U.S. patent application number 14/093000 was filed with the patent office on 2014-06-26 for fgf-9 and its use relating to blood vessels.
This patent application is currently assigned to UNIVERSITY OF WESTERN ONTARIO. The applicant listed for this patent is Matthew Frontini, Zengxuan Nong, Geoffrey J. Pickering. Invention is credited to Matthew Frontini, Zengxuan Nong, Geoffrey J. Pickering.
Application Number | 20140179601 14/093000 |
Document ID | / |
Family ID | 41254747 |
Filed Date | 2014-06-26 |
United States Patent
Application |
20140179601 |
Kind Code |
A1 |
Pickering; Geoffrey J. ; et
al. |
June 26, 2014 |
FGF-9 AND ITS USE RELATING TO BLOOD VESSELS
Abstract
There is provided a composition for controlling formation and/or
stabilization of a blood vessel comprising a first isolated nucleic
acid molecule that encodes a FGF-9 polypeptide and optionally one
or more isolated nucleic acid molecule that encodes another
angiogenic polypeptide. There is provided a composition for
controlling formation and/or stabilization of a blood vessel
comprising administering an effective amount of a composition
comprising an isolated FGF-9 polypeptide and one or more other
angiogenic polypeptides. The compositions provided herein may be
useful for controlling angiogenesis and/or vasculogenesis.
Inventors: |
Pickering; Geoffrey J.;
(London, CA) ; Nong; Zengxuan; (London, CA)
; Frontini; Matthew; (London, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pickering; Geoffrey J.
Nong; Zengxuan
Frontini; Matthew |
London
London
London |
|
CA
CA
CA |
|
|
Assignee: |
UNIVERSITY OF WESTERN
ONTARIO
London
CA
|
Family ID: |
41254747 |
Appl. No.: |
14/093000 |
Filed: |
November 28, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12990745 |
Jan 6, 2011 |
8623820 |
|
|
14093000 |
|
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Current U.S.
Class: |
514/8.1 ;
514/8.2; 514/8.5; 514/9.1 |
Current CPC
Class: |
A61K 38/1825 20130101;
C07K 14/50 20130101; A61P 35/00 20180101; A61K 38/1858 20130101;
A61K 45/06 20130101; A61P 9/10 20180101; A61P 9/00 20180101; A61K
31/7088 20130101; A61K 48/00 20130101 |
Class at
Publication: |
514/8.1 ;
514/8.2; 514/8.5; 514/9.1 |
International
Class: |
A61K 38/18 20060101
A61K038/18; A61K 45/06 20060101 A61K045/06; C07K 14/50 20060101
C07K014/50 |
Foreign Application Data
Date |
Code |
Application Number |
May 1, 2009 |
CA |
PCT/CA2009/000586 |
Claims
1. A composition for promoting therapeutic angiogenisis in an
animal comprising a first isolated nucleic acid molecule that
encodes a FGF-9 polypeptide and a second isolated nucleic acid
molecule that encodes an angiogenic polypeptide.
2. The composition of claim 1, wherein the PGF-9 polypeptide
comprises an amino acid sequence selected from the group consisting
of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, or a
variant thereof.
3. The composition of claim 2, wherein the FGF 9 polypeptide
comprises an amino acid sequence of SEQ ID NO:1 or a variant
thereof that is at least 80% identical to the amino acid sequence
of FIG. 1 (SEQ ID NO:1).
4. The composition of claim 2, wherein the angiogenic polypeptide
is selected from the group consisting of FGF2, FGF4, FGF5,
PDGF-.beta. FGF-1, VEGF (and all its variant), P1GF, Hedgehog
family member (Sonic, Indian and Desert), SDF-1 and IGF.
5. The composition of claim 2, wherein the variant comprises an
amino acid sequence having at least 80% identity with the amino
acid sequence selected from the group consisting of: SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
6. The composition of claim 1, wherein the first isolated nucleic
acid molecule comprises a nucleic acid sequence that hybridizes to
the complement of the nucleic acid sequence that encodes an amino
acid sequence selected from the group consisting of: SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, or a variant thereof.
7. The composition of claim 5, wherein the hybridization occurs
under stringent hybridization and wash conditions.
8. The composition of any one of claims 1 to 7, wherein the first
isolated nucleic acid molecule is comprised within a vector.
9. The composition of any one of claims 1 to 7, wherein the first
isolated nucleic acid molecule is comprised within a recombinant
cell.
10. The composition of claim 9, wherein the first isolated nucleic
acid molecule is integrated into the chromosome of the recombinant
cell.
11. The composition of claim 9, wherein the first isolated nucleic
acid molecule is extrachromosomal within the recombinant cell.
12. The composition of any one of claims 9 to 11, wherein the
recombinant cell is a stem cell.
13. The composition of any one of claims 1 to 12, wherein the
animal is a mammal.
14. The composition of any one of claims 1 to 12, wherein the
animal is a mammal selected from the group consisting of mouse,
pig, dog, rat, and human.
15. The composition of claim 14, wherein the mammal is a human.
16. A composition for promoting therapeutic angiogenesis in an
animal comprising an isolated FGF-9 polypeptide and one or more
other angiogenic polypeptides.
17. The composition of claim 16, wherein the FGF-9 polypeptide
comprises an amino acid sequence selected from the group consisting
of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, or a
variant thereof.
18. The composition of claim 17, wherein the FGF-9 polypeptide
comprises an amino acid sequence of SEQ ID NO:1 or a variant
thereof that is at least 80% identical to the amino acid sequence
of SEQ ID NO:1.
19. The composition of claim 17, wherein the other angiogenic
polypeptide is selected from the group consisting of FGF2, FGF4,
FGF5, and PDGF-.beta..
20. The composition of claim 17, wherein the variant comprises an
amino acid sequence having at least 80% identity with the amino
acid sequence selected from the group consisting of: SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
21. The composition of claim 17, wherein the FGF-9 polypeptide
comprises an amino acid sequence of SEQ ID NO:1.
22. The composition of claim 17, wherein the other angiogenic
polypeptide is FGF2.
23. The composition of any one of claims 17 to 22, wherein the
animal is a mammal.
24. The composition of any one of claims 17 to 22, wherein the
animal is a mammal selected from the group consisting of mouse,
pig, dog, rat, and human.
25. The composition of claim 24, wherein the mammal is human.
26. A composition for promoting therapeutic angiogenesis in an
animal comprising a recombinant cell producing an isolated FGF-9
polypeptide and another isolated angiogenic polypeptide.
27. The composition of claim 26, wherein the FGF-9 polypeptide
comprises an amino acid sequence selected from the group consisting
of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, or a
variant thereof.
28. The composition of claim 27, wherein the FGF-9 polypeptide
comprises an amino acid sequence of SEQ ID NO:1 or a variant
thereof that is at least 80% identical to the amino acid sequence
of SEQ ID NO:1.
29. The composition of claim 27, wherein the other angiogenic
polypeptide is selected from the group consisting of FGF2, FGF4,
FGF5, and PDGF-.beta..
30. The composition of claim 27, wherein the variant comprises an
amino acid sequence having at least 80% identity with the amino
acid sequence selected from the group consisting of: SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
31. The composition of claim 27, wherein the FGF-9 polypeptide
comprises an amino acid sequence of SEQ ID NO:1.
32. The composition of claim 27, wherein the other angiogenic
polypeptide is FGF2.
33. The composition of any one of claims 27 to 32, wherein the
animal is a mammal.
34. The composition of any one of claims 27 to 32, wherein the
animal is a mammal selected from the group consisting of mouse,
pig, dog, rat, and human.
35. The composition of claim 34, wherein the mammal is a human.
36. The composition of any one of claims 27 to 35, wherein the
recombinant cell is a stem cell.
37. The composition of any one of claims 1 to 36, further
comprising a pharmaceutically acceptable carrier.
38. The composition of any one of claims 1 to 36, further
comprising a chelating agent.
39. The composition of any one of claims 1 to 36, further
comprising a preservative.
40. A method of promoting formation of mature blood vessels in a
subject comprising administering an effective amount of the
composition of any one of claims 1 to 39 to the subject.
41. A method of treating ischemia in a subject comprising
administering an effective amount of the composition of any one of
claims 1 to 39 to the subject.
42. A use of the composition of any one of claims 1 to 39 for
preparation of a medicament.
43. A use of the composition of any one of claims 1 to 39 for
preparation of a medicament for promoting formation of mature blood
vessels in a subject.
44. A use of the composition of any one of claims 1 to 39 for
preparation of a medicament for treating ischemia in a subject.
45. A use of the composition of any one of claims 1 to 39 for
promoting formation of mature blood vessels in a subject.
46. A use of the composition of any one of claims 1 to 39 for
treating ischemia in a subject.
47. A kit comprising the composition of any one of claims 1 to 39,
and instructions for administering the composition to a
subject.
48. A method of promoting formation of mature blood vessels in a
subject comprising administering an effective amount of FGF-9 to
the subject.
49. A method of treating ischemia in a subject comprising
administering an effective amount of FGF-9 to the subject.
50. A use of FGF-9 for preparation of a medicament.
51. A use of FGF-9 for preparation of a medicament for promoting
for on of mature blood vessels in a subject.
52. A use of FGF-9 for preparation of a medicament for treating
ischemia in a subject.
53. A use of FGF-9 for promoting formation of mature blood vessels
in a subject.
54. A use of FGF-9 for treating ischemia in a subject.
55. A kit comprising FGF-9, and instructions for administering the
composition to a subject.
56. A kit comprising FGF-9 and a matrix material, and instructions
for preparing an in vitro vasculogenesis assay.
57. An in vitro vasculogenesis assay comprising a matrix material,
FGF-9, endothelial cells, smooth muscle cells, and a matrix
material for supporting growth and proliferation of all cells.
58. Use of FGF-9 for stabilization of blood vessels in vitro or in
vivo.
59. The use of claim 58, wherein the blood vessels exhibit vascular
leakage or are at risk of developing vascular leakage.
60. The use of claim 58, wherein the blood vessels are within a
tumour.
61. The use of FGF-9 for treatment of cancer.
62. A composition for improving the condition of a blood vessel in
an animal, the composition comprising an isolated FGF-9 polypeptide
and one or more other angiogenic polypeptides.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to controlling formation
and/or stabilization of blood vessels. More particularly, the
present invention relates to use of a FGF9 molecule for controlling
formation and/or stabilization of blood vessels
BACKGROUND OF THE INVENTION
[0002] The development of new blood vessels (angiogenesis) is
fundamental not only during embryogenesis but also as a protective
response in adult tissue subjected to ischemia. To be productive,
the formation of endothelial lined vessels is typically followed by
the recruitment of perivascular cells. This maturation process
produces functional vessels which persist over time and are
responsive to physiological stimuli. The production of mature
vessels is of therapeutic importance in the treatment of ischemic
disease. However, there has been little success in stimulating the
formation of mature microvessels in adults.
[0003] Angiogenesis is a physiological process involving the growth
of new blood vessels from pre-existing vessels. Angiogenesis
entails the proliferation and migration of endothelial cells to
form immature vascular networks. There is also a maturation phase
of angiogenesis that entails the recruitment of mesenchymal cells,
including pericytes and/or smooth muscle cells (SMCs), which wrap
the newly formed vessels to stabilize them. This is referred to as
angiogenic maturation. Such angiogenic maturation may be the
process of a recent angiogenic event, or include maturation,
maintenance, and stabilization of vessels generated through
angiogenesis at any point in development and post-natally.
