658, 2003).
Example 4
Model Assay for Angiogenesis
[0252] An endothelial tube assay can be used an in vitro model of
angiogenesis. Growth factor reduced Matrigel (7 mg/mL,
Collaborative Biomedical Products, Bedford, Mass.) is placed in
wells (100 .mu.l/well) of a pre-chilled 48-well cell culture plate
and is incubated at 37.degree. C. for 25-30 minutes to allow
polymerization. Human umbilical vein endothelial cells (30,000+in
300 .mu.l of endothelial basal medium with no serum, Clonetics,
Walkersville, Md.) at passages 3-5 are treated with 10% patient
serum, plated onto the Matrigel coated wells, and are incubated at
37.degree. C. for 12-16 hours. Tube formation is then assessed
through an inverted phase contrast microscope at 4.times. (Nikon
Corporation, Tokyo, Japan) and is analyzed (tube area and total
length) using the Simple PCI imaging analysis software.
Example 5
Soluble Endoglin Protein Levels as a Diagnostic Indicator of
Pre-Eclampsia and Eclampsia in Women (Romero Study)
[0253] This study was designed to evaluate whether soluble endoglin
is altered during clinical pre-eclampsia and whether it can be used
to predict pre-eclampsia and eclampsia in women.
[0254] This study was done under collaboration with Dr. Roberto
Romero, at the Wayne State University/NICHD Perinatology Branch,
Detroit, Mich. A retrospective longitudinal case-control study was
conducted using a banked biological sample database as previously
described in Chaiworapongsa et al. (The Journal of Maternal-Fetal
and Neonatal Medicine, Jan. 17, 2005 (1):3-18). All women were
enrolled in the prenatal clinic at the Sotero del Rio Hospital,
Santiago, Chile, and followed until delivery. Prenatal visits were
scheduled at 4-week intervals in the first and second trimester,
and every two weeks in the third trimester until delivery. Plasma
samples were selected from each patient only once for each of the
following six intervals: (1) 7-16 weeks, (2) 16-24 weeks, (3) 24-28
weeks, (4) 28-32 weeks, (5) 32-37 weeks, and (6) >37 weeks of
gestation. For each pre-eclamptic case, one control was selected by
matching for gestational age (.+-.2 weeks) at the time of clinical
diagnosis of pre-eclampsia. The clinical criteria for the diagnosis
of pre-eclampsia were the same as previously described in
Chaiworapongsa et al, supra.
Measurement of Plasma Endoglin Levels
[0255] The plasma samples stored at -70.degree. C. were thawed and
plasma soluble endoglin levels were measured in one batch using the
commercially available ELISA kits from R&D systems,
Minneapolis, Minn.(Catalog # DNDG00).
Stastistical Analysis
[0256] Analysis of covariance was used to assess the difference in
plasma concentrations of soluble endoglin between patients destined
to develop pre-eclampsia and in normal pregnancy after adjusting
for gestational age at blood sampling and intervals of sample
storage. Chi-square or Fisher's exact tests were employed for
comparisons of proportions. The statistics package used was SPSS
V.12 (SPSS Inc., Chicago, Ill.). Significance was assumed for a p
value of less than 0.05.
Results
[0257] The clinical characteristics of the study population are
described in Table 1. The group with pre-eclampsia included more
nulliparous women and delivered earlier than the control group.
Importantly, the birth weights of the fetuses were smaller in the
pre-eclamptic group and there were a higher proportion of women
carrying small-for-gestational-age (SGA) infants. TABLE-US-00001
TABLE 1 Clinical characteristics of the study population Normal
pregnancy Pre-eclampsia n = 44 n = 44 p Age (y) 29 .+-. 6 26 .+-. 6
0.04* Nulliparity 11 30 <0.001* (25%) (68.2%) Smoking 10 1
0.007* (22.7%) (2.3%) GA at delivery (weeks) 39.7 .+-. 1.1 36.9
.+-. 2.7 <0.001* Birthweight (grams) 3,372 .+-. 383 2,710 .+-.
766 <0.001* Birthweight <10.sup.th percentile 0 16 <0.001*
(36.4%) Value expressed as mean .+-. sd or number (percent) GA:
gestational age
[0258] The clinical characteristics of patients with pre-eclampsia
are described in Table 2. Thirty-two (72%) of the patients had
severe pre-eclampsia, while 10 patients had severe early-onset
pre-eclampsia defined as onset <34 weeks. TABLE-US-00002 TABLE 2
Clinical characteristics of patients with pre-eclampsia Blood
pressure (mmHg) Systolic 155 .+-. 15 Diastolic 100 .+-. 8 Mean
arterial pressure 118 .+-. 9 Proteinuria (dipstick) 3 .+-. 0.8
Aspartate aminotransferase.sup..alpha. (SGOT) (U/L) 29 .+-. 31
Platelet count.sup..beta. (.times.10.sup.3) (.mu./L) 206 .+-. 59
Severe pre-eclampsia 32 (72.7%) GAat pre-eclampsia diagnosed
.ltoreq.34 weeks 10 (22.7%) GAat pre-eclampsia diagnosed .gtoreq.37
weeks 27 (61.4%) Value expressed as mean .+-. sd or number
(percent) .sup..alpha.(n = 26); .sup..beta.(n = 42)
[0259] The serum soluble endoglin levels in the controls and the
pre-eclamptic women measured in the 6 gestational age windows are
shown in Table 3. Amongst the pre-eclamptics, their specimens were
divided into two groups--clinical pre-eclampsia (samples taken at
the time of symptoms of pre-eclampsia) and preclinical
pre-eclampsia (samples taken prior to clinical symptoms). The data
shows that at mid-pregnancy (24-28 weeks of gestation), serum
soluble endoglin concentrations start rising in women destined to
develop pre-eclampsia and become at least 3 fold higher than
controls by 28-32 weeks of gestation. Blood samples taken from
women with clinical pre-eclampsia show a very dramatic (nearly
10-15 fold) elevation when compared to gestational age matched
controls. TABLE-US-00003 TABLE 3 Plasma soluble endoglin
concentrations in normal pregnancy and pre-eclampsia Pre-clinical
Clinical Normal samples samples pregnancy p Pre-eclampsia p
Pre-eclampsia p.sup..beta. 1.sup.st blood sampling (7.1-16 weeks)
Soluble Endoglin (ng/ml) 3.89 .+-. .928 0.9 3.96 .+-. 1.28
Gestational age (weeks) 12.3 .+-. 2.2 0.2 11.6 .+-. 2.4 Range
8.4-15.9 7.7-15.1 n = 37 n = 34 2.sup.nd blood sampling (16.1-24
weeks) Soluble Endoglin (ng/ml) 3.36 .+-. 1.11 0.1 3.79 .+-. 1.37
Gestational age (weeks) 19.4 .+-. 1.7 0.06 20.2 .+-. 2.1 Range
16.3-23.4 16.7-24.0 n = 44 n = 36 3.sup.rd blood sampling (24.1-28
weeks) Soluble Endoglin (ng/ml) 3.18 .+-. .729 0.009* 5.27 .+-.
