U.S. patent application number 13/597626 was filed with the patent office on 2013-04-11 for artery- and vein-specific proteins and uses therefor.
This patent application is currently assigned to CALIFORNIA INSTITUTE OF TECHNOLOGY. The applicant listed for this patent is David J. Anderson, Zhoufeng Chen, Hai U. Wang. Invention is credited to David J. Anderson, Zhoufeng Chen, Hai U. Wang.
Application Number | 20130091591 13/597626 |
Document ID | / |
Family ID | 46123732 |
Filed Date | 2013-04-11 |
United States Patent
Application |
20130091591 |
Kind Code |
A1 |
Wang; Hai U. ; et
al. |
April 11, 2013 |
ARTERY- AND VEIN-SPECIFIC PROTEINS AND USES THEREFOR
Abstract
Arterial and venous endothelial cells are molecularly distinct
from the earliest stages of angiogenesis. This distinction is
revealed by expression on arterial cells of a transmembrane ligand,
called EphrinB2 whose receptor EphB4 is expressed on venous cells.
Targeted disruption of the EphrinB2 gene prevents the remodeling of
veins from a capillary plexus into properly branched structures.
Moreover, it also disrupts the remodeling of arteries, suggesting
that reciprocal interactions between pre-specified arterial and
venous endothelial cells are necessary for angiogenesis. This
distinction can be used to advantage in methods to alter
angiogenesis, methods to assess the effect of drugs on artery cells
and vein cells, and methods to identify and isolate artery cells
and vein cells, for example.
Inventors: |
Wang; Hai U.; (Folsom,
CA) ; Chen; Zhoufeng; (St. Louis, MO) ;
Anderson; David J.; (Altadena, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wang; Hai U.
Chen; Zhoufeng
Anderson; David J. |
Folsom
St. Louis
Altadena |
CA
MO
CA |
US
US
US |
|
|
Assignee: |
CALIFORNIA INSTITUTE OF
TECHNOLOGY
Pasadena
CA
|
Family ID: |
46123732 |
Appl. No.: |
13/597626 |
Filed: |
August 29, 2012 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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13078150 |
Apr 1, 2011 |
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13597626 |
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12567547 |
Sep 25, 2009 |
7939071 |
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13078150 |
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11028803 |
Jan 4, 2005 |
7595044 |
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12567547 |
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09687652 |
Oct 13, 2000 |
6887674 |
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11028803 |
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PCT/US99/08098 |
Apr 13, 1999 |
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09687652 |
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09085820 |
May 28, 1998 |
6864227 |
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PCT/US99/08098 |
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09083546 |
May 22, 1998 |
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09085820 |
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60081757 |
Apr 13, 1998 |
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Current U.S.
Class: |
800/3 ; 424/1.49;
424/172.1; 424/178.1; 424/9.1; 424/9.6; 800/14; 800/18 |
Current CPC
Class: |
C07K 2317/76 20130101;
A61K 39/3955 20130101; G01N 2333/52 20130101; G01N 33/6863
20130101; A01K 2267/0375 20130101; A61K 49/00 20130101; A61P 9/10
20180101; C12N 5/0691 20130101; A61K 49/0002 20130101; G01N 33/573
20130101; A01K 2227/105 20130101; C07K 14/705 20130101; A01K
2217/075 20130101; A01K 67/0275 20130101; A61K 49/0008 20130101;
C07K 16/2866 20130101; A01K 2267/03 20130101; C12N 2503/02
20130101; A61P 35/00 20180101; A61P 9/00 20180101; C07K 16/30
20130101; C12N 15/8509 20130101; A01K 67/0276 20130101 |
Class at
Publication: |
800/3 ; 800/14;
800/18; 424/172.1; 424/178.1; 424/9.1; 424/1.49; 424/9.6 |
International
Class: |
A61K 49/00 20060101
A61K049/00; A61K 39/395 20060101 A61K039/395; A61K 47/48 20060101
A61K047/48; A01K 67/027 20060101 A01K067/027 |
Claims
1-15. (canceled)
16. A method for selectively delivering an agent to veins in a
mammal, comprising administering to the mammal a complex
comprising: (a) the agent, and (b) a component which binds EphB4
under conditions appropriate for the component of (b) to bind
EphB4, whereby the agent is delivered to veins.
17-22. (canceled)
23. A transgenic nonhuman mammal having an indicator gene which is
detectably expressed in cells of arteries but not cells of
veins.
24. The transgenic nonhuman mammal of claim 23 wherein the
indicator gene is inserted in an artery-specific Ephrin family
ligand gene.
25-28. (canceled)
29. A transgenic nonhuman mammal having an indicator gene which is
expressed in venous endothelial cells but not in arterial
endothelial cells.
30. The transgenic nonhuman mammal of claim 29 wherein the
indicator gene is inserted in a vein-specific Eph family receptor
gene.
31. The transgenic nonhuman mammal of claim 30 wherein the
vein-specific Eph family receptor gene encodes EphB4.
32-76. (canceled)
77. The transgenic nonhuman mammal of claim 24 wherein the
artery-specific Ephrin family ligand gene encodes EphrinB2.