Angiogenic maturation may also include the process of maturation,
supporting, and stabilizing of blood vessels generated through the
process of vasculogenesis as defined by the de novo condensation of
appropriate stem, progenitor, or more differentiated cell types
into tubular vessels.
[0004] A number of proteins, typically referred to as angiogenic
proteins, are known to promote angiogenesis. Such angiogenic
proteins include members of the fibroblast growth factor (FGF)
family, the vascular endothelial growth factor (VEGF) family, the
platelet-derived growth factor (PDGF) family, or the insulin-like
growth factor (IGF) family. For example, certain FGF and VEGF
family members have been recognized as regulators of angiogenesis
during growth and development. Their role in promoting angiogenesis
in adult animals has also been examined.
[0005] Angiogenic proteins, such as FGF family members, have been
disclosed in many patent documents, for example U.S. Pat. No.
4,956,455 (titled Bovine fibroblast growth factor, issued Sep. 11,
1990), U.S. Pat. No. 5,155,214 (titled Basic fibroblast growth
factor, issued Oct. 13, 1992), U.S. Pat. No. 5,302,702 (titled
Chimeric fibroblast growth factors, issued Apr. 12, 1994), U.S.
Pat. No. 5,314,872 (titled: Glucan sulfate, stabilized fibroblast
growth factor composition, issued May 24, 1994), U.S. Pat. No.
5,352,589 (titled Deletion mutant of basic fibroblast growth factor
and production thereof, issued Oct. 4, 1994), U.S. Pat. No.
5,371,206 (titled DNA encoding chimeric fibroblast growth factors,
issued Dec. 6, 1994), U.S. Pat. No. 5,387,673 (titled Active
fragments of fibroblast growth factor, issued Feb. 7, 1995), U.S.
Pat. No. 5,439,818 (titled DNA encoding human recombinant basic
fibroblast growth factor, issued Aug. 8, 1995), U.S. Pat. No.
5,491,220 (titled Surface loop structural analogues of fibroblast
growth factors, issued Feb. 13, 1996), U.S. Pat. No. 5,514,566
(titled Methods of producing recombinant fibroblast growth factors,
issued May 7, 1996), U.S. Pat. No. 5,604,293 (titled Recombinant
human basic fibroblast growth factor, issued Feb. 18, 1997).
[0006] The fibroblast growth factors (FGF) are a family of at least
twenty-three structurally related polypeptides (named FGF1 to
FGF23) that are characterized by a high degree of affinity for
proteoglycans, such as heparin. The various FGF molecules range in
size from 15-23 kD, and exhibit a broad range of biological
activities in normal and malignant conditions. Activities that have
been characterized for FGF molecules include nerve cell adhesion
and differentiation; wound healing; as mitogens toward many
mesodermal and ectodermal cell types, as trophic factors, as
differentiation inducing or inhibiting factors: and as an
angiogenic factor. For example, PCT Publication WO98/50079 (titled
Techniques And Compositions For Treating Heart Failure And
Ventricular Remodeling By In Vivo Delivery Of Angiogenic
Transgenes, published Dec. 30, 2004) describes the use of FGF2,
FGF4, or FGF5 to ameliorate regional myocardial contractile
dysfunction in an animal model of heart failure. The therapeutic
mechanism of action is stated to be angiogenesis.
[0007] Angiogenesis entails the proliferation and migration of
endothelial cells from the existing vasculature in order to create
new blood vessels. These nascent vessels are incomplete as they
lack supporting layers of mature smooth muscle cells (SMCs). As a
result, immature vascular beds are prone to regression due to the
fact that endothelial cells retract and eventually undergo
apoptosis. Stabilization of newly or previously formed blood
vessels through angiogenic maturation by SMCs both prevents
regression while also conferring the critical ability to regulate
blood pressure. While a number of factors that stimulate the
recruitment of SMCs to blood vessels during development have been
identified, these pathways are poorly understood with respect to
postnatal angiogenesis.
[0008] Currently, blood vessel formation stimulated by established
soluble angiogenic cytokines either in vivo or simulated in vitro
are short-lived due to the fact that they lack complete layers of
supporting SMCs and are therefore of limited therapeutic or
experimental value.
SUMMARY OF THE INVENTION
[0009] The invention provides compositions and methods to promote
angiogenesis maturation and stabilization. It is herein first
demonstrated that compositions comprising FGF-9 and one or more
other angiogenic polypeptides can be used in vitro and in vivo to
promote, control and stabilize blood vessel formation. This
includes the improvement of blood vessel condition of existing
blood vessels. As such, the invention has wide application and
utility as a culture supplement for in vitro culture of cells
and/or tissues. The invention also has wide therapeutic application
in clinical conditions where angiogenesis is required or is
beneficial to provide stabilized blood vessels, in any of the
embodiments of the invention the invention can be provided a
composition for systemic or local administration with or without a
carrier or carrier matrix.
[0010] According to an aspect of the present invention there is
provided a method of promoting blood vessel formation in hypoxic or
ischemic tissues in a patient in need thereof, comprising
contacting said tissue with a composition comprising FGF-9
polypeptide and one or more other isolated angiogenic
polypeptide.
[0011] According to another aspect of the present invention is a
method of treating a condition amenable to treatment by promoting
angiogenesis, said method comprising administering to a subject in
need thereof an amount of a FGF-9 polypeptide composition
comprising FGF-9 and one or more other angiogenic polypeptides,
effective for promoting angiogenesis in said subject.
[0012] According to a further aspect of the present invention is a
method for improving the condition of a blood vessel, such
improvement comprising the proliferation and migration of
endothelial cells to form immature vascular networks, and the
recruitment of mesenchymal cells, including pericytes and/or smooth
muscle cells (SMCs) to wrap the vessels to stabilize them and thus
improve their condition, the method comprising contacting the blood
vessel with a composition comprising FGF-9 and one or more other
angiogenic factor(s).
[0013] In an aspect, there is provided a composition for
controlling formation and/or stabilization of a blood vessel
comprising a first isolated nucleic acid molecule that encodes a
FGF-9 polypeptide and optionally a second isolated nucleic acid
molecule that encodes another angiogenic polypeptide. In aspects,
the nucleic acid molecule can be DNA, RNA, single or
double-stranded.
[0014] In another aspect, there is provided a composition for
controlling formation and/or stabilization of a blood vessel
comprising an isolated FGF-9 polypeptide and optionally one or more
other angiogenic polypeptides.
[0015] In another aspect, there is provided a composition for
stabilizing existing blood vessels in need of such treatment, the
composition comprising an isolated FGF-9 polypeptide and one or
more angiogenic polypeptides.
[0016] In yet another aspect, there is provided a composition for
controlling formation and/or stabilization of a blood vessel
comprising a recombinant cell producing an isolated FGF-9
polypeptide and optionally another isolated angiogenic
polypeptide.
[0017] In a further aspect, there is provided a composition for
promoting therapeutic angiogenesis in an animal comprising a first
isolated nucleic acid molecule that encodes a FGF-9 polypeptide and
a second isolated nucleic acid molecule that encodes an angiogenic
polypeptide.
[0018] In still a further aspect, there is provided a composition
for promoting therapeutic angiogenesis in an animal comprising an
isolated FGF-9 polypeptide and another angiogenic polypeptide.
[0019] In an even further aspect, there is provided a composition
for promoting therapeutic angiogenesis in an animal comprising a
recombinant cell producing an isolated FGF-9 polypeptide and
another isolated angiogenic polypeptide.
[0020] In another aspect, there is provided a method of promoting
formation of mature blood vessels in a subject comprising
administering an effective amount of FGF-9 to the subject.
[0021] In still another aspect, there is provided a method of
treating ischemia in a subject comprising administering an
effective amount of FGF-9 to the subject.
[0022] In yet another aspect, there is provided a use of FGF-9 for
preparation of a medicament.
[0023] In still yet another aspect, there is provided a use of
FGF-9 for preparation of a medicament for promoting formation of
mature blood vessels in a subject or improving the condition of an
existing blood vessel
[0024] In another aspect, there is provided a use of FGF-9 for
preparation of a medicament for treating ischemia in a subject.
[0025] In even another aspect, there is provided a use of FGF-9 for
promoting formation of mature blood vessels in a subject.
[0026] In still another aspect, there is provided a use of FGF-9
for treating ischemia in a subject.
[0027] In a further aspect, there is provided a kit comprising
FGF-9 and a matrix material, and instructions for preparing an in
vitro vasculogenesis assay.
[0028] In accordance with a further aspect, there is provided a kit
comprising a FGF-9 containing composition and a matrix material,
with instructions for use and for preparing an in vitro
angiogenesis assay.
[0029] In still a further aspect, there is provided an in vitro
vasculogenesis assay comprising a matrix material, FGF-9,
endothelial cells, smooth muscle cells, and a matrix material for
supporting growth and proliferation of cells.
[0030] In yet a further aspect, there is provided a use of FGF-9
for stabilization of blood vessels in vitro or in vivo.
[0031] In yet a further aspect of the invention, there is provided
a FGF-9 containing composition for the promotion of the
angiogenenic maturation of blood vessels in vitro or in vivo.
[0032] In another aspect, there is provided a use of FGF-9 for
treatment of cancer.
[0033] Other features and advantages of the present invention will
become apparent from the following detailed description. It should
be understood, however, that the detailed description and the
specific examples while indicating embodiments of the invention are
given by way of illustration only, since various changes and
modifications within the spirit and scope of the invention will
become apparent to those skilled in the art from said detailed
description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] The present invention will become more fully understood from
the detailed description given herein and from the accompanying
drawings, which are given by way of illustration only and do not
limit the intended scope of the invention as herein described in
its various embodiments.
[0035] FIG. 1 shows mRNA coding region sequence and corresponding
amino acid sequence for mammalian FGF-9A) Homo sapiens (NCBI
gi:391718), B) Mus musculus (NCBI gi:1161346), C) Rattus norvegicus
(NCBI gi:391852);
[0036] FIG. 2 shows a multiple sequence alignment of Human, Mouse,
Rat, and Pig FGF-9 amino acid sequences;
[0037] FIG. 3 shows steps in a high throughput screen for secreted
by SMCs as they acquire specialized functions;
[0038] FIG. 4 shows that FGF-9 is upregulated as SMCs acquired
specialized functions;
[0039] FIG. 5 shows that FGF-9 does not initiate the angiogenic
process in subcutaneously implanted matrigel in mice;
[0040] FIG. 6 shows that FGF-9 stimulates recruitment of SM
.alpha.-actin expressing mural cells to nascent microvessels during
angiogenesis;
[0041] FIG. 7 shows that FGF-9 stimulates SM .alpha.-actin
expressing mural cells recruitment along continuous lengths of
blood vessels;
[0042] FIG. 8 shows that FGF-9 stimulates circumferential wrapping
of blood vessels by SM .alpha.-actin expressing mural cells;
[0043] FIGS. 9A-C shows that FGF-9 modified microvessels are
responsive to vasoactive stimuli and can vasoconstrict and
vasodilate;
[0044] FIG. 10 shows that FGF-9-stimulated wrapping may be
dependent on the upregulation of PDGFR-.beta.;
[0045] FIG. 11 shows vasculogenesis in an in vitro matrigel assay
with endothelial and smooth muscle cells;
[0046] FIG. 12 shows that FGF-9 stabilizes the neovasculature;
[0047] FIG. 13 shows that FGF-9 stimulates the recruitment of
nerves to the neovasculature;
[0048] FIG. 14 shows that FGF-9-stimulated wrapping is dependent on
the upregulation of PDGFR-.beta.;
[0049] FIG. 15 shows that FGF-9-mediated PDGFR-.beta. upregulation
and vessel maturation requires Sonic Hedgehog signaling;
[0050] FIG. 16 shows the in vitro direct effects of FGF-9 on SMCs
in culture;
[0051] FIG. 17 shows the proposed mechanism of action for FGF-9
during angiogenesis;
[0052] FIG. 18 shows a cancer model using a kidney carcinoma cell
line to demonstrate the "tightening" of the blood vessel to prevent
metastasis from primary tumors to distal sites.