4.12 Gestational age (weeks) 25.9 .+-. 1.3 0.2 26.4 .+-. 1.1 Range
24.1-28.0 24.6-28.0 n = 38 n = 29 4.sup.th blood sampling (28.1-32
weeks) Soluble Endoglin (ng/ml) 3.7 .+-. 1.1 <0.001* 10.2 .+-.
9.8 0.01* 96.1 .+-. 25.7 0.05 Gestational age (weeks) 29.9 .+-. 1.1
1.0 30.2 .+-. 1.0 1.0 30.4 .+-. 1.4 1.0 Range 28.3-32.0 28.7-32.0
29.4-31.4 n = 42 n = 33 n = 2.sup..delta. 5.sup.th blood sampling
(32.1-36.9 weeks) Soluble Endoglin (ng/ml) 5.79 .+-. 2.42 0.003*
10.51 .+-. 6.59 <0.00 43.14 .+-. 25.6 <0.00 Gestational age
(weeks) 34.7 .+-. 1.3 1.0 34.8 .+-. 1.5 1* 34.5 .+-. 1.2 1* Range
32.4-36.6 32.6-36.7 1.0 32.6-36.6 1.0 n = 37 n = 20 n = 13 6.sup.th
blood sampling (>=37 weeks) Soluble Endoglin (ng/ml) 8.9 .+-.
4.5 -- 15.23 .+-. 10.61 0.006* Gestational age (weeks) 39.4 .+-.
1.0 38.8 .+-. 1.1 Range 37.0-40.7 37.6-41.4 0.05 n = 27 n = 27
p.sup..beta.: compared between samples at clinical manifestation of
pre-eclampsia and normal pregnancy Value expressed as mean .+-. sd
.sup..delta.2 pre-eclamptic patients had no blood samples available
at clinical manifestation
[0260] To examine the relationship between plasma soluble endoglin
concentrations and the interval to clinical diagnosis of
pre-eclampsia, plasma samples of pre-eclamptic patients at
different gestational ages were stratified according to the
interval from blood sampling to clinical diagnosis into five
groups: (1) at clinical diagnosis, (2) 2-5.9 weeks before clinical
manifestation, (3) 6-10.9 weeks before clinical manifestation, (4)
11-15.9 weeks before clinical manifestation, and (5) 16-25 weeks
before clinical manifestation. The data shown in Table 4
demonstrates that the plasma soluble endoglin levels start going up
at 6-10.9 weeks before onset of symptoms in pre-eclamptics and are
at least 3 fold higher at 2-5.9 weeks before symptoms in women
destined to develop pre-eclampsia. TABLE-US-00004 TABLE 4 Plasma
soluble endoglin concentrations in normal and pre-eclamptic
pregnant women. Normal Pre- Blood sampling pregnancy eclampsia p At
clinical manifestation Soluble Endoglin (ng/ml) 7.63 .+-. 4.22
27.72 .+-. 26.20 <0.001* Gestational age (weeks) 37.2 .+-. 3.0
37.1 .+-. 2.7 0.9 Range 28.9-40.7 29.4-41.4 n = 42 .sup. n = 42
.sup..delta. 2-5.9 weeks before clinical manifestation Soluble
Endoglin (ng/ml) 4.67 .+-. 2.32 15.07 .+-. 10.15 <0.001*
Gestational age (weeks) 31.6 .+-. 3.8 32.8 .+-. 2.8 0.2 Range
24.1-36.3 27.1-36.7 n = 27 n = 27 Interval before clinical 3.8 .+-.
1.1 manifestation (weeks) 6-10.9 weeks before clinical
manifestation Soluble Endoglin (ng/ml) 3.61 .+-. 1.05 5.89 .+-.
3.07 <0.001* Gestational age (weeks) 28.5 .+-. 2.9 28.5 .+-. 2.9
0.9 Range 19.7-32.6 19.6-34.4 n = 37 n = 37 Interval before
clinical 8.3 .+-. 1.4 manifestation (weeks) 11-15.9 weeks before
clinical manifestation Soluble Endoglin (ng/ml) 3.35 .+-. 0.77 3.57
.+-. 0.92 0.5 Gestational age (weeks) 24.5 .+-. 3.1 24.2 .+-. 3.3
0.8 Range 17.6-27.9 17.7-28.0 n = 19 n = 19 Interval before
clinical 13.2 .+-. 1.3 manifestation (weeks) 16-25 weeks before
clinical manifestation Soluble Endoglin (ng/ml) 3.44 .+-. 1.07 3.69
.+-. 1.18 0.3 Gestational age (weeks) 17.6 .+-. 3.5 16.5 .+-. 4.5
0.2 Range 9.1-23.4 8.0-22.7 n = 42 n = 42 Interval before clinical
20.6 .+-. 3.6 manifestation (weeks) Value expressed as mean .+-. sd
.sup..delta. 2 pre-eclamptic patients had no blood samples
available at clinical manifestation
[0261] To examine the diagnostic potential of plasma soluble
endoglin concentrations to identify those destined to develop
pre-eclampsia, patients were stratified into early onset
pre-eclampsia (PE<34 weeks) and late onset pre-eclampsia
(PE>34 weeks). For patients with early-onset pre-eclampsia, the
mean plasma soluble endoglin levels was significantly higher in
pre-eclampsia (before clinical diagnosis) than in normal pregnancy
starting around 16-24 weeks of gestation (Table 5) with very
dramatic differences in 24-28 week and 28-32 week gestational
windows. In contrast, for patients with late-onset pre-eclampsia,
plasma soluble endoglin concentrations in pre-clinical
pre-eclampsia was significantly higher than in normal pregnancy
only at 28-32 weeks with very dramatic differences at 32-36 week of
gestation (Table 6). TABLE-US-00005 TABLE 5 Plasma soluble endoglin
concentrations in normal pregnant women and patients who developed
clinical Pre-eclampsia at 34 weeks of gestation or less.
Pre-clinical Clinical Normal samples samples pregnancy p
Pre-eclampsia p pre-eclampsia.sup..delta. p.sup..beta. 1.sup.st
blood sampling (7.1-16 weeks) Soluble Endoglin (ng/ml) 3.89 .+-.
.928 0.7 3.81 .+-. 1.11 Gestational age (weeks) 12.3 .+-. 2.2 0.4
11.6 .+-. 2.6 Range 8.4-15.9 8.0-15.1 n = 37 n = 8 2.sup.nd blood
sampling (16.1-24 weeks) Soluble Endoglin (ng/ml) 3.36 .+-. 1.11
0.02* 4.60 .+-. 1.72 Gestational age (weeks) 19.4 .+-. 1.7 0.7 19.8
.+-. 2.9 Range 16.3-23.4 17.3-23.9 n = 44 n = 7 3.sup.rd blood
sampling (24.1-28 weeks) Soluble Endoglin (ng/ml) 3.189 .+-. .729
<0.001* 10.22 .+-. 6.17 Gestational age (weeks) 25.9 .+-. 1.3
0.03* 26.8 .+-. 0.6 Range 24.1-28.0 26.0-27.3 n = 38 n = 6 4.sup.th
blood sampling (28.1-32 weeks) Soluble Endoglin (ng/ml) 3.70 .+-.