78. A method for testing an effect of a drug on growth of arteries,
comprising administering the drug to a transgenic nonhuman mammal
of claim 23, observing the effect of the drug on the growth of
arteries, and comparing the effect to that produced in a suitable
control mammal.
79. A method for testing an effect of a drug on growth of veins,
comprising administering the drug to a nonhuman mammal of claim 29,
observing the effect of the drug on the growth of veins, and
comparing the effect to that produced in a suitable control
mammal.
80. The transgenic nonhuman mammal of claim 23 wherein the mammal
is a mouse.
81. The transgenic nonhuman mammal of claim 29 wherein the mammal
is a mouse.
82. An article of manufacture, comprising: a container; a label;
and a composition comprising an Eph receptor antagonist contained
within the container; wherein the label indicates that the
composition can be used to treat a disease or disorder
characterized by undesirable or excessive vascularization or
vascular permeability.
83. The article of manufacture of claim 82 further comprising
instructions for administering the Eph receptor antagonist to a
mammal to treat a disease or disorder in the mammal.
84. An article of manufacture, comprising: a container; a label;
and a composition comprising an Eph receptor agonist contained
within the container; wherein the label indicates that the
composition can be used to stimulate angiogenesis.
85. The article of manufacture of claim 84 further comprising
instructions for administering the Eph receptor agonist to a mammal
to treat a disease or disorder in the mammal.
86. The method of claim 16, wherein the agent is a diagnostic
agent.
87. The method of claim 16, wherein the agent is an imaging
agent.
88. The method of claim 16, wherein the agent is a drug.
89. The method of claim 86, wherein the diagnostic agent comprises
a label selected from the group consisting of a radioactive label,
a fluorescent label, a calorimetric label, an enzyme label, an
antigenic label, an epitopic label and a biotin label.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of PCT
Application Number PCT/US99/08098 filed Apr. 13, 1999, which is a
continuation-in-part of U.S. application Ser. No. 09/085,820 filed
on May 28, 1998, which is a continuation-in-part of U.S.
application Ser. No. 09/083,546 filed on May 22, 1998 (abandoned).
This application also claims the benefit of U.S. Provisional
Application No. 60/081,757 filed on Apr. 13, 1998. The teachings of
each of these applications arc incorporated herein by reference in
their entirety.
BACKGROUND OF THE INVENTION
[0002] The process of blood vessel formation is fundamental in both
development and disease. The circulatory system is the first organ
system to emerge during embryogenesis, and is necessary to nourish
the developing fetus. Disorders of the circulatory system, such as
coronary artery disease, are a major cause of morbidity and
mortality in modern society. Thus, repairing, replacing and
promoting the growth. of blood vessels is a major target of
clinical research and of pharmaceutical development. Conversely,
the ingrowth of new capillary networks into developing tumors is
essential for the progression of cancer. Thus, the development of
drugs that inhibit this process of tumor angiogenesis is an equally
important therapeutic goal. Little attention has been paid to the
problem of how arteries and veins acquire their distinct
identities. Indeed, many people have assumed that the anatomical
and functional differences between arteries and veins simply
reflect physiological influences, such as blood pressure,
oxygenation and shear forces. Additional knowledge of how arteries
and veins acquire their respective identities would be valuable in
both research and clinical settings.
SUMMARY OF THE INVENTION
[0003] The present invention relates to a method of distinguishing
between arterial cells (including arterial endothelial cells) and
venous cells based on the expression of a protein on arterial cells
(arterial endothelial cells) and not on venous cells, and to a wide
variety of processes, methods and compositions of matter, including
those useful in research and clinical settings, which are based on
the difference in expression between the two cells types. As
described herein, it has been shown that there is a molecular
distinction between arterial endothelial cells (arteries) and
venous endothelial cells (veins) and that arterial endothelial
cells and venous endothelial cells bear molecular markers which can
be used to identify, separate, target, manipulate or otherwise
process each cell type specifically (separate from the other). As a
result, arteries and veins can now be distinguished from one
another, and cell types that make up arteries and veins can be
assessed for other genetic molecular or functional differences and
targeted, manipulated or otherwise processed individually or
separately for research, diagnostic and therapeutic purposes.
[0004] The present invention relates to methods of distinguishing
and separating arterial cells from vein cells, and more
specifically, distinguishing and separating arterial endothelial
cells from venous endothelial cells based on their respective
molecular markers; methods of selectively targeting or delivering
drugs or agents to arteries or veins; methods of altering
(enhancing or inhibiting, where "inhibiting" includes partially or
completely inhibiting) the function of artery-specific or
vein-specific molecular markers or interaction between them (and,
thus, enhancing or inhibiting the effect such functions or
interactions have on arterial endothelial cells or venous
endothelial cells); and methods of screening for drags which act
selectively on arterial cells (and more specifically, on arterial
endothelial cells) or venous cells (and more specifically, on
venous endothelial cells).