[0053] FIG. 19 shows in vitro co-culture and tubule formation using
FGF-9; and
[0054] FIG. 20 shows that FGF-9 results in increased association of
SMCs with endothelial tubes.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0055] For convenience, before further description of the present
invention, certain terms employed in the specification, examples,
and appended claims are provided herein. These definitions should
be read in light of the remainder of the disclosure and understood
as by a person of skill in the art. Also, the terms "including"
(and variants thereof), "such as", "e.g." as used herein are
non-limiting and are for illustrative purposes only. The articles
"a" and "an" are used herein to refer to one or to more than one
(i.e. to at least one) of the grammatical object of the article. By
way of example, "an element" means one element or more than one
element. The term "angiogenesis" is an art-recognized term, and
refers to the process and creation of new blood vessels formed from
pre-existing blood vessels. The term "restenosis" refers to a
re-narrowing of a blood vessel, thereby restricting blood flow.
This re-narrowing can be caused by, for example, a vessel's
response to an injury inflicted during balloon angioplasty. The
term "hypoxic tissue" refers to tissue with an insufficient amount
of oxygen. The term "ischemic tissue" refers to tissue with
insufficient blood flow.
[0056] All publications and patents mentioned herein, including
those items listed below, are hereby incorporated by reference in
their entirety as if each individual publication or patent was
specifically and individually indicated to be incorporated by
reference.
[0057] Methods and compositions for controlling formation and/or
stabilization of blood vessels are described herein. More
specifically, methods and compositions for controlling angiogenesis
and/or vasculogenesis are described herein.
[0058] Angiogenesis is a physiological process involving the growth
of new blood vessels from pre-existing vessels. One aspect of
angiogenesis entails the proliferation and migration of endothelial
cells to form immature vascular networks. Another aspect of
angiogenesis is a maturation phase that entails the recruitment of
mesenchymal cells, including pericytes and/or smooth muscle cells
(SMCs) that wrap vessels to stabilize them. This is referred to as
angiogenic maturation. Such angiogenic maturation may be the
process of a recent angiogenic event, or include maturation,
maintenance, and stabilization of vessels generated through
angiogenesis at any point in development and post-natally. Thus,
angiogenic maturation may occur independently of the aspect of
angiogenesis pertaining to formation of nascent or immature blood
vessels, and therefore may be beneficial in stabilizing any blood
vessel, nascent or otherwise. For example existing blood vessels in
an adult animal that exhibit vascular leakage, or are at risk of
exhibiting vascular leakage, can be stabilized by the process of
angiogenic maturation. Angiogenic maturation may also include the
process of maturation, supporting, and stabilizing of blood vessels
generated through the process of vasculogenesis in vitro and/or in
vivo. Vasculogenesis comprises de novo condensation of appropriate
stem, progenitor, or more differentiated cell types into tubular
vessels.
[0059] An angiogenic molecule is meant to encompass nucleic acids,
polypeptides, small molecule chemical compounds or any other
molecule that can be used to control at least one aspect of
angiogenesis. For example, an angiogenic polypeptide is a
polypeptide that can be used to control at least one aspect of
angiogenesis. Angiogenic polypeptides have been identified from
many naturally occurring sources and variants thereof have also
been produced and characterized. Such angiogenic polypeptides
include members of the fibroblast growth factor (FGF) family (for
example FGF-1, FGF-2, FGF-4, FGF-5, FGF-6, or FGF-9), the vascular
endothelial growth factor (VEGF) family (for example VEGF-121,
VEGF-145, VEGF-165, VEGF-167, VEGF-186, VEGF-189, VEGF-206, or
VEGF-C), the platelet-derived growth factor (PDGF) family (for
example PDGF-.alpha. or PDGF-.beta.), or the insulin-like growth
factor (IGF) family. Other non-limiting examples of angiogenic
peptides include PIGF, Hedgehog family member (Sonic, Indian and
Desert), and SDF-1.
[0060] Angiogenic molecules may be used to prepare compositions
that are useful for controlling at least one aspect of
angiogenesis. Such compositions may be useful in any method, in
vivo or in vitro, where formation and/or stabilization of a blood
vessel is desired. Stabilization of blood vessels may be useful in
forming or maintaining blood vessels, for example, by reducing
regression of newly formed vessels, maintaining or conferring
responsiveness to vasoactive stimuli, reduction of vascular leakage
or formation of tight junctions. Stabilization of blood vessels may
be useful in respect of both angiogenesis or vasculogenesis.
[0061] Compositions and methods described herein will comprise a
FGF molecule, and more typically a FGF polypeptide.
[0062] In one example, compositions comprising a FGF-9 molecule are
provided.
[0063] Without wishing to be bound by theory FGF-9 is believed to
control stabilization and/or formation of blood vessels or control
angiogenesis and/or vasculogenesis by influencing migration of
mesenchymal cells to blood vessels. The mesenchymal cells can
provide stability to a blood vessel by physical contact and more
specifically by circumferential wrapping of blood vessels. The
blood vessels may be nascent or may be at any level of development
or maturity.
[0064] Compositions comprising a FGF-9 molecule may be useful to
control angiogenesis. Compositions comprising a FGF-9 molecule may
be useful to promote maturation of blood vessels during
angiogenesis. Compositions comprising a FGF-9 molecule may also be
useful to promote formation of functional blood vessels during
angiogenesis. Compositions comprising a FGF-9 molecule may also be
useful for recruitment of smooth muscle alpha-actin positive cells
to nascent blood vessels during angiogenesis. Compositions
comprising a FGF-9 molecule may also be useful for inducing
expression of smooth muscle alpha-actin in mural cell precursors.
Compositions comprising a FGF-9 molecule may also be useful to
promote the formation and/or maturation and/or stabilization of
functional blood vessels during vasculogenesis including
stimulation of re-innervaton of blood vessels. Compositions
comprising a FGF-9 molecule may also be useful for therapeutic
angiogenesis, for example in treatment of ischemia by stimulating
creation of new functional blood vessels in ischemic organs,
tissues or parts to increase the level of oxygen-rich blood
reaching these areas.
[0065] Compositions comprising a FGF-9 molecule may be used to
treat any disease or condition where the formation of new blood
vessels provides a prophylactic and/or therapeutic benefit in
absence of existing extracellular structure. Accordingly, a method
for controlling angiogenesis in a subject comprises administering a
composition comprising a FGF-9 molecule and another angiogenic
agent in combination with a natural or synthetic or
natural-synthetic combination of extracellular matrix as a
therapeutic biomaterial. The FGF-9 molecule may be administered
systemically or locally in a region where therapeutic angiogenesis
is needed to facilitate angiogenesis within the appropriate
extracellular structure as would be required for therapeutic tissue
engineering.
[0066] Compositions comprising a FGF-9 molecule may be used to
treat any disease or condition where the formation of new blood
vessels provides a prophylactic and/or therapeutic benefit.
Accordingly, a method for controlling angiogenesis in a subject
comprises administering a composition comprising a FGF-9 molecule.
In another example, a method for treating ischemia in a subject
comprises administering a composition comprising a FGF-9 molecule.
The FGF-9 molecule may be administered systemically or locally in a
region where therapeutic angiogenesis is needed to treat
ischemia.
[0067] Ischemia can be described as an inadequate flow of blood to
a part of the body, caused by constriction or blockage of the blood
vessels supplying it, with resultant damage or dysfunction to this
part of the body. Ischemia is a well known feature of heart
diseases, transient ischemic attacks, cerebrovascular accidents,
ruptured arteriovenous malformations, and peripheral artery
occlusive disease. The heart, the kidneys, and the brain are among
the organs that are the most sensitive to inadequate blood supply.
Ischemia in brain tissue, for example due to stroke or head injury,
causes a process called the ischemic cascade to be unleashed, in
which proteolytic enzymes, reactive oxygen species, and other
harmful chemicals damage and may ultimately kill brain tissue.
Ischemia to the heart can cause angina (CIP), which is sometimes
debilitating and refractory to current forms of therapy. Ischemia
to the heart can also lead to heart muscle injury and muscle death
(myocardial infarction).
[0068] Ischemia to the lower limbs is also common in diabetes and
can cause refractory lower limb pain (claudication) and lead to
gangrene and amputation.
[0069] Compositions comprising a FGF-9 molecule may be used to
treat any disease or condition where the stabilization or
angiogenic maturation of existing blood vessels provides a
prophylactic and/or therapeutic benefit. Accordingly, a method for
controlling angiogenesis in a subject comprises administering a
composition comprising a FGF-9 molecule. In another example, a
method for treating vascular leakage in a subject comprises
administering a composition comprising a FGF-9 molecule. The FGF-9
molecule may be administered systemically or locally in a region
where therapeutic angiogenic maturation is needed to treat or
mitigate risk in a patient.
[0070] Vascular leakage can be described as the unregulated
movement of molecules, such as proteins, toxins, and oxidized
molecules, out of the blood to the nearby tissues. This leakage can
lead to the damage and death of local cells. This is a prominent
feature of diabetic retinopathy, which can lead to blindness. It
may also be a feature of neurological disorders such as Lou
Gehrig's disease and Alzheimer's disease. When severe vascular
leakage is accompanied by extravasation of red blood cells and
other circulating cells. This is referred to as hemorrhage.
Hemorrhage from small vessels can have severe consequences for
tissues such as the brain and the eye.
[0071] In a further embodiment of the invention, the compositions
as described herein have utility in the field of cancer for the
treatment thereof. The administration of FGF-9 compositions of the
invention may be used for stabilizing angiogenesis in tumours and
effectively causing a localization of the cancer and less
metastases such that the tumour can be more readily excised from
the patient. In this aspect, the tumour may be directly targeted
with local administration of the FGF-9 containing compositions of
the invention. Alternatively, the FGF-9 containing compositions of
the invention may be administered systemically and/or locally for
reduction in metastases of cancerous cells throughout the body. It
is expected that tumours so treated will be more readily excisable
from the patient with less chance of tumour metastases.
[0072] Compositions comprising a FGF-9 molecule and another
angiogenic molecule may be used in combination with vascular
endothelial cells and vascular smooth muscle cells with or without
appropriate other cell types to generate individual, a system, or a
network of stabilized blood vessels in vitro. In such cases, the
existence of stabilized blood vessels would be of benefit for
research and development purposes, evaluation of potential
therapeutic compounds, or in assessing the activity of modified
cells in blood vessel formation or angiogenesis for experimental
purposes. Accordingly, a method for controlling angiogenesis in
vitro comprises administering a composition comprising a FGF-9
molecule and another angiogenic molecule in a range of
concentrations appropriate for inducing vessel formation and
maturation with a suitable culture medium.