1.10 0.01* 17.66 .+-. 8.9 0.008* 96.10 .+-. 25.76 0.05 Gestational
age (weeks) 29.9 .+-. 1.1 1.0 29.7 .+-. 1.1 1.0 30.4 .+-. 1.4 1.0
Range 28.3-32.0 28.7-31.3 29.4-31.4 n = 42 n = 6 n = 2.sup..delta.
5.sup.th blood sampling (32.1-36.9 weeks) Soluble Endoglin (ng/ml)
5.79 .+-. 2.42 53.38 .+-. 32.09 0.001* Gestational age (weeks) 34.7
.+-. 1.3 33.5 .+-. 0.5 <0.001* Range 32.4-36.6 32.6-34.0 n = 37
n = 6 p.sup..beta.: compared between samples at clinical
manifestation of pre-eclampsia and normal pregnancy Value expressed
as mean .+-. sd .sup..delta.2 pre-eclamptic patients had no blood
samples available at clinical manifestation
[0262] TABLE-US-00006 TABLE 6 Plasma soluble endoglin
concentrations in normal pregnant women and pre-eclamsptics (34
weeks of gestation) Pre-clinical Clinical Normal samples samples
pregnancy p Pre-eclampsia p Pre-eclampsia p.sup..beta. 1.sup.st
blood sampling (7.1-16 weeks) Soluble Endoglin (ng/ml) 3.89 .+-.
.928 0.9 4.01 .+-. 1.35 Gestational age (weeks) 12.3 .+-. 2.2 0.2
11.6 .+-. 2.4 Range 8.4-15.9 7.7-15.1 n = 37 n = 26 2.sup.nd blood
sampling (16.1-24 weeks) Soluble Endoglin (ng/ml) 3.36 .+-. 1.11
0.4 3.59 .+-. 1.23 Gestational age (weeks) 19.4 .+-. 1.7 0.04* 20.3
.+-. 1.9 Range 16.3-23.4 16.7-24.0 n = 44 n = 29 3.sup.rd blood
sampling (24.1-28 weeks) Soluble Endoglin (ng/ml) 3.18 .+-. .729
0.1 3.98 .+-. 2.13 Gestational age (weeks) 25.9 .+-. 1.3 0.4 26.3
.+-. 1.1 Range 24.1-28.0 24.6-28.0 n = 38 n = 23 4.sup.th blood
sampling (28.1-32 weeks) Soluble Endoglin (ng/ml) 3.70 .+-. 1.10
0.001* 8.57 .+-. 9.45 Gestational age (weeks) 29.9 .+-. 1.1 0.2
30.3 .+-. 1.0 Range 28.3-32.0 28.7-32.0 n = 42 n = 27 5.sup.th
blood sampling (32.1-36.9 weeks) Soluble Endoglin (ng/ml) 5.79 .+-.
2.42 <0.001* 10.51 .+-. 6.59 <0.001* 34.36 .+-. 16.30
<0.001* Gestational age (weeks) 34.7 .+-. 1.3 1.0 34.8 .+-. 1.5
0.9 35.4 .+-. 0.9 0.7 Range 32.4-36.6 32.6-36.7 34.3-36.6 n = 37 n
= 20 n = 7 6.sup.th blood sampling (>=37 weeks) Soluble Endoglin
(ng/ml) 8.98 .+-. 45.12 -- 15.23 .+-. 10.61 0.006* Gestational age
(weeks) 39.4 .+-. 1.0 38.8 .+-. 1.1 0.05 Range 37.0-40.7 37.6-41.4
n = 27 n = 27 p.sup..beta.: compared between samples at clinical
manifestation of pre-eclampsia and normal pregnancy Value expressed
as mean .+-. sd
Summary
[0263] The results of these experiments demonstrate that women with
clinical pre-eclampsia have very high levels of circulating soluble
endoglin when compared to gestational age matched controls. The
results also demonstrate that women destined to develop
pre-eclampsia (pre-clinical pre-eclampsia) have higher plasma
soluble endoglin levels than those who are predicted to have a
normal pregnancy. The increase in soluble endoglin levels is
detectable at least 6-10 weeks prior to onset of clinical symptoms.
Finally, these results demonstrate that both early onset and late
onset pre-eclampsia have elevated circulating soluble endoglin
concentrations, but the alterations are more dramatic in the early
onset pre-eclampsia.
Example 6
Soluble Endoglin Protein Levels as a Diagnostic Indicator of
Pre-Eclampsia and Eclampsia in Women (CPEP Study)
[0264] As described above, we have discovered that soluble
endoglin, a cell surface receptor for the pro-angiogenic protein
TGF-.beta. and expressed on endothelium and syncytiotrophoblast, is
upregulated in pre-eclamptic placentas. In the experiments
described above, we have shown that in pre-eclampsia excess soluble
endoglin is released from the placenta into the circulation through
shedding of the extracellular domain; soluble endoglin may then
synergize with sFlt1, an anti-angiogenic factor which binds
placental growth factor (PlGF) and VEGF, to cause endothelial
dysfunction. To test this hypothesis, we compared serum
concentrations of soluble endoglin, sFlt1, and free PlGF throughout
pregnancy in women who developed pre-eclampsia and in those women
with other pregnancy complication such as gestational hypertension
(GH) and pregnancies complicated by Small-for-gestational (SGA)
infants to those of women with normotensive control pregnancies.
This study was done in collaboration with the Dr. Richard Levine at
the NIH.
[0265] There were two principal objectives of this study. The firs
objective was to determine whether, in comparison with normotensive
controls, elevated serum concentrations of soluble endoglin, sFlt1,
and reduced levels of PlGF can be detected before the onset of
pre-eclampsia and other gestational disorders such as gestational
hypertension or pregnancies complicated by small-for-gestational
(SGA) infants. The second objective was to describe the time course
of maternal serum concentrations of soluble endoglin, sFlt-1, and
free PlGF with respect to gestational age in women with
pre-eclampsia, gestational hypertension, or SGA with separate
examination of specimens obtained before and after onset of
clinical symptoms, and in normotensive controls.
Methods
[0266] Clinical Information
[0267] This study was a case control study of pregnancy
complications (premature pre-eclampsia, term pre-eclampsia,
gestational hypertension, pregnancies with SGA infants,
normotensive control pregnancies) nested within the cohort of 4,589
healthy nulliparous women who participated in the Calcium for
Pre-eclampsia Prevention trial (CPEP). 120 random cases were
selected from each of the study groups. The study methods were
identical to the nested case control study recently performed for
pre-eclampsia (Levine et al, N. Eng. J. Med. 2004, 350:672-83).