[0005] The invention also relates to transgenic nonhuman mammals,
such as transgenic mice, in which genes encoding an arterial cell
molecular marker or a venous cell molecular marker are altered,
either physically or functionally, and their use as "indicator
mice" to specifically visualize either arteries or veins, to assess
the function of the molecular marker which has been altered and to
identify drugs which affect (enhance or inhibit) their function. It
further relates to antibodies which bind an arterial cell-specific
marker or a venous cell-specific marker; viral or other vectors
targeted to arteries or veins by virtue of their containing and
expressing, respectively, an arterial cell-specific marker or a
venous cell-specific marker; cDNAs useful for preparing libraries
to be screened for additional artery- or vein-specific genes, and
immortalized cell lines derived from isolated. arterial endothelial
cells, from venous endothelial cells, or from transgenic animals
(e.g., mice) of the present invention.
[0006] A molecular marker for an arterial cell or a venous cell is
any gene product (protein or RNA or combination thereof) expressed
by one of these cell types and not by the other. Such a marker can
be arterial endothelial cell-specific (artery-specific) or venous
endothelial cell-specific (vein-specific) products or proteins. In
specific embodiments, these can be referred to, respectively, as
arterial endothelial cell-specific (artery-specific) ligands and
venous endothelial cell-specific (vein-specific receptors. Such
molecular markers can be expressed on cell types in addition to
arterial or venous endothelial cells, but are not expressed on both
arterial and venous endothelial cells. Molecular markers can
include, for example, mRNAs, members of ligand-receptor pairs, and
any other proteins such as adhesion proteins, transcription,
factors or antigens which are not expressed on both cell types. In
one embodiment, the molecular marker is a membrane receptor which
is the receptor for a growth factor which acts on arteries or
veins. In another embodiment the molecular marker is a member of an
endothelial cell surface ligand-receptor pair which is expressed on
arterial or venous endothelial cells, but not on both. For example,
as described in detail herein, a member of the Ephrin family of
ligands and a member of the Eph family of receptors which is its
receptor are molecular markers for arterial endothelial cells and
venous endothelial cells, respectively and are useful to
distinguish the two cell types. Any Ephrin family ligand which is
expressed on arterial endothelial cells, but not on venous
endothelial cells and a venous endothelia cell-specific Eph family
receptor which binds the arterial endothelial cell-specific ligand
can be used to distinguish between arteries and veins.
[0007] In one embodiment, the present invention relates to the
discovery that arterial endothelial cells express an Ephrin family
ligand and venous endothelial cells express an Eph family receptor
which is a receptor of the Ephrin family ligand expressed on the
arterial endothelial cells; methods of distinguishing or separating
arterial cells (arteries) from venous cells (veins); methods of
selectively targeting or delivering drugs or agents to arteries or
veins; methods of enhancing or inhibiting angiogenesis, including
angiogenesis in tumors, such as by altering (increasing, decreasing
or prolonging) activity of at least one member of an Ephrin family
ligand-cognate Eph family receptor pair and drugs useful in the
methods; and methods of screening for drugs which selectively act
on arteries or veins.
[0008] It further relates to transgenic nonhuman mammals, such as
transgenic mice, which have altered genes encoding an Ephrin family
ligand or altered genes encoding an Eph family receptor, such as
EphrinB2 knockout mice which contain a tau-lacZ (tlacZ) insertion
that marks arteries but not veins or EphB4 knockout mice which
contain a reporter construct (e.g., lacZ or alkaline phosphatase
gene) in the EphB4 locus; methods of using these mice as "indicator
mice" to define and visualize angiogenic processes (e.g., tumor
angiogenesis and ischemia-associated cardiac neovascularization) or
to screen drugs for their angiogenic or anti-angiogenic effects on
arteries or veins in vivo; and cells, such as immortalized cells,
derived from the transgenic mice. The present invention also
relates to antibodies which bind an artery-specific Ephrin family
receptor (e.g., antibodies which bind EprhinB2); antibodies which
bind a venous-specific Eph family receptor (e.g., antibodies which
bind EphB4); viral or other vectors which are targeted to arteries
or veins for vessel-specific gene therapy by virtue of their
containing and expressing DNA encoding, respectively, an Ephrin
family ligand (e.g., EphrinB2) or an Eph family receptor (e.g.,
EphB4); cDNAs useful for preparing libraries to be screened for
additional artery-specific or vein-specific genes (whose gene
products, in turn, might be artery-or vein-specific drug targets)
and methods of repairing or replacing damaged arteries or veins by
transplantation of isolated arterial or venous endothelial cells,
immortalized cell lines derived from them, or synthetic vessels
configured from these cells.
[0009] As described herein and as is known to those of skill in the
art, Ephrin family ligands are divided into two subclasses (EphrinA
and EphrinB) and Eph family receptors are divided into two groups
(EphA and EphB). As is also known, within each subclass or group,
individual members are designated by an arabic number. The
invention is described herein with specific, reference to EphrinB2
and EphB4, However, other Ephrin family ligand-Eph family receptor
pairs which show similar artery-and vein-specific expression and
their uses are also the subject of this invention. Similar artery-
and vein-specific pairs can be identified by methods known to those
of skill in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1A is a diagram of the wild e locus of the EphrinB2
gene showing the Exon-1 structure. The filled box represents 5
untranslated region. The hatched box starts at the ATG, and
includes the signal sequence. H=HindIII; X=XbaI; N=NcoI;
E=EcoRI.