[0073] In another non-limiting example, such a method for
generating stabilized blood vessels in vitro for the described
purposes may benefit from the formation of 3-dimensional
structures. Accordingly, for the generation of such 3 dimensional
vascular structures such culture medium may include a composition
of FGF-9 and another angiogenic molecule at an appropriate
concentration to generate stabilized blood vessels; an
extracellular matrix that may include but are not limited to such
factors as fibrin, Matrigel, collagen, hyaluronic acid,
proteoglycans, derivatives thereof or synthetic structural agents;
and may or may not contain a natural or synthetic cross linking
agent to generate the three dimensional structure.
[0074] Without wishing to be limited by theory, FGF-9 molecules
appear to promote formation of functional blood vessels by
influencing a contractile phenotype in smooth muscle cells and
stimulating their recruitment to nascent blood vessels. A
fundamental characteristic of vascular smooth muscle cells is their
ability to convert between immature, proliferative and mature,
contractile phenotypes. One process that requires this plasticity
is angiogenesis where smooth muscle cells must proliferate and
migrate to nascent microvessels. Smooth muscle cells must then wrap
around these microvessels and reacquire the ability to contract in
order to stabilize the nascent culture. Using a unique smooth
muscle cell-line that reversibly converts between immature and
mature phenotypes, secreted factors that may regulate the
stabilization of neovessels have been identified. High density
microarray analysis of maturing smooth muscle cells revealed only
27 secreted factors were upregulated with FGF-9 being the most
upregulated. In contrast other FGFs showed no change
(FGF-7,-11,-12,-14,-18) or were downregulated (FGF-1,-2,-5) as
smooth muscle cells matured.
[0075] Without limitation, the FGF-9 molecule may be a full-length
naturally occurring polypeptide or a variant thereof, or may be a
nucleic acid molecule encoding a FGF-9 polypeptide or variant
thereof. Furthermore, a recombinant cell producing the FGF-9
molecule is provided.
[0076] An FGF-9 polypeptide may be provided by any source or
method, for example, natural isolate or recombinant or synthetic
origin or suitable combinations thereof. Administration of the
FGF-9 polypeptide to a subject can be used to control angiogenesis,
and more specifically to promote recruitment of smooth muscle cells
during angiogenesis. The FGF-9 polypeptide may be of any length
provided that its angiogenic activity is maintained. The sequence
of the FGF-9 polypeptide may be based on a complete or partial
naturally occurring amino acid sequence. An FGF-9 polypeptide may
be used either singly or in combination with other polypeptides,
angiogenic or otherwise, in the preparation of a composition that
controls angiogenesis. A polypeptide refers to a chain of amino
acids, for example peptides, oligopeptides, or proteins, having a
biological function, and does not refer to a specific length of the
chain.
[0077] An isolated FGF-9 polypeptide is a polypeptide that has been
identified and separated and/or recovered from at least one
component of its natural environment. The isolated polypeptide will
typically have been purified by at least one purification step,
and, in some embodiments purification may be achieved (1) to a
degree sufficient to obtain at least 15 residues of N-terminal or
internal amino acid sequence by use of a sequenator, or (2) to
homogeneity by SDS-PAGE under non-reducing or reducing conditions
using Coomassie blue or, preferably, silver stain. Isolated
polypeptide includes polypeptide in situ within recombinant cells,
since at least one component of the FGF-9 polypeptide natural
environment will not be present. An isolated polypeptide may be
produced by synthetic or recombinant techniques, for example as
described in J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989,
Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3,
Cold Spring Harbor Laboratory Press. An isolated polypeptide
produced as a result of recombinant techniques may be referred to
as a recombinant polypeptide.
[0078] A nucleic acid encoding an FGF-9 polypeptide may be any
nucleic acid molecule of, for example. cDNA, genomic DNA, synthetic
DNA or RNA origin or suitable combinations thereof. Administration
of the nucleic acid encoding an FGF-9 polypeptide to a subject can
be used to control angiogenesis, and more specifically to promote
recruitment of smooth muscle cells during angiogenesis. The nucleic
acid may be of any length provided that the angiogenic activity is
maintained by the encoded FGF-9 polypeptide. The sequence of the
nucleic acid encoding an FGF-9 polypeptide may be based on a
complete or partial naturally occurring nucleic acid sequence. A
nucleic acid sequence encoding an FGF-9 polypeptide may be used
either singly or in combination with other nucleic acid sequences,
encoding angiogenic polypeptides or encoding any other desired
polypeptide, in the preparation of a composition to control
angiogenesis.
[0079] An isolated nucleic acid molecule encoding a FGF-9
polypeptide is a nucleic acid molecule that is identified and
separated from at least one contaminant nucleic acid molecule with
which it is ordinarily associated in the natural source of the
nucleic acid. Such an isolated nucleic acid molecule is other than
in the form or setting in which it is found in nature. Isolated
nucleic acid molecules therefore are distinguished from the nucleic
acid molecule as it exists in natural cells. An isolated nucleic
acid molecule encoding a FGF-9 polypeptide includes nucleic acid
molecule encoding a FGF-9 polypeptide contained in cells that
ordinarily express the FGF-9 polypeptide where, for example, the
nucleic acid molecule is in a chromosomal or extrachromosomal
location different from that of natural cells. The isolated nucleic
acid molecule may be referred to as a recombinant nucleic acid
molecule where the isolated nucleic acid molecule has been
manipulated using recombinant techniques, for example, as described
in J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular
Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold
Spring Harbor Laboratory Press.
[0080] Variants include, without limitation, analogs, derivatives,
fragments, truncations, splice variants, mutants, deletions,
substitutions, insertions, fusions and the like.
[0081] A FGF-9 polypeptide or a nucleic acid encoding a FGF-9
polypeptide may be mutated or changed or derivatised in any manner
desired (for example, any number or combination of deletions,
insertions, or substitutions) to produce a corresponding variant.
Use of such variants in controlling angiogenesis is contemplated,
and such a variant nucleic acid or variant polypeptide may be
mutated or changed or derivatised any manner in comparison to a
naturally occurring nucleic acid or polypeptide sequence,
respectively, provided that the angiogenic activity is maintained.
Similarly, nucleic acids or polypeptides having varying degrees of
sequence identity to a corresponding naturally occurring nucleic
acid or polypeptide sequence may be tolerated without eliminating
an angiogenic activity. For example, a composition may comprise an
FGF-9 polypeptide having a sequence that is identical to a
naturally-occurring form of the FGF-9 polypeptide or a variant
thereof that has a sequence that is at least 80% identical to a
naturally-occurring form of the FGF-9 polypeptide. As another
example, a composition may comprise a nucleic acid molecule having
a coding sequence that is identical to a naturally-occurring form
of the coding sequence or a variant thereof that has a sequence
that is at least 70% identical to a naturally-occurring form of the
coding sequence. Determination of sequence identity of proteins and
nucleic acids by computer based methods, as well as nucleic acid
hybridization techniques using high stringency conditions for
determining or identifying nucleic acid sequences that share high
(eg., at least 70%) sequence identity are well known to the skilled
person.
[0082] Stringency of hybridization reactions is readily
determinable by one of ordinary skill in the art, and generally is
an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes
require higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured DNA to reanneal when complementary strands
are present in an environment below their melting temperature. The
higher the degree of sequence identity between the probe and
hybridizable sequence, the higher the relative temperature which
can be used. High stringency conditions may be identified by those
that: (1) employ low ionic strength and high temperature for
washing, for example 0.015 M sodium chloride/0.0015 M sodium
citrate/0.1% sodium dodecyl sulfate at 50.degree. C.; (2) employ
during hybridization a denaturing agent, such as formamide, for
example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%
Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at
pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at
42.degree. C.; or (3) employ 50% formamide, 5.times.SSC (0.75 M
NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),
0.1% sodium pyrophosphate, 5.times.Denhardt's solution, sonicated
salmon sperm DNA (50 .mu.g/m)), 0.1% SDS, and 10% dextran sulfate
at 42.degree. C., with washes at 42.degree. C. in 0.2.times.SSC
(sodium chloride/sodium citrate) and 50% formamide at 55.degree.
C., followed by a high-stringency wash consisting of 0.1.times.SSC
containing EDTA 55.degree. C. Hybridization and wash times should
be sufficient for achieving equilibrium.
[0083] Percent (%) sequence identity of amino acid or nucleic acid
sequences with respect to FGF-9 polypeptides and nucleic acid
sequences encoding FGF-9 polypeptides is the percentage of residues
in a candidate sequence that are identical with the FGF-9
polypeptide amino acid sequence or the FGF-9 polypeptide-encoding
nucleic acid sequence, as the case may be, after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity. Alignment for purposes of
determining percent amino acid sequence identity or percent nucleic
acid sequence identity can be achieved in various ways that are
within the skill in the art, for instance, using publicly available
computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or
Megalign (DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over a desired
length of sequence, for example, at least 20, 30, 40, 50, 60, 70,
80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200
residues or even the full-length of the sequences being
compared.
[0084] When considering a FGF-9 polypeptide or variant thereof, the
variant FGF-9 polypeptide will typically have an amino acid
sequence that is at least 80, 82, 84, 86, 88, 90, 92, 94, 96, or 98
percent identical to the corresponding FGF-9 polypeptide.
[0085] When considering a nucleic acid sequence encoding a FGF-9
polypeptide or variant thereof, the variant nucleic acid sequence
will typically be at least 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,
90, 92, 94, 96, or 98 percent identical to the corresponding
nucleic acid encoding the FGF-9 polypeptide.
[0086] Techniques and strategies for producing variants are well
known in the art. In one example, with regard to polypeptides, a
FGF-9 polypeptide may be modified in vivo or in vitro by,
glycosylation, amidation, phosphorylation, carboxylation,
truncation, fragmentation, substitution, and the like without
eliminating angiogenic activity. In another example, with regard to
nucleic acids, substitution mutations can be made in a nucleic acid
encoding a FGF-9 polypeptide such that a particular codon is
changed to a codon which codes for a different amino acid. A
substitution mutation of this sort can be made to change an amino
acid in the resulting protein in a non-conservative manner (i.e.,
by changing the codon from an amino acid belonging to a grouping of
amino acids having a particular size or characteristic to an amino
acid belonging to another grouping) or in a conservative manner
(i.e. by changing the codon from an amino acid belonging to a
grouping of amino acids having a particular size or characteristic
to an amino acid belonging to the same grouping). Such a
conservative change generally leads to less change in the structure
and function of the resulting protein. A non-conservative change is
more likely to alter the structure, activity or function of the
resulting protein. Groupings of amino acids are known to the
skilled person. For example, the nonpolar (hydrophobic) amino acids
include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan and methionine. Amino acids containing
aromatic ring structures are phenylalanine, tryptophan, and
tyrosine. The polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine. The
positively charges (basic) amino acids include arginine, lysine and
histidine. The negatively charged (acidic) amino acids include
aspartic acid and glutamic acid. Any number of such substitutions
or any other type of alteration (e.g., deletion or insertion) or
modification may be tolerated provided that the angiogenic effect
is not eliminated.
[0087] Recombinant cells, comprising a FGF-9 polypeptide or a
nucleic acid sequence that encodes a FGF-9 polypeptide may be used
for controlling angiogenesis. Recombinant cell types may include
any cell type that is compatible with the physiology of an intended
subject for therapeutic angiogenesis or for the application of
generating in vitro models of angiogenesis, vasculogenesis, or any
other in vitro model employing intact and stabilized blood
vessels.