From each woman blood specimens were obtained before study
enrollment (13-21 wks), at 26-29 wks, at 36 wks, and on suspicion
of hypertension or proteinuria. All serum specimens collected at
any time during pregnancy before onset of labor and delivery were
eligible for the study. Cases included 120 women who developed term
pre-eclampsia, gestational hypertension, or SGA and who delivered a
liveborn or stillborn male baby without known major structural or
chromosomal abnormalities, and from whom a baseline serum specimen
was obtained. For premature pre-eclampsia, defined as (PE<37
weeks) all 72 patients from the CPEP cohort were studied. The
clinical criterion for the diagnosis of pre-eclampsia is described
in Levine et al., (2004), surpa. All cases of gestational
hypertension were required to have a normal urine protein
measurement within the interval from 1 day prior to onset of
gestational hypertension through 7 days following. SGA was defined
as <10th and <5th (severe SGA) percentile, using Zhang &
Bowes' tables of birthweight for gestational age, specific for
race, nulliparity, and infant gender. Controls were randomly
selected from women without pre-eclampsia or gestational
hypertension or SGA who delivered a liveborn or stillborn baby
without known major structural malformations or chromosomal
anomalies and matched, one control to one case, by the clinical
center, gestational age at collection of the first serum specimen
(.+-.1 wk), by freezer storage time (.+-.1 year), and by number of
freeze-thaws. A total of 1674 serum specimens were studied.
Matching by gestational age was done to control for gestational
age-related differences in levels of sFlt-1, VEGF, and PlGF.
Matching for freezer storage time was done to minimize differences
due to possible degradation during freezer storage. Matching by
clinical center was done to control for the fact that pre-eclampsia
rates differed significantly between centers, perhaps due to
differences in the pathophysiology of the disease. In addition, the
centers may have used slightly different procedures for collecting,
preparing, and storing specimens. Matching by number of thaws was
also performed to ensure that cases and controls will have been
subjected equally to freeze thaw degradation.
[0268] ELISA Measurements
[0269] ELISA for the various angiogenic markers were performed at
the Karumanchi laboratory by a single research assistant that was
blinded to the clinical outcomes.
[0270] Commercially available ELISA kits for soluble endoglin
(DNDG00), sFlt1 (DVR100), PlGF (DPG00) were obtained from R&D
systems, (Minneapolis, Minn.).
[0271] Statistical Analysis
[0272] T-test was used for the comparison of the various
measurements after logarithmatic transformation to determine
significance. P<0.05 was considered as statistically
significant.
Results
[0273] The mean soluble endoglin (FIG. 6), sFlt1 (FIG. 7) and PlGF
(FIG. 8) concentrations for the five different study groups of
pregnant women throughout pregnancy during the various gestational
age group windows as described in the methods are shown in FIGS.
6-8. For the pre-eclampsia groups and gestational hypertensive
groups, specimens taken after onset of clinical symptoms are not
shown here. Compared with gestational age-matched control
specimens, soluble endoglin and sFlt1 increased and free PlGF
decreased beginning 9-11 wks before preterm pre-eclampsia, reaching
levels 5-fold (46.4 vs 9.8 ng/ml, P<0.0001) and 3-fold higher
(6356 vs 2316 pg/ml, P<0.0001) and 4-fold lower (144 vs 546
pg/ml, P<0.0001), respectively, after pre-eclampsia onset. For
term pre-eclampsia, soluble endoglin increased beginning 12-14 wks,
free PlGF decreased beginning 9-11 wks, and sFlt1 increased <5
wks before pre-eclampsia onset. Serum concentrations of sFlt1 and
free PlGF did not differ significantly between pregnancies with SGA
or average for gestation age/large for gestation age (AGA/LGA)
infants from 10-42 wks of gestation. Serum soluble endoglin was
modestly increased in SGA pregnancies beginning at 17-20 wks (7.2
vs 5.8 ng/ml, P=0.03), attaining concentrations of 15.7 and 43.7
ng/ml at 37-42 wks for mild and severe SGA, respectively, as
compared with 12.9 ng/ml in AGA/LGA pregnancies (severe SGA vs
AGA/LGA, P=0.002). In gestation hypertensive study, compared with
GA-matched control specimens, modest increases in soluble endoglin
were apparent <1-5 wks before gestational hypertension, reaching
levels 2-fold higher for soluble endoglin (29.7 vs 12.5 ng/ml,
P=0.002) after onset of gestational hypertension. The adjusted odds
ratio for subsequent preterm PE for specimens obtained at 21-32 wks
which were in the highest quartile of control soluble endoglin
concentrations (>7.2 ng/ml), as compared to all other quartiles,
was 9.8 (95% CI 4.5-21.5).
[0274] The soluble endoglin anti-angiogenic index for pre-eclampsia
was defined as (sFlt1+0.25 soluble endoglin)/PlGF. The index was
calculated throughout the various gestational age groups for the
five different study groups. The soluble endoglin anti-angiogenic
index for pre-eclampsia anti-angiogenesis for samples taken prior
to clinical symptoms is shown in FIG. 9. Elevated values for the
soluble endoglin anti-angiogenic index were noted as early as 17-20
weeks of pregnancies and seemed to get more dramatic with advancing
gestation in severe pre-mature pre-eclampsia. In term
pre-eclampsia, SGA and GH, there was a modest elevation during the
end of pregnancy (33-36 weeks) when compared to control women.
[0275] FIGS. 10 and 11 depict the mean concentrations of soluble
endoglin (FIG. 10) and soluble endoglin anti-angiogenic index (FIG.
11) according to the number of weeks before clinical premature
pre-eclampsia (PE<37 weeks). Even as early 9-11 weeks prior to
the onset of premature pre-eclampsia, there was a 2-3 fold
elevation in soluble endoglin and soluble endoglin anti-angiogenic
index in women destined to develop pre-eclampsia with dramatic
elevations (>5 fold) in 1-5 weeks preceding clinical
symptoms.
[0276] FIGS. 12 and 13 show the alteration in soluble endoglin
(FIG. 12) and the soluble endoglin anti-angiogenic index (FIG. 13)
throughout pregnancy for term pre-eclampsia (PE>37 weeks) before
and after symptoms. Elevation in soluble endoglin and the soluble
endoglin anti-angiogenic index are noted starting at 33-36 weeks of
pregnancy reaching on average 2-fold higher levels at the time of
clinical pre-eclampsia.
[0277] FIGS. 14 and 15 show a modest elevation in soluble endoglin
(FIG. 14) and the soluble endoglin anti-angiogenic index (FIG. 15)
detected in women during gestational hypertension, and 1-5 weeks
preceding gestational hypertension (during 33-36 week of pregnancy)
when compared to normotensive controls.
[0278] FIGS. 16 and 17 show a modest elevations in soluble endoglin
(FIG. 16) and the soluble endoglin anti-angiogenic index (FIG. 17)
detected during the 33-36 week gestational windows in women with
severe SGA and not in all women with SGA when compared to control
pregnancies.
Summary
[0279] The results of this study show that the soluble endoglin
levels and soluble endoglin anti-angiogenic index levels, when
measured prior to 33 weeks of pregnancy, was dramatically elevated
in women destined to develop premature pre-eclampsia and in women
with clinical premature pre-eclampsia (PE<37 weeks) when
compared to normal control pregnancy. Therefore, soluble endoglin
levels and soluble endoglin anti-angiogenic index levels (prior to
33 weeks) can not only be used for the diagnosis of premature
pre-eclampsia, but also for the prediction of pre-eclampsia. It
appears that elevations in soluble endoglin levels and soluble
endoglin anti-angiogenic index levels start as early as 10-12 weeks
prior to symptoms of pre-eclampsia.