[0011] FIG. 1B is a diagram of the targeting vector used to disrupt
the EphrinB2 gene.
[0012] FIG. 1C is a schematic representation of the mutated
EphrinB2 locus.
[0013] FIG. 2 is a bar graph indicating the binding activity to
GPI-ephrin-B2 of EphB2Fc in the presence of hamster anti-ephrin-B2
hybridoma supernatants.
DETAILED DESCRIPTION OF THE INVENTION
[0014] As described herein, it has been shown that arteries and
veins are genetically distinct from the earliest stages of
embryonic development and that reciprocal interactions between
arteries and veins are essential for proper vessel formation. This
finding not only changes dramatically our view of the basic
ontogenetic anatomy of embryonic vasculature, but also provides the
means to distinguish between arterial endothelial cells and venous
endothelial cells, both physically and functionally. As a result,
means of separating the two cell types from one another; of
identifying other artery- or vein-specific genes; of assessing the
selective effects of drugs or other agents on arteries or veins
and, thus, identifying those which are artery- or vein-specific;
and of selectively delivering or targeting substances to either
cell type, are now available. In addition, the work described
herein makes it possible to modulate (enhance or inhibit) or
control vasculogenesis and angiogenesis and to do so, if desired,
in an artery-specific or vein-specific manner.
[0015] As described in the examples, a gene which encodes a cell
membrane-associated ligand which is present in the nervous system
and the vascular system has been shown, in adult mice to be
expressed by arterial endothelial cells, and not by venous
endothelial cells. Further, the gene which encodes the receptor for
the ligands has been shown to be expressed by venous endothelial
cells, but not by artery cells. Thus, for the first time, a marker
found on arterial endothelial cells (an artery-specific marker) and
a venous endothelial cell- (vein-specific) marker are available,
making it possible to distinguish between arteries and veins for a
variety of purposes, such as further study and understanding of the
mechanisms of blood vessel formation; selective targeting of
treatments or therapies to arteries or veins (targeting to arteries
but not veins or vice versa) and selective modulation (enhancement
or inhibition) of formation, growth and survival of arteries and/or
veins.
[0016] In addition, the work presented in the examples demonstrates
that reciprocal signaling between arteries and veins is crucial for
vessel morphogenesis (development/formation of arteries and veins).
As described, deletion of the ligand-encoding gene in mice
prevented the proper development of both arterial and venous
vessels. Since the ligand is present on arteries (but not veins),
the occurrence of the venous defect is evidence that veins require
a signal from arteries for vessel morphogenesis. Conversely, since
the arteries are also defective in the mutant mice, the ligand must
have a function in the arterial cells themselves, in addition to
its role in signaling to the veins. In view of the fact the ligand
present on arterial endothelial cells is a transmembrane structure,
it most likely functions to receive and transduce to arterial cells
a reciprocal signal from venous cells.
[0017] Specifically, a ligand which is a member of the Ephrin
family of Eph family receptor interactive proteins (Eph family of
transmembrane ligands) has been shown to be expressed by arterial
endothelial cells, but not by venous endothelial cells. Thus, it is
now possible to distinguish between or target arteries and veins by
relying on the presence or absence of an Ephrin family ligand and
its receptor, which is a member of the Eph family of receptor
protein-tyrosine kinases. As described herein, arterial endothelial
cells have been shown to express EphrinB2 and venous endothelial
cells have been shown to express EphB4, which is an EphrinB2
receptor. EphrinB2 is not expressed on venous endothelial cells and
EphB4 is not expressed on arterial endothelial cells, providing a
means by which the two cell types can be identified or
distinguished and, thus, a means by which arterial endothelial
cells and venous arterial cells can be, for example, separated from
one another, targeted specifically or acted upon in a selective
manner (e.g., by a drug or agent which acts upon one cell type to
the exclusion of the other). For example, antibodies that bind to
EphrinB2 or to its extracellular domain can be fluorescently
labeled and allowed to bind to a mixture of cells, which are then
subjected to fluorescent activated cell sorting to select cells of
arteries from the mixture.
[0018] The work described herein, particularly in the examples,
refers to EphrinB2 and EphB4. However, any ligand-receptor pair
from the Ephrin/Eph family, any other ligand-receptor pair or any
gene product produced by one cell type and not the other (e.g., an
Ephrin ligand is expressed by arterial endothelial cells but not by
venous endothelial cells and an Eph receptor is expressed by venous
endothelial cells but not by arterial endothelial cells) can be
used to distinguish between or identify and, thus, selectively act
upon, arterial endothelial cells and venous arterial cells.