[0088] A cell may be altered or modified to comprise a nucleic acid
sequence that does not naturally occur in the cell, and as such the
cell will be considered recombinant. In other examples, a cell may
be altered or modified to comprise an additional copy of a nucleic
acid sequence that naturally occurs in the cell, and such cells
will also be considered recombinant. As is understood by one of
skill in the art, a nucleic acid encoding a FGF-9 polypeptide may
be introduced into a cell using any known technique, for example,
microinjection, electroporation, viral transfection, lipofectamine
transfection, calcium phosphate precipitation and the like. In
certain non-limiting examples, a stem cell may be modified by
introduction of a nucleic acid molecule encoding a FGF-9
polypeptide, and then the modified cells may be administered to a
subject. In certain other examples, a nucleic add molecule encoding
a FGF-9 polypeptide may be incorporated into an appropriate
construct or vehicle, for example a viral construct, and
administered to a subject such that the nucleic add molecule
encoding the FGF-9 polypeptide is introduced and expressed in at
least a portion of the cells of the subject.
[0089] A nucleic acid encoding a FGF-9 polypeptide may be operably
linked to control sequences, typically in the context of a suitable
vector. A useful control sequence may be any nucleic add element
that is necessary or advantageous for expression of the coding
sequence of the nucleic acid sequence. Each control sequence may be
native or foreign to the nucleic acid sequence encoding the FGF-9
polypeptide. Such control sequences include, but are not limited
to, a leader, a polyadenylation sequence, a propeptide sequence, a
promoter, a signal sequence, or a transcription terminator.
Alternatives for incorporating control sequences are readily
available to the skilled person. For example, a nucleic acid
encoding a FGF-9 polypeptide may be under the control of an
endogenous upstream promoter, or it may be put under control of a
heterologous upstream promoter. Examples of suitable promoters for
directing the transcription of the modified nucleotide sequence,
such as PS4 nucleic acids, in a bacterial host include the promoter
of the lac operon of E. coli, the Streptomyces coelicolor agarase
gene dagA promoters, the promoters of the Bacillus licheniformis
alpha-amylase gene (amyL), the promoters of the Bacillus
stearothermophilus maltogenic amylase gene (amyM), the promoters of
the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the
promoters of the Bacillus subtilis xylA and xylB genes, the
promoter of the Bacillus subtilis aprE gene and a promoter derived
from a Lactococcus sp.-derived promoter including the P170
promoter. When the gene encoding the PS4 variant polypeptide is
expressed in a bacterial species such as E. coli, a suitable
promoter can be selected, for example, from a bacteriophage
promoter including a T7 promoter and a phage lambda promoter.
[0090] For transcription in a fungal species, examples of useful
promoters are those derived from the genes encoding the,
Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic
proteinase, Aspergillus niger neutral alpha-amylase, A. niger add
stable alpha-amylase, A. niger glucoamylase, Rhizornucar miehei
lipase, Asperginus oryzae alkaline protease, Aspergillus oryzae
triose phosphate isomerase or Aspergillus nidulans acetamidase.
[0091] Examples of suitable promoters for the expression in a yeast
species include but are not limited to the Gal 1 and Gal 10
promoters of Saccharomyces cerevisiae and the Pichia pastoris AOX1
or AOX2 promoters.
[0092] Still further suitable promoters are available to the
skilled person, for example, cytomegalovirus, Rous Sarcoma Virus,
synthetic pox viral promoter, pox synthetic late promoter 1, pox
synthetic late promoter 2 early promoter 2, pox 011 promoter, pox
141 promoter, pox 13L promoter; pox 12L promoter, pox 11L promoter,
pox DIOR promoter, PRV gX, HSV-1 alpha 4, chicken beta-actin
promoter, HCMV immediate early, MDV gA, MDV gB, MDV gD, MDV gB,
BHV-1.1 VP8 and ILT gD and internal ribosomal entry site
promoter.
[0093] A suitable vector may be any vector (for example, a plasmid
or virus) which can incorporate a nucleic acid sequence encoding a
FGF-9 polypeptide and any desired control sequences and can bring
about the expression of the nucleic acid sequence. The choice of
the vector will typically depend on the compatibility of the vector
with a host cell into which the vector is to be introduced. In
certain examples, the vector may exist as an extrachromosomal
entity, with replication being independent of chromosomal
replication, for example, a plasmid, an extrachromosomal element, a
minichromosome, or an artificial chromosome. In other examples, the
vector may be one which, when introduced into the host cell, is
integrated into the genome and replicated together with the
chromosome(s) into which it has been integrated. Still other
examples of vectors and techniques for manipulating vectors will be
known and apparent to the skilled person.
[0094] Recombinant cells may comprise a FGF-9 polypeptide or a
nucleic acid sequence encoding a FGF-9 polypeptide, either singly
or in combination, with other desired polypeptide or nucleic acid
molecules, respectively, for example to optimize or enhance
efficacy. Furthermore, a nucleic add sequence may be mutated or
altered prior to introduction into the cells as desired, for
example for codon optimization for expression in a particular cell
type. In addition, a nucleic acid sequence may be altered to
encoded a fusion of a FGF-9 polypeptide with one or more other
polypeptide as desired in an application, for example fusion with a
targeting polypeptide or a carrier polypeptide.
[0095] The skilled person will recognize that variants described
herein with respect FGF-9 molecules and cells comprising FGF-9
molecules can apply equally to other polypeptides, nucleic acid
molecules, and cells that are used in combination with FGF-9
molecules and cells comprising FGF-9 molecules. In certain
examples, angiogenic polypeptides, nucleic acid molecules encoding
angiogenic polypeptides or cells producing angiogenic polypeptides
may be used in combination with FGF-9 molecules or cells producing
FGF-9 molecules. Such angiogenic polypeptides include other members
of the fibroblast growth factor (FGF) family, the vascular
endothelial growth factor (VEGF) family, the platelet-derived
growth factor (PDGF) family, or the insulin-like growth factor
(IGF) family. In certain examples, an FGF-9 molecule is used in
combination with FGF2. In certain examples other polypeptides,
nucleic acid molecules, or cells that are used in combination with
FGF-9 molecules and cells comprising FGF-9 molecules may include
appropriate extracellular matrix proteins to comprise a
3-dimensional structure in vitro or a biomaterial for therapeutic
purposes. Such extracellular matrix proteins may include but are
not limited to naturally occurring molecules such as collagen,
fibrin, and proteoglycans.
[0096] As is understood by the skilled person, administration of
polypeptides, nucleic acid molecules, or cells can be done in a
variety of manners and combinations thereof. For example,
administration may be done intramuscularly, subcutaneously,
intravenously, intranasally, intradermaly, intrabursally, in ova,
ocularly, orally, intra-tracheally or intra-bronchially, as well as
combinations of such modalities. The dose may vary with the size of
the intended subject. Methods of administration are known to the
skilled person, for example, U.S. Pat. Nos. 5,693,622; 5,589,466;
5,580,859; and 5,566,064. The amounts of polypeptide, nucleic acid
sequence, or recombinant cell needed for preparation of a
composition is well understood by one of skill in the art.
[0097] The polypeptides, nucleic acids, or recombinant cells as
described herein, may be used in combination with a
pharmaceutically acceptable carrier for preparation of a
composition for controlling angiogenesis. Pharmaceutically
acceptable carriers are well known to those skilled in the art and
include but are not limited to proteins, sugars, and the like. One
example of such a suitable carrier is a physiologically balanced
culture medium containing one or more stabilizing agents such as
hydrolyzed proteins, lactose, and the like. Another example of an
acceptable carrier is 0.01-0.1M, and preferably 0.05M, phosphate
buffer or 0.8% saline. Acceptable carriers may be aqueous or
non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol,
vegetable oils such as olive oil, and injectable organic esters
such as ethyl oleate. Examples of aqueous carriers are water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Preservatives and other additives for
pharmaceutical compositions are also well known to the skilled
person, for example antimicrobials, antioxidants, chelating agents,
inert gases, organic acids and the like. Another example of such a
suitable carrier is a biomaterial comprising natural or synthetic
extracellular matrix material.
[0098] The phrases "parenteral administration" and "administered
parenterally" are art-recognized terms, and include modes of
administration other than enteral and topical administration, such
as injections, and include, without limitation, intravenous,
intramuscular, intrapleural, intravascular, intrapericardial,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intra-articular, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0099] The term "treating" is an art-recognized term which includes
curing as well as ameliorating at least one symptom of any
condition or disease.
[0100] The phrase "pharmaceutically acceptable" is art-recognized.
In certain embodiments, the term includes compositions, polymers
and other materials and/or dosage forms which are, within the scope
of sound medical judgment, suitable for use in contact with the
tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
[0101] The phrase "pharmaceutically acceptable carrier" is
art-recognized, and includes, for example, pharmaceutically
acceptable materials, compositions or vehicles, such as a liquid or
solid filler, diluent, excipient, solvent or encapsulating
material, involved in carrying or transporting any subject
composition from one organ, or portion of the body, to another
organ, or portion of the body. Each carrier must be "acceptable" in
the sense of being compatible with the other ingredients of a
subject composition and not injurious to the patient. In certain
embodiments, a pharmaceutically acceptable carrier is
non-pyrogenic. Some examples of materials which may serve as
pharmaceutically acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, sunflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
formulations.
[0102] The term "pharmaceutically acceptable salts" is
art-recognized, and includes relatively non-toxic, inorganic and
organic acid addition salts of compositions of the present
invention, including without limitation, therapeutic agents,
excipients, other materials and the like. Examples of
pharmaceutically acceptable salts include those derived from
mineral acids, such as hydrochloric acid and sulfuric acid, and
those derived from organic acids, such as ethanesulfonic acid,
benzenesulfonic acid, p-toluenesulfonic acid, and the like.
Examples of suitable inorganic bases for the formation of salts
include the hydroxides, carbonates, and bicarbonates of ammonia,
sodium, lithium, potassium, calcium, magnesium, aluminum, zinc and
the like. Salts may also be formed with suitable organic bases,
including those that are non-toxic and strong enough to form such
salts. For purposes of illustration, the class of such organic
bases may include mono-, di-, and trialkylamines, such as
methylamine, dimethylamine, and triethylamine; mono-, di- or
trihydroxyalkylamines such as mono-, di-, and triethanolamine;
amino acids, such as arginine and lysine; guanidine;
N-methylglucosamine; N-methylglucamine; L-glutamine;
N-methylpiperazine; morpholine; ethylenediamine;
N-benzylphenethylamine; (trihydroxymethyl)aminoethane; and the
like. See, for example, J. Pharm. Sci., 66:1-19 (1977).
[0103] A "patient," "subject," or "host" to be treated by the
subject method may mean either a human or non-human animal, such as
primates, mammals, and vertebrates.
[0104] The term "prophylactic or therapeutic" treatment is
art-recognized and includes administration to the host of one or
more of the subject compositions. If it is administered prior to
clinical manifestation of the unwanted condition (e.g., disease or
other unwanted state of the host animal) then the treatment is
prophylactic, i.e., it protects the host against developing the
unwanted condition, whereas if it is administered after
manifestation of the unwanted condition, the treatment is
therapeutic (i.e., it is intended to diminish, ameliorate, or
stabilize the existing unwanted condition or side effects
thereof).
[0105] The phrases "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" are art-recognized, and include the administration of
a subject composition or other material other than directly into
the central nervous system, e.g., by subcutaneous administration,
such that it enters the patient's system and, thus, is subject to
metabolism and other like processes.