[0280] The soluble endoglin levels and soluble endoglin
anti-angiogenic index levels were also significantly elevated in
term pre-eclampsia (PE>37 weeks) and modestly elevated in
gestational hypertension and severe SGA when measured late in
pregnancy (33-36 week gestational windows). Therefore, soluble
endoglin levels and soluble endoglin anti-angiogenic index levels
can also be used to identify other pregnancy complications such as
SGA and gestation hypertension when measured after 33 weeks of
pregnancy.
Example 7
Involvement of Soluble Endoglin in the Pathogenesis of
Pre-Eclampsia
[0281] We have shown that endoglin, a cell surface receptor for the
pro-angiogenic protein TGF-.beta. and expressed on endothelium and
syncytiotrophoblast, is upregulated in pre-eclamptic placentas. We
have also shown that in pre-eclampsia, excess soluble endoglin is
released from the placenta into the circulation through shedding of
the extracellular domain. The experiments described below were
designed to test the hypothesis that soluble endoling may synergize
with sFlt1, an anti-angiogenic factor which binds placental growth
factor (PlGF) and VEGF, to cause endothelial dysfunction.
Materials and Methods
[0282] Reagents
[0283] Recombinant Human endoglin, human sFlt1, mouse endoglin,
mouse sFlt1, human TGF-.beta.1, human TGF-.beta.3, mouse VEGF were
obtained from R&D systems (Minneapolis, Minn.). Mouse
monoclonal antibody (catalog # sc 20072) and polyclonal antibody
(sc 20632) against the N-terminal region of human endoglin was
obtained from Santa Cruz Biotechnology, Inc. ELISA kits for human
sFlt1, mouse sFlt1 and human soluble endoglin were obtained from
R&D systems, MN.
[0284] Generation of Adenoviruses
[0285] Adenoviruses against sFlt1 and control adenovirus (CMV) have
been previously described (Maynard et al, J. Clin. Invest. 111:
649:658 (2003)) and were generated at the Harvard Medical Core
facility in collaboration with Dr. Richard Mulligan. To create the
soluble endoglin adenovirus, we used the Adeasy Kit (Stratagene).
Briefly, human soluble endoglin (Thr 27-Leu 586) was PCR amplified
using human cDNA full length endoglin clone (Invitrogen, CA) as the
template and the following oligonucleotides as primers: forward
5'-ACG AAG CTT GAA ACA GTC CAT TGT GAC CTT-3' (SEQ ID NO: 3) and
reverse 5'TTA GAT ATC TGG CCT TTG CTT GTG CAA CC-3' (SEQ ID NO: 4).
Amplified PCR fragments were initially subcloned into pSecTag2-B
(Invitrogen, CA) and the DNA sequence was confirmed. A mammalian
expression construct encoding His-tagged human soluble endoglin was
PCR amplified using pSecTag2 B-soluble endoglin as the template and
subcloned into pShuttle-CMV vector (Stratagene; Kpn1 and Sca1
sites), an adenovirus transfer vector, for adenovirus generation.
Adenovirus expressing soluble endoglin (sE) was then generated
using the standard protocol per manufacturer instructions and
confirmed for expression by western blotting. The confirmed clone
was then amplified on 293 cells and purified on a CsCl2 density
gradient as previously described (Kuo et al, Proc. Natl. Acad. Sci.
USA 98:4605-4610 (2001)). The final products were titered by an
optical absorbance method (Sweeney et al, Virology, 2002,
295:284-288). The titer is expressed as plaque forming units
(pfu)/mL based on a formula derived from previous virus preps that
were titered using the standard plaque dilution based titration
assay kit (BD Biosciences Clontech, Palo Alto, Calif., Cat. No.
K1653-1) and the optical absorbance method.
Patients
[0286] All the patients for this study were recruited at the Beth
Israel Deaconess Medical Center after obtaining appropriate
IRB-approved consents. Pre-eclampsia was defined as (1) Systolic
BP>140 and diastolic BP>90 after 20 weeks gestation in a
previously normotensive patient, (2) new onset proteinuria (1+by
dipstick on urinanalysis or >300 mg of protein in a 24 hr urine
collection or random urine protein/creatinine ratio >0.3), and
(3) resolution of hypertension and proteinuria by 12 weeks
postpartum. Patients with baseline hypertension, proteinuria, or
renal disease were excluded. For the purposes of this study,
patients were divided into mild and severe pre-eclampsia based on
the absence or presence of nephrotic-range proteinuria (>3 g of
protein on a 24 hour urine collection or urine protein to
creatinine ratio greater than 3.0). HELLP syndrome was defined when
patients had evidence of thrombocytopenia (<100000 cells/.mu.l),
increased LDH (>600 IU/L) and increased AST (>70 IU/L).
Healthy pregnant women were included as controls. 8 patients with
pre-term deliveries for other medical reasons were included as
additional controls. Placental samples were obtained immediately
after delivery. Serum was collected from pregnant patients at the
time of delivery (0-12 hours prior to delivery of the placenta)
after obtaining informed consent. These experiments were approved
by the Institutional Review Board at the Beth Israel Deaconess
Medical Center.
[0287] ELISA and Western Blots
[0288] ELISA for various proteins (sFlt1, soluble endoglin) was
done as per manufacturer's instructions using commercial kits from
R&D systems, MN.
[0289] Western blots and ELISA were used for checking the
expression of adenoviral-infected transgenes in the rat plasma as
described elsewhere (Maynard et al, supra).
[0290] Immunoprecipitation (IP) Experiments
[0291] IP followed by western blots were used to identify and
characterize soluble endoglin in the placental tissue and serum
specimens from patients with pre-eclampsia. Human placental tissue
was washed with cold PBS and lysed in homogenization buffer [10 mM
Tris-HCl, pH 7.4; 15 mM NaCl; 60 mM KCl; 1 mM EDTA; 0.1 mM EGTA;
0.5% Nonidet P-40; 5% sucrose; protease mixture from Roche
(Indianapolis, Ind.)] for 10 minutes. Placental lysates were then
subjected to immunoprecipitation with an anti-human monoclonal
mouse endoglin antibody (Santa Cruz Biotechnology, Inc., Santa
Cruz, Calif.). Immunoaffinity columns were prepared by the
directional coupling of 3-5 mg of the purified antibody to 2ml
protein A-Sepharose using an immunopure IgG orientation kit (Pierce
Chemical Co., Rockford, Ill., USA) according to the manufacturer's
instructions. Columns were then washed extensively with RIPA buffer
containing protease mixture, and bound proteins were eluted with
0.1 mol/L glycine-HCl buffer, pH 2.8. The eluent was collected in
0.5-ml fractions containing 1 mol/L Tris-HCl buffer.