[0019] The ephrins ands are of two structural types, which can be
further subdivided on the basis of sequence relationships and,
functionally, on the basis of the preferential binding they exhibit
for two corresponding receptor subgroups. Structurally, there are
two types of ephrins: those which are membrane-anchored by a
glycerophosphatidylinositol (GPI) linkage and those anchored
through a transmembrane domain. Conventionally, the ligands are
divided into the Ephrin-A subclass, which are GPI-linked proteins
which bind preferentially to EphA receptors, and the ephrinB
subclass, which are transmembrane proteins which generally bind
preferentially EphB receptors.
[0020] The Eph family receptors are a family of receptor
protein-tyrosine kinases which are related to Eph, a receptor named
for its expression in an erythropoietin-producing human
hepatocellular carcinoma cell line. They are divided into two
subgroups on the basis of the relatedness of their extracellular
domain sequences and their ability to bind preferentially to
ephrinA proteins or ephrinB proteins. Receptors which interact
preferentially with ephrinA proteins are EphA receptors and those
which interact preferentially with ephrinB proteins are EphB
receptors.
[0021] As used herein, the terms Ephrin and Eph are used to refer,
respectively, to ligands and receptors. They can be from any of a
variety of animals (e.g., mammals/nonmammals,
vertebrates/nonvertebrates, including humans). The nomenclature in
this area has changed rapidly and the terminology used herein is
that proposed as a result of work by the Eph Nomenclature
Committee, which can be accessed, along with previously-used names
at web site http://www.eph-nomenclature.com. For convenience, eph
receptors and their respective ligand(s) are given in the
Table.
TABLE-US-00001 EPH RECEPTORS AND LIGAND SPECIFICITIES Eph Receptors
Ephrins EphA1 Ephrin-A1 EphA2 Ephrin-A3, -A1, A5, -A4 EphA3
Ephrin-A5, -A2, A3, -A1 EphA4 Ephrin-A5, -A1, A3, -A2 -B2, -B3
EphA5 Ephrin-A5, -A1, A2, -A3, -A4 EphA6 Ephrin-A2, -A1, A3, -A4,
-A5 EphA7 Ephrin-A2, -A3, A1 EphA8 Ephrin-A5, -A3, A2 EphB1
Ephrin-B2, -B1, A3 EphB2 Ephrin-B1, -B2, B3 EphB3 Ephrin-B1, -B2,
B3 EphB4 Ephrin-B2, -B1 EphB5 Unknown EphB6 Unknown Ligand
specificities are arranged in order of decreasing affinity. Adapted
from Pasquale, E.B. (1997) Curr. Opin. Cell Biol. 9(5)608.
The work described herein has numerous research and clinical
applications, which are discussed below.
[0022] As used herein, a transgenic mouse is one which has,
incorporated into the genome of some or all its nucleated cells, a
genetic alteration which has been introduced into the mouse or at
least one of its ancestors, by the manipulations of man. A
transgenic mouse can result, for example, from the introduction of
DNA into a fertilized mouse ovum or from the introduction of DNA
into embryonic stem cells.
[0023] One embodiment of the present invention is a transgenic
mouse, which because of its particular genotype, expresses only in
cells of veins or only in cells of arteries a gene whose RNA
transcript or polypeptide gene product can be detected, for
example, by in situ hybridization of RNA, by fluorescence, by
detection of enzymatic activity, or by detection of a gene product
by antibody binding and a detection system for the bound
antibodies.
[0024] A particular embodiment of the present invention is a
transgenic mouse of genotype EphrinB2.sup.+/-, wherein the "minus"
allele denotes an allele in which a naturally occurring allele has
been deleted, modified or replaced with a mutant allele, including
a mutant allele which can have an insertion of an indicator gene.
Such a "minus" allele can encode an EphrinB2 ligand which has wild
type, altered or no ligand function. A mouse of genotype
EphrinB2.sup.+/tlacZ has been produced as described in Example 1
and used to demonstrate that arterial endothelial cells and venous
endothelial cells differ genetically from early stages of
development and that reciprocal interactions, essential for proper
capillary bed formation, occur between the two types of vessels. A
transgenic mouse of the same phenotype can be produced by other
methods known to those of skill in the art. Such methods are
illustrated below using the EphrinB2 gene as an example, but can
also be used for any other vein- or artery-specific gene.
[0025] For example, it is possible to produce a vector carrying an
insertion, a deletion, or one or more point mutations in the
EphrinB2 gene. The EprhinB2 transgene can be introduced into the
genome, via a vector carrying a mutagenized EphrinB2 allele, either
by introducing the transgene into a fertilized ovum, by the method
of Wagner et al., U.S. Pat. No. 4,873,191 (1989), or by introducing
the transgene into embryonic stem (ES) cells (see, for example,
Capecchi, M. R., Science 244:1288-1292, 1989), or by other
methods.
[0026] An insertion of DNA used to construct a transgenic knockout
mouse can have within it a gene whose presence can be readily
tested, such as neo, which confers upon its host cells resistance
to G418. It is an advantage of an EphrinB2.sup.+/- indicator mouse
(e.g., EphrinB2.sup.+/taulacZ to be able to express, under the
control of the EphrinB2 promoter, an indicator gene, which can be
any gene riot endogenously expressed by mice. A particularly
advantageous indicator gene is one which facilitates the detection
of EphrinB2 expression, presumably as it is occurring in the wild
type allele, by the production of a gene product that is
detectable, for example, by its own light absorbance properties,
its ability to act upon a substrate to yield a colored product, or
its ability to bind to an indicator or dye which is itself
detectable.