[0106] The phrase "therapeutically effective amount" is an
art-recognized term. In certain embodiments, the term refers to an
amount of the therapeutic agent that, when used alone or in
combination with a suitable matrix of the present invention,
produces some desired effect at a reasonable benefit/risk ratio
applicable to any medical treatment. In certain embodiments, the
term refers to that amount necessary or sufficient to control the
formation and/or stabilization of blood vessels. The effective
amount may vary depending on such factors as the disease or
condition being treated, the particular targeted constructs being
administered, the size of the subject or the severity of the
disease or condition. One of ordinary skill in the art may
empirically determine the effective amount of a particular compound
without necessitating undue experimentation. The exact formulation,
route of administration and dosage for the composition and
pharmaceutical compositions disclosed herein can be chosen by the
individual physician in view of the patient's condition. (See.
e.g., Fingi et al. 1975, in "The Pharmacological Basis of
Therapeutics", Chapter 1, which is hereby incorporated herein by
reference in its entirety). The dosage may be a single one or a
series of two or more given in the course of one or more days, as
is needed by the patient. Where no human dosage is established, a
suitable human dosage can be inferred from ED.sub.50 or ID.sub.50
values, or other appropriate values derived from in vitro or in
vivo studies, as qualified by toxicity studies and efficacy studies
in animals. Dosage intervals can also be determined using MEC
value. Compositions should be administered using a regimen which
maintains plasma levels above the MEC for approximately 10% and
approximately 90% of the time, preferably between approximately 30%
and approximately 90%, and most preferably between approximately
50% and approximately 90%.
[0107] The amount of FGF-9 composition of the invention required
for administration to provide a therapeutic effect varies as is
understood by one of skill in the art and may vary depending on the
embodiment of the invention; used in vitro, in vivo systemically,
in vivo locally; in conjunction with a biomaterial; in an in vivo
extended release format. Therefore, in some aspects the dosage may
be from about 0.1 ng/ml to 100 ng/ml. In other aspects the dosage
may be up to about 500 ng/ml. In still other aspects the amount may
range up to about 1 mg/kg to about 100 mg/kg. It is understood by
those of skill in the art that the amount of FGF-9 composition of
the invention may be selected from any sub-range of the therapeutic
dosages described herein: up to 0.01 ng/ml, up to 500 ng/ml.
Dosages expressed by weight may also cover up to about 1 mg/kg to
about 1000 mg/kg range, such as for example but not limited to; 1
mg/kg-500 mg/kg; 1 mg/kg-250 mg/kg; 1 mg/kg-200 mg/kg; 1 mg/kg-150
mg/kg; 1 mg/kg-75 mg/kg; 1 mg/kg-50 mg/kg; and 1 mg/kg-25 mg/kg and
any sub-ranges of any of these ranges. Again, it is also possible
that the amount may be greater than 1000 mg/kg and in some aspects
less than 1 mg/kg.
[0108] In one embodiment, the pharmaceutical FGF-9 compositions
provided herein may be formulated as controlled-release
compositions, i.e. compositions in which the FGF-9 and other
angiogenic polypeptide(s) is released over period of time after
administration. Controlled- or sustained-release compositions
include formulation in lipophilic depots (e.g. fatty acids, waxes,
oils). In another embodiment, the composition is an
immediate-release composition, i.e. a composition in which all of
the FGF-9 and other angiogenic polypeptide(s) is released
immediately after administration.
[0109] In yet another embodiment, the pharmaceutical compositions
of the invention can be delivered in a controlled release system.
For example, the FGF-9 composition comprising angiogenic
polypeptide(s) may be administered using intravenous infusion, an
implantable osmotic pump, a transdermal patch, liposomes, or other
modes of administration. In one embodiment, a pump may be used (see
Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);
Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Eng1.1.
Med. 321:574 (1989). In another embodiment, polymeric materials can
be used. In yet another embodiment, a controlled release system can
be placed in proximity to the therapeutic target, i.e., the brain,
thus requiring only a fraction of the systemic dose (see, e.g.,
Goodson, in Medical Applications of Controlled Release, supra, vol.
2, pp. 115-138 (1984). Other controlled-release systems are
discussed in the review by Langer (Science 249:1527-1533 (1990).
The compositions of the invention may also include incorporation of
FGF-9 active into or onto particulate preparations of polymeric
compounds such as polylactic add, polyglycolic add, hydrogels, etc,
or onto liposomes, microemulsions, micelles, unilamellar or
multilamellar vesicles, erythrocyte ghosts, or spheroplasts.) Such
compositions will influence the physical state, solubility,
stability, rate of in vivo release, and rate of in vivo
clearance.
[0110] Also comprehended by the invention are particulate
compositions coated with polymers (e.g. poloxamers or poloxamines)
and the compound coupled to antibodies directed against
tissue-specific receptors, ligands or antigens or coupled to
ligands of tissue-specific receptors.
[0111] Also embodied by the invention are the FGF-9 compounds
modified by the covalent attachment of water-soluble polymers such
as polyethylene glycol, copolymers of polyethylene glycol and
polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl
alcohol, polyvinylpyrrolidone or polyproline. The modified
compounds are known to exhibit substantially longer half-lives in
blood following intravenous injection than do the corresponding
unmodified compounds (Abuchowski et al., 1981; Newmark et al.,
1982; and Katre et al., 1987). Such modifications may also increase
the compound's solubility in aqueous solution, eliminate
aggregation, enhance the physical and chemical stability of the
compound, and greatly reduce the immunogenicity and reactivity of
the compound. As a result, the desired in vivo biological activity
may be achieved by the administration of such polymer-compound
abducts less frequently or in lower doses than with the unmodified
compound.
[0112] In a non-limiting embodiment, a localized medical device or
biodegradable implant may be used to includes the functionality of
time-course release of the compositions of the invention. The
medical device may be composed of a solid casing with internal
gel-like fluid containing the FGF-9 compositions of the invention.
The gel-like fluid may be a cryoprecipitate, an administration
matrix, or a composition of various polymers suitable for the
sustained release of the composition. The biodegradable implant
contains a biodegradable delivery means, or carrier, as well as the
FGF-9 compositions of the invention. The carrier may be chosen so
as to remain within the implanted site for a prolonged period and
slowly release the angiogenic factors contained therein to the
surrounding environment. This mode of delivery allows the FGF-9
compositions of the invention to remain in therapeutically
effective amounts within the site for a prolonged period.
[0113] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific Examples. These Examples are
described solely for purposes of illustration and are not intended
to limit the scope of the invention. Changes in form and
substitution of equivalents are contemplated as circumstances may
suggest or render expedient. Although specific terms have been
employed herein, such terms are intended in a descriptive sense and
not for purposes of limitation.
EXAMPLES
Example 1
FGF-9 Variants
[0114] FGF-9 sequences and variants thereof may be derived from
various naturally occurring sources. For example, FIG. 1 shows
nucleic acid sequences encoding Human, Mouse and Rat FGF-9
retrieved from GenBank. As another example, FIG. 2 shows a multiple
sequence alignment of Human, Mouse, Rat, and Pig FGF-9 amino acid
sequences. Species specific amino acid sequences of FGF-9 retrieved
from GenBank were aligned using JalView 2.3. A high degree of
conservation in the amino acid sequence can be observed.
[0115] Variants of the FGF-9 sequences shown in FIG. 1 or 2 can be
produced using known methods such as those described in Sambrook J
et al. 2000. Molecular Cloning: A Laboratory Manual (Third
Edition). Variants may be tested for the ability of wrapping and/or
stabilizing a blood vessel.
Example 2
High Throughout Screening for the Factors Secreted by SMCs as they
Acquire Specialized Functions
[0116] Human SMCs were subjected to serum deprivation for 8 days.
This process converts SMCs to a state whereby they migrate in a
specialized pattern. SMCs acquiring this specialized phenotype
typically possess the ability of wrapping and stabilizing blood
vessels. RNA harvested at days 0 and 8 was subsequently subjected
to high density microarray analysis using Affymetrix U133 arrays.
Three biological replicates were performed.
[0117] Analysis of microarray data revealed that of 1087 secreted
factors expressed by SMCs only 27 were statistically upregulated as
SMCs acquired the specialized phenotype. Of the 27 upregulated
genes, FGF-9 was the most upregulated. The upregulation of FGF-9
was confirmed at the protein level as shown in FIG. 3.
Example 3
FGF-9 is Upregulated as SMCs Acquired Specialized Functions
[0118] Human SMCs were subjected to serum deprivation for 8 days.
This process converts SMCs to a state whereby they migrate in a
specialized pattern. SMCs acquiring this specialized phenotype
typically possess the ability of wrapping and stabilizing blood
vessels. RNA harvested at days 0 and 8 was subsequently subjected
to high density microarray analysis.
[0119] As shown in FIG. 4 expression of FGF-3, 6, 8, 10, 13, 17,
19, 20, 22 and FGFR-4 was not detected. Expression of FGF-7, 11,
12, 14, 18 and FGFR-1, 2, and 3 was detected but did not change in
response to serum withdrawal. FGF-1, 2 and 5 were downregulated in
response to serum withdrawal while FGF-9 was upregulated as SMCs
acquired the specialized phenotype.
Example 4
FGF-9 does not Initiate the Angiogenic Process in Subcutaneously
Implanted Matrigel in Mice
[0120] Matrigel plugs were mixed with either 500 ng/mL FGF-9, 500
ng/mL FGF-2 or 500 ng/ml. FGF-2 and 20 ng/mL FGF-9 and
subcutaneously injected into 3 month old C57/Bl6 mice. Mice were
sacrificed and matrigel plugs were harvested 8 days after
implantation and immunostained for CD31 (brown), to identify
endothelial cell-lined microvessels
[0121] FIG. 5 shows the results of this experiment. FGF-9 alone did
not induce angiogenesis as indicated by an absence of CD31
immunoreactivity inside the matrigel (FIG. 5A). FGF-2 (FIG. 5B) and
FGF-2+FGF-9 (FIG. 5C) both induce angiogenesis in the matrigel as
indicated by CD31 immunoreactivity (brown).
[0122] CD31-positive microvessels immunostained in FIGS. 5B and 5C
are quantified. Graphical representation of the area of
CD31-positive microvessels as shown in FIG. 5D indicates that
equivalent levels of angiogenesis occur in matrigel plugs
containing FGF-2 (FIG. 5B) and FGF-2 FGF-9 (FIG. 5C). 5E FGF-9
stimulates the formation of perfusable vessels. Mice bearing
matrigel plugs containing FGF-2 or FGF-2 FGF-9 for 7 days were
sacrificed and perfused with microfil and subjected to
Three-dimensional (3D) micro-computed tomography (micro CT). The
graph depicts the depth to which perfusable vessels penetrated the
matrigel plug and demonstrates that vessels formed in matrigel
containing FGF-2+FGF-9 were more functional and had a higher degree
of perfusion compared to FGF-2 alone.
Example 5
FGF-9 Stimulates Recruitment of SM .alpha.-Actin Expressing Mural
Cells to Nascent Microvessels During Angiogenesis
[0123] Photomicrographs of 8 .mu.m-thick sections of Matrigel plugs
harvested 8 days after implantation into C57/Bl6 mice and
immunostained for CD31 (brown) and smooth muscle .alpha.-actin
(red) and quantification of the percentage of microvessels
supported by smooth muscle (5M) .alpha.-actin positive cells.