Protein-containing fractions were pooled and concentrated 9- to
10-fold with CENTRICON Centrifugal Concentrator (Millipore Corp.,
Bedford, Mass., USA). The immunoprecipitated samples were separated
on a 4-12% gradient gel (Invitrogen) and proteins were transferred
to polyvinylidene difluoride (PVDF) membranes. Endoglin protein was
detected by western blots using polyclonal anti-human rabbit
endoglin primary antibody (Santa Cruz Biotechnology, Inc., Santa
Cruz, Calif.).
[0292] Endothelial Tube Assay
[0293] Growth factor reduced matrigel (7 mg/mL, Collaborative
Biomedical Products, Bedford, Mass.) was placed in wells
(1001/well) of a pre-chilled 48-well cell culture plate and
incubated at 37.degree. C. for 30 minutes to allow polymerization.
HUVEC cells (30,000+in 300 .mu.l of endothelial basal medium with
no serum, Clonetics, Walkersville, Md.) were treated with various
combinations of recombinant protein (soluble endoglin, sFlt1, or
both) and plated onto the Matrigel coated wells, and incubated at
37.degree. C. for 12-16 hours. Tube formation was then assessed
through an inverted phase contrast microscope at 4.times. (Nikon
Corporation, Tokyo, Japan) and quantitatively analyzed (tube area
and total length) using the Simple PCI imaging analysis
software.
[0294] Microvascular Permeability Experiments
[0295] Balb-C mice were injected through the retro-orbital venous
plexus with 1.times.10.sup.8 pfu of adenovirus expressing GFP or
soluble endoglin or sFlt1 or combinations and microvascular
permeability assay was performed 48 hours later. Mice were
anesthetized by IP injection of 0.5 ml Avertin. 100 ml of 1% Evans
blue dye (in PBS) was injected into the tail vein. 40 minutes
later, mice were perfused via heart puncture with PBS containing 2
mM EDTA for 20 minutes. Organs (brain, lung, liver, kidney) were
harvested and incubated in formamide for 3 days to elute Evans blue
dye. OD of formamide solution was measured using 620 nm wave
length.
[0296] Renal Microvascular Reactivity Experiments
[0297] Microvascular reactivity experiments were done as described
previously (Maynard et al., supra) using rat renal microvessels
(70-170 .mu.m internal diameter). In all experimental groups, the
relaxation responses of kidney microvessels were examined after
pre-contraction of the microvessels with U46619 (thromboxane
agonist) to 40-60% of their baseline diameter at a distending
pressure of 40 mmHg. Once the steady-state tone was reached, the
responses to various reagents such as TGF-.beta.1 or TGF-.beta.3 or
VEGF were examined in a standardized order. All drugs were applied
extraluminally.
[0298] Animal Models
[0299] Both pregnant and non-pregnant Sprague-Dawley rats were
injected with 2.times.10.sup.9 pfu of adenoviruses (Ad CMV or Ad
sFlt1 or Ad sE or Ad sFlt1+Ad sE) by tail vein injections. Pregnant
rats were injected at day 8-9 of pregnancy (early second trimester)
and blood pressure measured at day 16-17 of pregnancy (early third
trimester). Blood pressures were measured in the rats after
anesthesia with pentobarbital sodium (60 mg/kg, i.p.). The carotid
artery was isolated and cannulated with a 3-Fr high-fidelity
microtip catheter connected to a pressure transducer (Millar
Instruments, Houston, Tex.). Blood pressure was recorded and
averaged over a 10-minute period. Blood, tissue and urine samples
were then obtained before euthanasia. Plasma levels were measured
on the day of blood pressure measurement (day 8 after injection of
the adenoviruses), recognizing that 7-10 days after adenoviral
injection corresponds to the peak level of expression of these
proteins. Circulating sFlt-1 and soluble endoglin levels were
confirmed initially by western blotting and then quantified using
commercially available murine ELISA kits (R & D Systems,
Minneapolis, Minn.). Urinary albumin was measured both by both
standard dipstick and quantified by competitive enzyme-linked
immunoassay using a commercially available rat albumin ELISA kit
(Nephrat kit, Exocell Inc, Philadelphia, Pa.). Urinary creatinine
was measured by a picric acid colorimetric procedure kit (Metra
creatinine assay kit, Quidel Corp, San Diego, Calif.). AST and LDH
were measured using the commercially available kits (Thermo
Electron, Louisville, Colo.). Platelet counts from rat blood were
measured using an automated hemocytometer (Hemavet 850, Drew
Scientific Inc, Oxford, Conn.). A peripheral smear of the blood
with Wright's stain was performed for the detection of schistocytes
in circulating blood. After the measurement of blood pressure and
collection of specimens, the rats were sacrificed and organs
harvested for histology. The litter was counted and individual
placentas and fetuses weighed. Harvested kidneys were placed in
Bouin's solution, paraffin embedded, sectioned and stained with
H&E, PAS or Masson's trichrome stain.
[0300] Statistical Comparisons
[0301] Results are presented as mean .+-. standard error of mean
(SEM) and comparisons between multiple groups were made by analysis
of variance using ANOVA. Significant differences are reported when
p<0.05.
Results
Elevated Soluble Endoglin in Patients with Pre-Eclampsia
[0302] Using the serum specimens from patients described in Table
7, we measured the circulating concentrations of soluble endoglin
in the various groups of pre-eclamptic patients and control
pregnant patients. TABLE-US-00007 TABLE 7 Clinical characteristics
and circulating soluble endoglin in the various patient groups
Severe pre- Mild pre- Severe pre- eclampsia Normal eclampsia
eclampsia, no with HELLP Pre-term (n = 30) (n = 11) HELLP (n = 17)
(n = 11) (n = 8) Maternal age (yrs) 32.43 33.18 29.5 33.73 31.88
Gestational age (wks) 38.65 31.91* 29.06* 26.52* 30.99* Primiparous
(%) 43.3 63.6 47.1 90.9 62.5 Systolic blood pressure (mmHg) 122
157* 170* 166* 123 Diastolic blood pressure (mmHg) 72 99* 104* 103*
77 Proteinuria (g protein/g creatinine) 0.37 2.5* 8.64* 5.16* 0.6
Uric acid (mg/dl) 5.27 6.24 7.29* 6.31 7.35 Hematocrit (%) 35.5
33.6 33.7 33.5 34.3 Platelet count 238 230 249 69.4* 229 Creatinine
(mg/dl) 0.55 0.62 0.62 0.64 0.67 Soluble endoglin in (ng/ml) 18.73
36.12* 52.55** 99.83** 10.9 *P < 0.05 **P < 0.005
[0303] The average serum concentrations of soluble endoglin was at
least two fold higher in mild pre-eclampsia and 3-4 fold higher in
patients with severe pre-eclampsia. In pre-eclamptic patients
complicated with the HELLP syndrome, the concentration of soluble
endoglin was at least 5-10 fold higher than gestational age matched
control specimens. Additionally, the levels of soluble endoglin in
pregnant patients correlate with the levels of sFlt1 (FIG. 18). The
R2 value for correlation was 0.6. (Note that the circulating
concentrations of sFlt-1 reported here are at least 4-5 fold higher
than previously published (Maynard et al., supra). This is due to a
difference in the sensitivity of a new ELISA kit from R&D
systems which lacks urea in the assay diluent and therefore gives
consistently higher values than previously published.) In other
words, patients with the highest levels of soluble endoglin also
had the highest circulating levels of sFlt1. The origin of soluble
endoglin is most likely the syncitiotrophoblast of the placenta as
evidenced by the enhanced staining seen on our placental
immunohistochemistry (FIG. 19 and 20). These figures show that
endoglin protein is expressed by the syncitiotrophoblasts and is
vastly upregulated in pre-eclampsia. Our western blot data (FIGS.