[0027] Further, alternative methods are available to produce
conditional knockouts or tissue specific knockouts of a gene
expressed specifically in veins or in arteries (i.e., a
vein-specific or artery-specific gene), for example by a
site-specific recombinase such as Cre (acting at loxP site) or FLP1
(acting at FRT site) of yeast.
[0028] The bacteriophage P1 Cre-loxP recombination system is
capable of mediating loxP site-specific recombination in both ES
cells and transgenic mice. The site-specific recombinase Cre can
also be used in a predefined cell lineage or at a certain stage of
development. See, for example, Cu, H. et al., Science 265:103-106,
1994, in which a DNA polymerase .beta. gene segment was deleted
from T cells; see also Tsien, J. Z. et al., Cell 87:1317-1326,
1996, in which Cre/loxP recombination was restricted to cells in
the mouse forebrain.) The impact of the mutation on these cells can
then be analyzed.
[0029] The Cre recombinase catalyzes recombination between 34 by
loxP recognition sequences (Sauer. B. and Henderson, N., Proc.
Natl. Acad. Sci. USA 85:5166.5170, 1988). The loxP sequences can be
inserted into the genome of embryonic stern cells by homologous
recombination such that they flank one or more exons of a gene of
interest (making a "floxed" gene). It is crucial that the
insertions do not interfere with normal expression of the gene.
Mice homozygous for the foxed gene are generated from these
embryonic stem cells by conventional techniques and are crossed to
a second mouse that harbors a Cre transgene under the control of a
tissue type- or cell type-specific transcriptional promoter. In
progeny that are homozygous for the foxed gene and that carry the
Cre transgene, the floxed, gene will be deleted by Cre/loxP
recombination, but only in those cell, types in which the Cre
gene-associated promoter is active.
[0030] A gene that encodes a protein which acts to have the effect
of mimicking the phenotype caused by mutations in a vein-specific
or artery-specific gene can also be used to achieve the same effect
as knockouts in vein-specific or artery-specific genes.
[0031] A mutation in a gene which encodes a product which prevents
binding of ligand to receptor or prevents the functional
consequences of such binding and thereby duplicates the phenotype
of a vein- or artery-specific gene knockout (e.g., a dominant
negative mutant) can be used as an alternative to a knockout. The
mutated gene can be put under the control of a tissue-specific
promoter to be expressed in vein or artery, depending on the
tissue-specific gene product whose function is to be inhibited.
[0032] In addition, one Or more dominant negative alleles of an
artery-specific or vein-specific gene can be put under the control
of an inducible promoter so that upon induction, the effect of the
inhibition of acne function can be studied. A dominant negative
mutant can be isolated or constructed by mutagenesis and methods to
make a transgenic mouse.
[0033] Testing to identify the desired mutant or wild type alleles,
or for the identification of other alleles, can be done by PCR on
isolated gnomic DNA, using appropriate primers, or by Southern
blots using appropriate hybridization probes, by a combination of
these procedures, or by other methods.
[0034] In addition to the uses of an indicator mouse described in
the Examples herein, one use of a mouse having an indicator gene
which can mark artery cells is a method for testing an effect of a
drag on growth of arteries. The method can comprise administering
the drug to a mouse (e.g., embryo, neonate, juvenile, adult, a
wound site, tumor, ischemic lesion or arteriovenous malformation in
any of the preceding) having an indicator gene inserted in a gene
specifically expressed in arteries, and observing the effect of the
drug on the growth of the arteries, compared to the effect in a
suitable control mouse having an indicator gene, not treated with
the drug, but maintained under identical conditions. Similar tests
may be performed on an indicator mouse having an indicator gene
which marks vein cells. The effect of the drug can be, for example,
to promote growth, to inhibit growth, or to promote aberrant
growth. Administration of the drug can be by any suitable route
known to those of skill in the art.
[0035] An indicator mouse having an indicator gene inserted in a
gene specifically expressed in artery cells can be crossed with a
mouse of another strain carrying a mutation in. a gene which is to
be tested for its effect on the growth and development of blood
vessels, to allow far easier visualization of the effects of the
mutation specifically on artery cells. In tests similar to those
described above, the effect of a drug can be assessed on the mouse
which results from this type of cross, to see, for example, whether
the effect of the mutation can be alleviated by the drug. In like
manner, an indicator mouse having an indicator gene inserted in a
gene specifically expressed in vein cells can be used in a cross
with a mouse with a mutation whose effect on growth of veins is to
be evaluated, and the resulting hybrid used in studies of the
growth of veins.