[0124] FGF-9 induced the recruitment of smooth muscle .alpha.-actin
positive cells around CD31-positive microvessels to a substantially
greater extent than FGF-2 alone. Quantification of investment of
nascent blood vessels by SM .alpha.-actin positive cells, as shown
in the graph in FIG. 6, confirmed that FGF-9 stimulated the
recruitment of smooth muscle around microvessels to a much greater
extent than FGF-2 alone (83.+-.7.7% vs. 37.+-.5.4% P<0.05).
Example 6
FGF-9 Stimulates SM .alpha.-Actin Expressing Mural Recruitment
Along Continuous Lengths of Blood Vessels
[0125] Photomicrographs of Matrigel plugs harvested 8 days after
implantation, cut into 200 .mu.m thick-sections and immunostained
for CD31 (left panel) and SM .alpha.-actin (central panel) and
quantitation of SM .alpha.-actin coverage of CD31-positive
vessels.
[0126] FGF-9 induced SM .alpha.-actin positive cells to associate
along large length of nascent microvessels. Quantification of SM
.alpha.-actin positive cell coverage of CD31 nascent blood vessels,
as shown in the graph in FIG. 7, revealed that FGF-9 induced
significantly more coverage compared to FGF-2 (45.4.+-.53% vs.
7.2.+-.1.8% P<0.05).
Example 7
FGF-9 Stimulates Circumferential Wrapping of Blood Vessels by SM
.alpha.-Actin Expressing Mural Cells
[0127] Confocal microscopy of FGF-2.+-.FGF-9 matrigel plugs
harvested 8 days after implantation. Sequential images, 1 .mu.m
thick, along the z-axis were collected to obtain 90 .mu.m thick
z-stack depicting the vessel.
[0128] FIG. 8A shows orthogonal views of CD31-positive endothelial
cells (left panel) and SM .alpha.-actin mural cells (central
panel). FIG. 88 shows a three dimensional reconstruction of a 90
.mu.m thick z-stack composed of 1 .mu.m thick images.
[0129] The orthogonal views depict tight association between
endothelial and supporting SM .alpha.-actin mural cells along the
length of the vessel. The three dimensional reconstruction of the
entire z-stack of this vessels illustrated that the actin positive
mural cells circumferentially wrapped around the blood vessel,
reminiscent of the organization found in vivo. This highlights the
intimate and structurally cohesive association of SM .alpha.-actin
expressing mural cells with blood vessels implying physiologic
stabilization.
Example 8
FGF-9-Modified Microvessels are Responsive to Vasoactive Stimuli
and can Vasodilate and Vasoconstrict
[0130] 14 days after matrigel injection mice were anesthetized, the
skin overlying a matrigel was surgically removed and a catheter to
deliver drugs was sutured in the region of the plug. FITC-labeled
dextran was injected via tail vein and the diameter of vessels in
the matrigel plug was visualized using an inverted fluorescent
microscope. Doses of vasoconstrictors indicated in FIG. 9 were
applied and images of vessels were acquired over 5 minutes.
[0131] Representative tracings from separate mice, of the change in
vessel diameter over time in response to phenylephrine (PE) and KCI
are shown in FIG. 9. FGF-9-modified microvessels exhibited larger
constrictions in response to PE and KCI compared to FGF-2 induced
microvessels.
[0132] The tracings in FIG. 9B depict the change in vessel diameter
in response to phenylephrine and KCI treatment. KCI will depolarize
SMCs and result in receptor independent constriction if they are
present on the blood vessels. FGF-2 induced vessels exhibit mild if
any response to vasoconstrictors while the diameter of FGF-9
vessels is reduced by up to 65% of their initial diameter.
[0133] In separate experiments, doses of the vasoconstrictor PE
followed by the vasodilator were applied to the blood vessels in
matrigel plugs. Micrographs in C demonstrates that blood vessels in
FGF-2 FGF-9 containing matrigel plugs constricted in response to PE
and dilated in response to SNP.
Example 9
FGF-9-Stimulated Wrapping is Dependent on the Upregulation of
PDGFR-.beta.
[0134] Mouse dermal fibroblasts were stimulated with the doses of
FGF-2 or FGF-9, indicated in FIG. 11A, for 24 h and then harvested.
Cellular protein was separated by SDS-PAGE and the abundance of
PDGFR-.beta. was assessed by western blot analysis. As shown in
FIG. 11A, FGF-9 induced the upregulation of PDGFR-.beta. protein in
mouse dermal fibroblasts.
[0135] Matrigel plugs containing either FGF-2 or FGF-2 FGF-9 were
harvested 8 days after implantation and immunostained for
PDGFR-.beta.. As shown in FIG. 11B, PDGFR-.beta. expression was
markedly increased in matrigel plugs containing FGF-2+FGF-9
compared to FGF-2 alone.
[0136] Matrigel plugs containing either FGF-2 or FGF-2 FGF-9 in the
presence of a PDGFR-.beta. blocking antibody were harvested 8 days
after implantation. The association of SM .alpha.-actin positive
mural cells at blood vessels was assessed by immunostaining for
CD31 and SM .alpha.-actin. The graph in FIG. 11C shows that
blockade of PDGFR-.beta. attenuates FGF-9-mediated recruitment of
SM .alpha.-actin positive mural cells to nascent blood vessels
compared to control antibody (18.+-.4.4% vs. 87.+-.4.3%
p<0.05).
Example 10
FGF-9 Upregulates SMC Recruitment Proteins In Vitro and In Vivo
[0137] A number of factors related to angiogenesis were screened in
culture in an effort to identify mediators of FGF-9 effects.
Several factors, including Vegf and TGF-beta1 were unaffected by
FGF-9 treatment or overexpression. In contrast PDGFR-.beta., a
mediator of SMC homing to endothelial cells was upregulated by
FGF-9 treatment and overexpression in human smooth muscle cells and
mouse dermal fibroblasts (FIG. 11A-C).
[0138] Also components of Sonic Hedgehog signaling, which are
required for formation of arteries in zebrafish were also
upregulated at the mRNA level. The upregulation of PDGFR-.beta. by
FGF-9 during angiogenesis was confirmed in vivo by
immunohistochemistry (FIG. 11B). The effect of a PDGFR-.beta.
blocking antibody on SMC recruitment to nascent vessels in vivo was
also tested.
Example 11
2- and 3-Dimensional Vasculogenesis Assay
[0139] Human SMCs were treated with increasing doses of FGF-9 for
24 hr. Human umbilical endothelial cells expressing eGFP (green
fluorescent protein) were plated onto growth factor-reduced
matrigel coated dishes at a density of 1.5.times.10.sup.4
cells/cm.sup.2. After 4 hr human smooth muscle cells expressing
mRFP (red fluorescent protein) were added at a density of
1.5.times.10.sup.4 cells/cm.sup.2 and the migration of SMCs to
endothelial tubules was tracked microscopically for 24 h optionally
in the presence of 100 ng/ml of FGF-9. Fluorescence
photomicrographs depict the association of mRFP expressing SMCs
with eGFP expressing endothelial tubules 10 h after addition of
SMCs (FIG. 12).
[0140] Subsequently, endothelial cells and human SMCs will be
co-cultured on matrigel, or within a matrigel or equivalent matrix
using methodologies well known to someone skilled in the art (for
example as described in Scaffolding in Tissue Engineering (eds. Ma,
P. X. and Eliseeff, J), 2005 CRC Press, Boca Raton, Fla.), to
produce two dimensional and 3-dimensional cultures respectively.
The formation, activity, and stabilization of blood vessels in the
cultures will be assayed. Supplemented in such cultures will be
variations of and combinations of optimized concentrations of FGF2
and FGF9. Over time, the assembly of blood vessels and the extent
of vasculogenesis will be tracked by time lapse microscopy, and
properties evaluated by confocal microscopy. The reactivity of such
vessels will be evaluated by methods already described herein.
Example 12
FGF-9 Stabilizes the Neovasculature and the Vessel Stabilization is
Persistent Over Time
[0141] A Photomicrographs of matrigel plugs (FIG. 12) harvested 1
year after implantation and immunostained for CD31 (brown) 1213
Quantification of angiogenesis assessed as the area containing CD31
positive microvessels. FGF-2+FGF-9-containing matrigel plugs had
significantly more vessels 1 year after implantation especially
vessels with a diameter greater than 15 .mu.M.
Example 13
FGF-9 Stimulates the Recruitment to the Neovasculature
[0142] A. Photomicrographs (FIG. 13) of matrigel plugs containing
either FGF-2 or FGF-2+FGF-9 harvested 28 days after implantation
and immunostained for the neurofilament protein marker NF-200 to
detect the presence of blood vessel-associated nerves. B. The graph
depicts the percentage of nerved-associated blood vessels at 14 and
28 days after implantation.
Example 14
Stimulated Wrapping is Dependent on the Upregulation of
PDGFR-.beta.
[0143] A. RT-PCR of human aortic SMCs (FIG. 14) stimulated with
increasing concentrations of recombinant FGF-9 for 24 h. A number
of genes involved in angiogenesis were assessed. With only
PDGFR-.beta. being upregulated B, RT-PCR of human aortic SMCs
overexpressing cDNA encoding GFP or FGF-9. C, Western Blots of
C57/Bl6 dermal fibroblasts treated with either FGF-2 or FGF-9 for
24 h.
[0144] Mouse dermal fibroblasts were stimulated with the doses of
FGF-2 or FGF-9, indicated in FIG. 15C, for 24 hand then harvested.
Cellular protein was separated by SDS-PAGE and the abundance of
PDGFR-.beta. was assessed by western blot analysis. As shown in
FIG. 14B, in contrast to FGF-2, FGF-9 induced the upregulation of
PDGFR-.beta. protein in mouse dermal fibroblasts.
[0145] Photomicrographs of Matrigel plugs containing FGF-2 FGF-9
with either control IgG or PDGFR-.beta. blocking antibody double
immunolabeled for CD31 (brown) and sm .alpha.-actin (red) harvested
8 days after implantation. The presence of PDGFR-.beta. blocking
antibody attenuated the FGF-9 induced recruitment of sm
.alpha.-actin positive cells to new blood vessels. E Quantitation
of SM .alpha.-actin coverage of CD31-positive vessels in mice
bearing FGF-2 or FGF-2 FGF-9 with either control IgG or
PDGFR-.beta. blocking antibody. The graph in FIG. 11E shows that
blockade of PDGFR-.beta. attenuates FGF-9-mediated recruitment of
SM .alpha.-actin positive mural cells to nascent blood vessels
compared to control antibody (18.+-.4.4% vs. 87.+-.4.3%
p<0.05).
Example 15
FGF-9-Mediated PDGFR-.beta. Upregulation and Vessel Maturation
Requires Sonic Hedgehog Signaling
[0146] FIG. 15A. RT-PCR of human aortic SMCs stimulated with
increasing concentrations of recombinant FGF-9 for 24 h. 15B,
RT-PCR of human aortic SMCs overexpressing cDNA encoding GFP or
FGF-9. 15C, Western Blots of C57/Bl6 dermal fibroblasts pretreated
with either DMSO or 500 nM cycloparnine and subsequently stimulated
with vehicle or FGF-9 for 24 hr, 15D. Photomicrographs of FGF-2 and
FGF-9 containing matrigel plugs with either DMSO or cyclopamine
double immunolabeled for CD31 (brown) and sm .alpha.-actin (red). E
Quantitation of SM .alpha.-actin coverage of CD31-positive vessels
representative of 6 mice bearing FGF-2 or FGF-2+FGF-9 with either
vehicle or 500 nM cyclopamine (79.+-.4 vs 38.+-.4.8,
p<0.05).