21 A and 21B) and northern blot further confirms that the soluble
endoglin is a shed form of the extracellular domain of endoglin
protein and is approximately 65 kDA in size and is expressed in
excess quantities in pre-eclamptic placentas and that it circulates
in excess quantities in pre-eclamptic placentas. The predicted
length of the protein is approximately 437 amino acids.
Soluble Endoglin is an Anti-Angiogenic Molecule and Induces
Vascular Dysfunction
[0304] We used an in vitro model of angiogenesis to understand the
function of the soluble endoglin. Soluble endoglin modestly
inhibits endothelial tube formation, that is further enhanced by
the presence of sFlt1 (FIG. 22). In pre-eclampsia, it has been
reported that in addition to endothelial dysfunction, there is also
enhanced microvascular permeability as evidenced by edema and
enhanced leakage of Evan's blue bound albumin extracellularly. In
order to see if soluble endoglin induces microvascular leak, we
used mice treated for 48 hours with soluble endoglin and sFlt. A
combination of soluble endoglin and sFlt1 induced a dramatic
increase in albumin leakage in the lungs, liver and the kidney and
a modest leakage in the brain as demonstrated using Evan's blue
assay (FIG. 23) soluble endoglin alone induced a modest leakage in
the liver. These data suggest that soluble endoglin and sFlt1
combination are potent anti-angiogenic molecules and can induce
significant vascular leakage.
[0305] To assess the hemodynamic effects of soluble endoglin, a
series of microvascular reactivity experiments in rat renal
microvessels were performed. We studied first the effects of
TGF-.beta.1 and TGF-.beta.3-two known ligands of endoglin. Both
TGF-.beta.1 and TGF-.beta.3 induced a dose-dependent increase in
vascular diameter. Importantly in the presence of excess soluble
endoglin, the effect of both the TGFs were significantly attenuated
(FIG. 24). Finally, the combination of VEGF and TGF-.beta.1 induced
vasodilation which was blocked by excess soluble endoglin and sFlt1
(FIG. 25). This suggests that the sFlt1 and soluble endoglin may
oppose the physiological vasodilation induced by angiogenic growth
factors such as VEGF and TGF-.beta.1 and induce hypertension.
In Vivo Effects of Soluble Endoglin and sFlt1
[0306] In order to assess the vascular effects of soluble endoglin
and sFlt1, we resorted to adenoviral expression system in pregnant
rats. Adenovirus encoding a control gene (CMV) or soluble endoglin
or sFlt1 or sFlt1+soluble endoglin were injected by tail vein on
day 8 of pregnancy in Sprague Dawley rats. On day 17, animals were
examined for pre-eclampsia phenotype. Table 8 includes the
hemodynamic and biochemical data. TABLE-US-00008 TABLE 8
Hemodynamic and biochemical data for adenovirus treated rat animal
models. Urine Platelet Fetal MAP in Alb/creat count .times. LDH AST
weight Groups N mm Hg .mu.g/mg 1000/.mu.l U/L U/L in gms Control 4
86.33 84.17 1378 257 43 4.56 (CMV) sFlt1 4 134* 3478.3* 1247 324 78
3.55 sE 4 112* 366.90 1406 463 95 3.20 sFlt1 + 4 145* 6478.2* 538*
1428* 187* 2.50* sE MAP--mean arterial pressure (diastolic pressure
+ 1/3 pulse pressure); Alb/Creat--Albumin/creatinine ratios;
LDH--Lactate dehyrogenase; AST--Aspartate Aminotransferase. *P <
0.05 when compared to control group. Fetal weight is the sum of 4
fetuses chosen randomly per group The average circulating
concentrations of sFlt1 was 410 ng/ml in the sFlt1 group and 430
ng/ml in the sFlt1 + sE group. Average circulating concentrations
of sE was 318 ng/ml in the sE group and 319 ng/ml in the sFlt1 + sE
group.
[0307] Soluble endoglin alone induced a mild hypertension. sFlt1
induced both hypertension and proteinuria, as previously reported.
Importantly, the combination of sFlt1 and soluble endoglin induced
severe hypertension, nephrotic range proteinuria, growth
restriction of the fetuses and biochemical evidence of the
development of the HELLP syndrome (elevated LDH, elevated AST and
decreasing platelet counts) (Table 8). Evidence of hemolysis in the
soluble endoglin+sFlt1 group was confirmed by peripheral smear
which revealed schistocytes and reticulocytosis (FIGS. 26A-B).
Finally, renal histology also confirmed a severe glomerular
endotheliosis in the soluble endoglin+sFlt1 group (FIGS.
27A-27D).
Summary
[0308] These results demonstrate that soluble endoglin is
up-regulated in pre-eclamptic placentas and is present at extremely
high levels in patients with pre-eclampsia. The highest levels of
soluble endoglin were present in patients with HELLP syndrome, one
of the most severe forms of pre-eclampsia. These results also
demonstrate that soluble endoglin levels correlated with the
elevated sFlt1 in pregnant patients and was higher in those
patients in whom there is a higher circulating sFlt1 levels. In
addition, the results indicate that soluble endoglin is an
anti-angiogenic molecule and disrupts endothelial function in
multiple endothelial assays such as angiogenesis assays,
microvascular permeability assays, and microvascular reactivity
experiments. Importantly, soluble endoglin can amplify the toxic
consequence of sFlt1 in these in vitro endothelial assays. Further,
in in vivo assays, adenoviral expression of soluble endoglin
induces mild hypertension without any significant proteinuria.
However, in the presence of sFlt1, soluble endoglin induces
significant vascular damage as evidenced by the presence of severe
hypertension, proteinuria, glomerular endotheliosis, development of
the HELLP syndrome and fetal growth restriction. These data
suggests that soluble endoglin plays an important role in the
causality of the maternal syndrome of pree-clampsia and underscore
the need for agents that neutralize soluble endoglin for the
treatment of pre-eclampsia.
[0309] The mechanism of soluble endoglin release is likely
proteolytic cleavage of the extracellular region of the endoglin
molecule. Specific proteases that are up-regulated in the
pre-eclamptic tissue may serve as candidate molecules. One example
would be the membrane type matrix metalloproteinase-1 (MT1-MMP)
that has been shown to cleave betaglycan, a molecule that shares
similarity to endoglin (Velasco-Loyden G et al, J. Biol. Chem.