[0036] As a result of the work described herein, it is possible to
differentiate between arterial endothelial cells (arteries) and
venous endothelial cells (veins) by taking advantage of the
presence of an artery-specific or vein-specific gene product on the
surface of the cells. Arterial endothelial cells and venous
endothelial cells can each be isolated from cells of other tissue
types by, for instance, excision of artery or vein tissue from a
sample of mammalian tissue, dissociation of the cells, allowing the
cells to bind, under appropriate conditions, to a substance which
has some property or characteristic (e.g., a molecule which
provides a label or tag, or molecule that has affinity for both the
an artery-specific cell surface protein and another type of
molecule) that facilitates separation of cells bound to the
substance from cells not bound to the substance. Separation of the
cells can take advantage of the properties of the bound substance.
For example, the substance can be an antibody (antiserum,
polyclonal or monoclonal) which has been raised against the protein
specific to arterial endothelial cells (or to a sufficiently
antigenic portion of the protein) and labeled with a fluorochrome,
with biotin, or with another label. Separation of cells bound to
the substance can be by fluorescence activated cell sorting (FACS),
for a fluorescent label, by streptavidin affinity column, for a
biotin label, by other affinity-based separation methods, or, for
example, by antibody-conjugated magnetic beads or solid supports.
"Isolated" as used herein for cells indicates that the cells have
been separated from other cell types so as to be a population
enriched for a certain cell type, compared to the starting
population, and is not limited to the case of a population
containing 100% one cell type.
[0037] Other means of separation can exploit, for blood vessel
cells bearing an indicator insertion in a gene encoding an artery-
or vein-specific protein, the properties of the indicator gene
product or portion of fusion protein encoded by the indicator
insertion. For example, cells producing an artery- or vein-specific
fusion protein with a green fluorescent protein portion or a blue
fluorescent protein portion can be separated from non-fluorescent
cells by a cell sorter. Cells producing a fusion protein having an
artery- or vein-specific protein portion and an indicator protein
or portion with binding or enzymatic activity can be detected by
the ability of the fusion protein to bind, to a fluorescent
substrate, for example a substrate for .beta.-galactosidase or
.beta.-lactamase, or to produce a fluorescent product in cells.
[0038] The isolation of arterial endothelial cells and the
isolation of venous endothelial cells allows for tests of these
cell types in culture to assess the effects of various drugs,
growth factors, ligands, cytokines, members of the Eph and Ephrin
families of receptors and ligands, molecules that bind to cell
surface proteins, or other molecules which can have effects on the
growth and development of arteries and veins. One or more of these
substances can be added to the culture medium, and the effects of
these additions can be assessed (e.g., by measurements of growth
rate or viability, enzyme assays, assays for the presence of cell
surface components, incorporation of labeled precursors into
macromolecules such as DNA, RNA or proteins).
[0039] Isolated arterial endothelial cells or isolated venous
endothelial cells can be maintained in artificial growth medium,
and an immortalized cell line can be produced from each such
isolated cell type (i.e., "transformation") by infection with one
of any number of viruses (e.g., retroviruses, by transduction of
immortalizing oncogenes such as v-myc or SV40 T antigen) known to
effectively transform cells in culture. The virus can be chosen for
its species specificity of infectivity (e.g., murine ecotropic,
virus for mouse cells; amphotropic or pseudotyped viruses for human
cells). As an alternative to viral transformation, cells can be
maintained in culture by propagating the cells in medium containing
one or more growth factors.
[0040] Immortalized cell lines derived from either vein or artery
cells can be used to produce cDNA libraries to facilitate study of
genes actively expressed in each of these tissues. Further, such
cell lines can be used to isolate and identify proteins expressed
in the cells, for instance, by purifying the proteins from
conditioned growth medium or from the cells themselves.
[0041] As one alternative to using immortalized cell lines of
arterial or venous origin, cells or cell lines of non-arterial
origin or non-venous origin (e.g., endothelial cells from other
tissues, or fibroblasts) can be genetically altered (by the
introduction of one or more not-endogenously expressed genes) to
express an artery-specific or vein-specific cell surface protein,
and used in methods to detect and identify substances that
interfere with receptor-ligand interaction.
[0042] Introduction of one or more genes into a cell line can be,
for instance, by transformation, such as by electroporation, by
calcium phosphate, DEAE-dextran, or by liposomes, using a vector
which has been constructed to have an insertion of one or more
genes. See Ausubel, F. M. et al, Current Protocols in Molecular
Biology, chapter 9, containing supplements through Supplement 40,
Fall, 1997, John Wiley & Sons, New York. The introduction of
one or more genes to be expressed in a cell line can also be
accomplished by viral infection, for example, by a retrovirus.
Retroviral gene transfer has been used successfully to introduce
genes into whole cell populations, thereby eliminating problems
associated with clonal variation.
[0043] The ability to differentiate and to isolate the cells of
veins and arteries allows for a wide variety of applications for a
wide variety of purposes. For example, it is now possible to assess
the effects of various agents, such as drugs, diagnostic reagents
and enviromnental/dietary factors, on arteries and veins and to
determine if the effects observed are common to both types of cells
or specific to one cell type.