Example 16
In Vitro Direct Effects on SMCs in Culture
[0147] FIG. 16A. Lysates of SMCs expressing either GFP or FGF-9
were subjected to Western blot analysis to assess levels of smooth
muscle cell markers. SMCs expressing FGF-9 exhibited a more
primitive and plastic phenotype compared to GFP expressing cultures
as indicates by decreased levels of smooth muscle .alpha.-actin and
calponin. 16B. WPCs expressing either GFP or FGF-9 were stained
with Annexin to detect apoptotic cells and subjected to flow
cytometry. Cultures of SMC expressing FGF-9 were less apoptotic as
indicated by the smaller proportion of Annexin positive cells. C.
SMCs expressing either GFP or FGF-9 were serially cultured and
counted to determine their cumulative population doubling and DNA
from each passage was isolated and subjected to real time PCR
analysis to assess the rate of telomere attrition. The graph shows
that cultures of SMCs expressing FGF-9 experienced a decreased rate
of telomere decay compared to control SMCs.
Example 17
Schematic of Proposal Mechanism of Action for FGF-9 During
Angiogenesis
[0148] FGF-9 signals to a pool of mesenchymal precursors and/or
immature stem cells to enhance their survival, prevent senescence.
Through either a the same or an unrelated pathway, FGF-9 activates
Sonic Hedgehog signaling to induce the upregulation of the
PDGFR-.beta. receptor on these mesenchymals cells. This increase
the competence of this pool of cells to migrate to the ligand for
this receptor, PDGF-bb which is secreted by endothelial cells
forming new blood vessels. The recruitment of mesenchymal cells to
blood vessels results in their maturation to SMC cells which are
capable of stabilizing the nascent microvasculature and imparting
vasoresponsiveness (FIG. 17).
Example 18
Orthotopic Renal Cancer Model of Metastases
[0149] An animal model of metastatic cancer was generated. FGF-9
alone, or optionally in combination with another angiogenic
molecule, was administered to mice to demonstrate utility in
treatment of cancer, it is established that angiogenesis drives the
growth of tumors and that the blood vessels formed in tumors lack
supporting mural cells and are prone to leakage. A negative
correlation has been established between vessels coverage and
metastasis and poor survival in human colorectal cancers. Recent
studies have functionally demonstrated that wrapping of tumor blood
vessels can limit tumor cell metastasis.
[0150] Female Balb/c mice were injected in the subcapsular space of
the left kidney with 5.times.10.sup.5 RENCA cells expressing either
GFP or FGF-9 suspended in growth factor reduced matrigel. After 14
days, both kidneys were excised and lungs were excised and
assessed. FIG. 18A. Photographs of excised lungs from Balb/c mice
bearing kidney tumors derived from either GFP- or FGF-9 expressing
RENCA cells. Surface metastases can be identified as irregular
translucent bulges/distentions/protruberance on the lung surface
(arrows) and were more prevalent in GFP-RENCA bearing mice. 18B.
Quantitation of kidney weight which indicates that there was no
difference in the size of primary tumors in the left kidney of GFP
expressing RENCA cells compared to FGF-9 expressing RENCA cells
(514.9 mg.+-.114.6 vs. 536.2 mg.+-.95.4 n=6 GFP vs. FGF-9). 18C.
The graph depicts the average number of surface metastases on the
lungs of Balb/c mice with a trend towards a reduction in metastasis
in mice bearing tumors derived from FGF-9 expressing RENCA cells
(74.+-.43 vs 2.+-.4.3 n=6)
Example 19
In Vitro Co-Culture
[0151] HUVECs expressing EGFP were plated on growth factor reduced
matrigel and allowed to adhere for 4 hr before the addition of
HITC6 SMCs expressing mRFP, pretreated with the indicated doses of
FGF-9 for 16 h, and fluorescent images were subsequently acquired
10 h later, arrows indicate RFP-positive SMCs aligned along
GFP-positive endothelial tubules (FIG. 19). In control conditions
few REP-positive cells were associated with GFP-positive
endothelial tubules while increasing concentrations of FGF-9
resulted in increasing association of SMCs with endothelial
tubules.
Example 20
In Vitro Endothelial Tubules
[0152] HUVECs expressing EGFP were plated on growth factor reduced
matrigel and allowed to adhere for 4 hr before the addition of
HITC6 SMCs expressing mRFP, pretreated with the indicated doses of
FGF-9 for 16 h, and fluorescent images were subsequently acquired
10 h later, arrows indicate REP-positive SMCs aligned along
GFP-positive endothelial tubules (FIG. 20). In control conditions
few REP-positive cells were associated with GFP-positive
endothelial tubules while increasing concentrations of FGF-9
resulted in increasing association of SMCs with endothelial
tubules. This together with the results shown in FIGS. 16B and 16C
demonstrates better cell survival resulting in vessel
formation.
[0153] The above-described embodiments are intended to be examples
and alterations and modifications may be effected thereto, by those
of skill in the art, without departing from the scope of the
invention which is defined by the claims appended hereto
Sequence CWU 1
1
41208PRTHomo sapiens 1Met Ala Pro Leu Gly Glu Val Gly Asn Tyr Phe
Gly Val Gln Asp Ala 1 5 10 15 Val Pro Phe Gly Asn Val Pro Val Leu
Pro Val Asp Ser Pro Val Leu 20 25 30 Leu Ser Asp His Leu Gly Gln
Ser Glu Ala Gly Gly Leu Pro Arg Gly 35 40 45 Pro Ala Val Thr Asp
Leu Asp His Leu Lys Gly Ile Leu Arg Arg Arg 50 55 60 Gln Leu Tyr
Cys Arg Thr Gly Phe His Leu Glu Ile Phe Pro Asn Gly 65 70 75 80 Thr
Ile Gln Gly Thr Arg Lys Asp His Ser Arg Phe Gly Ile Leu Glu 85 90
95 Phe Ile Ser Ile Ala Val Gly Leu Val Ser Ile Arg Gly Val Asp Ser
100 105 110 Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly
Ser Glu 115 120 125 Lys Leu Thr Gln Glu Cys Val Phe Arg Glu Gln Phe
Glu Glu Asn Trp 130 135 140 Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr Lys
His Val Asp Thr Gly Arg 145 150 155 160 Arg Tyr Tyr Val Ala Leu Asn
Lys Asp Gly Thr Pro Arg Glu Gly Thr 165 170 175 Arg Thr Lys Arg His
Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val 180 185 190 Asp Pro Asp
Lys Val Pro Glu Leu Tyr Lys Asp Ile Leu Ser Gln Ser 195 200 205
2208PRTMus sp. 2Met Ala Pro Leu Gly Glu Val Gly Ser Tyr Phe Gly Val
Gln Asp Ala 1 5 10 15 Val Pro Phe Gly Asn Val Pro Val Leu Pro Val
Asp Ser Pro Val Leu 20 25 30 Leu Ser Asp His Leu Gly Gln Ser Glu
Ala Gly Gly Leu Pro Arg Gly 35 40 45 Pro Ala Val Thr Asp Leu Asp
His Leu Lys Gly Ile Leu Arg Arg Arg 50 55 60 Gln Leu Tyr Cys Arg
Thr Gly Phe His Leu Glu Ile Phe Pro Asn Gly 65 70 75 80 Thr Ile Gln
Gly Thr Arg Lys Asp His Ser Arg Phe Gly Ile Leu Glu 85 90 95 Phe
Ile Ser Ile Ala Val Gly Leu Val Ser Ile Arg Gly Val Asp Ser 100 105
110 Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu
115 120 125 Lys Leu Thr Gln Glu Cys Val Phe Arg Glu Gln Phe Glu Glu
Asn Trp 130 135 140 Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr Lys His Val
Asp Thr Gly Arg 145 150 155 160 Arg Tyr Tyr Val Ala Leu Asn Lys Asp
Gly Thr Pro Arg Glu Gly Thr 165 170 175 Arg Thr Lys Arg His Gln Lys
Phe Thr His Phe Leu Pro Arg Pro Val 180 185 190 Asp Pro Asp Lys Val
Pro Glu Leu Tyr Lys Asp Ile Leu Ser Gln Ser 195 200 205
3208PRTRattus norvegicus 3Met Ala Pro Leu Gly Glu Val Gly Ser Tyr
Phe Gly Val Gln Asp Ala 1 5 10 15 Val Pro Phe Gly Asn Val Pro Val
Leu Pro Val Asp Ser Pro Val Leu 20 25 30 Leu Ser Asp His Leu Gly
Gln Ser Glu Ala Gly Gly Leu Pro Arg Gly 35 40 45 Pro Ala Val Thr
Asp Leu Asp His Leu Lys Gly Ile Leu Arg Arg Arg 50 55 60 Gln Leu
Tyr Cys Arg Thr Gly Phe His Leu Glu Ile Phe Pro Asn Gly 65 70 75 80
Thr Ile Gln Gly Thr Arg Lys Asp His Ser Arg Phe Gly Ile Leu Glu 85
90 95 Phe Ile Ser Ile Ala Val Gly Leu Val Ser Ile Arg Gly Val Asp
Ser 100 105 110 Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr
Gly Ser Glu 115 120 125 Lys Leu Thr Gln Glu Cys Val Phe Arg Glu Gln
Phe Glu Glu Asn Trp 130 135 140 Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr
Lys His Val Asp Thr Gly Arg 145 150 155 160 Arg Tyr Tyr Val Ala Leu
Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr 165 170 175 Arg Thr Lys Arg
His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val 180 185 190 Asp Pro
Asp Lys Val Pro Glu Leu Tyr Lys Asp Ile Leu Ser Gln Ser 195 200 205
4208PRTSus scrofa 4Met Ala Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly
Val Gln Asp Ala 1 5 10 15 Val Pro Phe Gly Asn Val Pro Val Leu Pro
Val Asp Ser Pro Val Leu 20 25 30 Leu Ser Asp His Leu Ser Gln Ser
Glu Ala Gly Gly Leu Pro Arg Gly 35 40 45 Pro Ala Val Thr Asp Leu
Asp His Leu Lys Gly Ile Leu Arg Arg Arg 50 55 60 Gln Leu Tyr Cys
Arg Thr Gly Phe His Leu Glu Ile Phe Pro Asn Gly 65 70 75 80 Thr Ile
Gln Gly Thr Arg Lys Asp His Ser Arg Phe Gly Ile Leu Glu 85 90 95
Phe Ile Ser Ile Ala Val Gly Leu Val Ser Ile Arg Gly Val Asp Ser 100
105 110 Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser
Glu 115 120 125 Lys Leu Thr Gln Glu Cys Val Phe Arg Glu Gln Phe Glu
Glu Asn Trp 130 135 140 Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr Lys His
Val Asp Thr Gly Arg 145 150 155 160 Arg Phe Tyr Val Ala Leu Asn Lys
Asp Gly Thr Pro Arg Glu Gly Thr 165 170 175 Arg Thr Lys Arg His Gln
Lys Phe Thr His Phe Leu Pro Arg Pro Val 180 185 190 Asp Pro Asp Lys
Val Pro Glu Leu Tyr Lys Asp Ile Leu Ser Gln Ser 195 200 205
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