279:7721-33 (2004)). Therefore inhibitors of such proteases may
serve as valuable targets for the treatment of pre-eclampsia.
Other Embodiments
[0310] The description of the specific embodiments of the invention
is presented for the purposes of illustration. It is not intended
to be exhaustive or to limit the scope of the invention to the
specific forms described herein. Although the invention has been
described with reference to several embodiments, it will be
understood by one of ordinary skill in the art that various
modifications can be made without departing from the spirit and the
scope of the invention, as set forth in the claims. All patents,
patent applications, and publications referenced herein are hereby
incorporated by reference.
[0311] Other embodiments are in the claims.
Sequence CWU 1
1
4 1 1311 DNA Homo sapiens 1 atggaccgcg gcacgctccc tctggctgtt
gccctgctgc tggccagctg cagcctcagc 60 cccacaagtc ttgcagaaac
agtccattgt gaccttcagc ctgtgggccc cgagagggac 120 gaggtgacat
ataccactag ccaggtctcg aagggctgcg tggctcaggc ccccaatgcc 180
atccttgaag tccatgtcct cttcctggag ttcccaacgg gcccgtcaca gctggagctg
240 actctccagg catccaagca aaatggcacc tggccccgag aggtgcttct
ggtcctcagt 300 gtaaacagca gtgtcttcct gcatctccag gccctgggaa
tcccactgca cttggcctac 360 aattccagcc tggtcacctt ccaagagccc
ccgggggtca acaccacaga gctgccatcc 420 ttccccaaga cccagatcct
tgagtgggca gctgagaggg gccccatcac ctctgctgct 480 gagctgaatg
acccccagag catcctcctc cgactgggcc aagcccaggg gtcactgtcc 540
ttctgcatgc tggaagccag ccaggacatg ggccgcacgc tcgagtggcg gccgcgtact
600 ccagccttgg tccggggctg ccacttggaa ggcgtggccg gccacaagga
ggcgcacatc 660 ctgagggtcc tgccgggcca ctcggccggg ccccggacgg
tgacggtgaa ggtggaactg 720 agctgcgcac ccggggatct cgatgccgtc
ctcatcctgc agggtccccc ctacgtgtcc 780 tggctcatcg acgccaacca
caacatgcag atctggacca ctggagaata ctccttcaag 840 atctttccag
agaaaaacat tcgtggcttc aagctcccag acacacctca aggcctcctg 900
ggggaggccc ggatgctcaa tgccagcatt gtggcatcct tcgtggagct accgctggcc
960 agcattgtct cacttcatgc ctccagctgc ggtggtaggc tgcagacctc
acccgcaccg 1020 atccagacca ctcctcccaa ggacacttgt agcccggagc
tgctcatgtc cttgatccag 1080 acaaagtgtg ccgacgacgc catgaccctg
gtactaaaga aagagcttgt tgcgcatttg 1140 aagtgcacca tcacgggcct
gaccttctgg gaccccagct gtgaggcaga ggacaggggt 1200 gacaagtttg
tcttgcgcag tgcttactcc agctgtggca tgcaggtgtc agcaagtatg 1260
atcagcaatg aggcggtggt caatatcctg tcgagctcat caccacagcg g 1311 2 437
PRT Homo sapiens 2 Met Asp Arg Gly Thr Leu Pro Leu Ala Val Ala Leu
Leu Leu Ala Ser 1 5 10 15 Cys Ser Leu Ser Pro Thr Ser Leu Ala Glu
Thr Val His Cys Asp Leu 20 25 30 Gln Pro Val Gly Pro Glu Arg Gly
Glu Val Thr Tyr Thr Thr Ser Gln 35 40 45 Val Ser Lys Gly Cys Val
Ala Gln Ala Pro Asn Ala Ile Leu Glu Val 50 55 60 His Val Leu Phe
Leu Glu Phe Pro Thr Gly Pro Ser Gln Leu Glu Leu 65 70 75 80 Thr Leu
Gln Ala Ser Lys Gln Asn Gly Thr Trp Pro Arg Glu Val Leu 85 90 95
Leu Val Leu Ser Val Asn Ser Ser Val Phe Leu His Leu Gln Ala Leu 100
105 110 Gly Ile Pro Leu His Leu Ala Tyr Asn Ser Ser Leu Val Thr Phe
Gln 115 120 125 Glu Pro Pro Gly Val Asn Thr Thr Glu Leu Pro Ser Phe
Pro Lys Thr 130 135 140 Gln Ile Leu Glu Trp Ala Ala Glu Arg Gly Pro
Ile Thr Ser Ala Ala 145 150 155 160 Glu Leu Asn Asp Pro Gln Ser Ile
Leu Leu Arg Leu Gly Gln Ala Gln 165 170 175 Gly Ser Leu Ser Phe Cys
Met Leu Glu Ala Ser Gln Asp Met Gly Arg 180 185 190 Thr Leu Glu Trp
Arg Pro Arg Thr Pro Ala Leu Val Arg Gly Cys His 195 200 205 Leu Glu
Gly Val Ala Gly His Lys Glu Ala His Ile Leu Arg Val Leu 210 215 220
Pro Gly His Ser Ala Gly Pro Arg Thr Val Thr Val Lys Val Glu Leu 225
230 235 240 Ser Cys Ala Pro Gly Asp Leu Asp Ala Val Leu Ile Leu Gln
Gly Pro 245 250 255 Pro Tyr Val Ser Trp Leu Ile Asp Ala Asn His Asn
Met Gln Ile Trp 260 265 270 Thr Thr Gly Glu Tyr Ser Phe Lys Ile Phe
Pro Glu Lys Asn Ile Arg 275 280 285 Gly Phe Lys Leu Pro Asp Thr Pro
Gln Gly Leu Leu Gly Glu Ala Arg 290 295 300 Met Leu Asn Ala Ser Ile
Val Ala Ser Phe Val Glu Leu Pro Leu Ala 305 310 315 320 Ser Ile Val
Ser Leu His Ala Ser Ser Cys Gly Gly Arg Leu Gln Thr 325 330 335 Ser
Pro Ala Pro Ile Gln Thr Thr Pro Pro Lys Asp Thr Cys Ser Pro 340 345
350 Glu Leu Leu Met Ser Leu Ile Gln Thr Lys Cys Ala Asp Asp Ala Met
355 360 365 Thr Leu Val Leu Lys Lys Glu Leu Val Ala His Leu Lys Cys
Thr Ile 370 375 380 Thr Gly Leu Thr Phe Trp Asp Pro Ser Cys Glu Ala
Glu Asp Arg Gly 385 390 395 400 Asp Lys Phe Val Leu Arg Ser Ala Tyr
Ser Ser Cys Gly Met Gln Val 405 410 415 Ser Ala Ser Met Ile Ser Asn
Glu Ala Val Val Asn Ile Leu Ser Ser 420 425 430 Ser Ser Pro Gln Arg
435 3 30 DNA Artificial Sequence primer 3 acgaagcttg aaacagtcca
ttgtgacctt 30 4 29 DNA Artificial Sequence Primer 4 ttagatatct
ggcctttgct tgtgcaacc 29
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