[0044] For example, it can no longer be assumed that angiogenic and
anti-angiogenic factors or drugs act equivalently on arterial and
venous cells. Isolation of these cell types of these tissues, which
is made possible by the present work, allows testing of these
angiogenic and anti-angiogenic factors for arterial or venous
specificity, which will provide more selective clinical indications
for these drugs. It will also allow the discovery of new artery- or
vein-selective drugs, such as by high-throughput screening of
immortalized arterial or venous endothelial cell lines. Existing
drugs can also be selectively targeted to arteries or veins by
using the proteins described herein as targeting devices (e.g.,
liposomes or viral vehicles having the protein or an extracellular
domain portion thereof on the viral surface) to deliver drugs
(e.g., chemically coupled drugs) to one type of vessel or the
other. For example, artery-specific agents can be used to promote
collateral growth of arteries to bypass coronary artery occlusions
or ischemic lesions.
[0045] There are numerous approaches to screening agents for their
selective effects (angiogenic or anti-angiogenic, affecting
vasotension, or inhibiting formation of atherosclerotic plaques) on
arteries and veins. For example, high-throughput screening of
compounds or molecules can be carried out to identify agents or
drugs which act selectively on arteries or veins or, in some cases,
on both. Test agents to be assessed for their effects on artery or
vein cells can be any chemical (element, molecule, compound), made
synthetically, made by recombinant techniques or isolated from a
natural source. For example, test agents can be peptides,
polypeptides, peptoids, sugars, hormones, or nucleic acid
molecules, such as antisense nucleic acid molecules. In addition,
test agents can be small molecules or molecules of greater
complexity made by combinatorial chemistry, for example, and
compiled into libraries. These libraries can comprise, for example,
alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers
and other classes of organic compounds. Test agents can also be
natural or genetically engineered products isolated from lysates or
growth media of cells--bacterial, animal or plant--or can be the
cell lysates or growth media themselves. Presentation of test
compounds to the test system can be in either an isolated form or
as mixtures of compounds, especially in initial screening
steps.
[0046] The compounds or molecules (referred to collectively as
agents or drugs) which are screened can be those already known to
have angiogenic, anti-angiogenic activity, anti-plaque activity or
vasoactivity, or those of unknown effectiveness. In the case of
those agents of known effect, the screening will be useful to
identify those drugs which act selectively on arterial endothelial
cells or on venous endothelial cells. In the case of those agents
of unknown effect, screening will be useful to newly identify drugs
which have angiogenic, antiangiogenic activity, anti-plaque
activity or vasoactivity, and to establish the cell type (arterial,
venous) on which they act. For example, immortalized cell lines of
arterial or venous origin can be used to screen libraries of
compounds to identify drugs with artery- or vein-specific drug
effects.
[0047] In one embodiment, an assay can be caned out to screen for
drugs that specifically inhibit binding of an Ephrin ligand to its
Eph receptor, such as binding of EphrinB2 to the EphB4 receptor, or
vice-versa, by inhibition of binding of labeled ligand- or
receptor-Fc fusion proteins to immortalized cells. Alternatively,
such libraries can be screened to identify members which enhance
binding of an Ephrin ligand to its Eph receptor by enhancing
binding of labeled ligand- or receptor-Fc fusion proteins to
immortalized cells. Drugs identified through this screening can
then be tested in animal models (e.g., models of cancer,
arteriovenous malformations or coronary artery disease) to assess
their activity in vivo.
[0048] A drug that inhibits interaction of an artery-specific cell
surface molecule e.g., an arterial endothelial cell-specific
surface molecule) with a vein-specific cell surface molecule (e.g.,
a veinous endothelial cell-specific surface molecule) can be
identified by a method in which, for example, the arterial
endothelial cell-specific surface molecule and the venous
endothelial cell-specific surface molecule are combined with a drug
to be assessed. for its ability to inhibit interaction between the
cell-specific molecules, under conditions appropriate for
interaction between the cell-specific molecules. The cell-specific
molecules may be used in the assay such that both are found on
intact cells in suspension isolated arterial or venous endothelial
cells, immortalized cells derived from these, or cells which have
been modified to express an artery- or vein-specific cells surface
molecule); one cell type is fixed to a solid support, and the other
molecule specific to the other cell type is in soluble form in a
suitable solution; or the molecule specific to one cell type is
fixed to a solid support while the molecule specific to the other
cell type is found free in a solution that allows for interaction
of the cell-specific molecules. Other variations are possible to
allow for the convenient assessment of the interaction between the
two different cell-specific molecules.
[0049] In further steps of the assay, the extent to which the
cell-specific molecules interact is determined, in the presence of
the drug, and in a separate test (control), in the absence of the
drug. The extent to which interaction of the cell-specific
molecules occurs in the presence and in the absence of the drug to
be assessed is compared. If the extent to which interaction of the
cell-specific molecules occurs is less in the presence of the drug
than in the absence of the drug, the drug is one which inhibits
interaction of the arterial endothelial cell-specific molecule with
the venous endothelial cell-specific molecule. If the extent to
which interaction of the cell-specific molecules occurs is greater
in the presence of the drug than in the absence of the drug, the
drug is one which enhances interaction of the arterial endothelial
cell-specific molecule with the venous endothelial cell-specific
molecule.
[0050] In one embodiment of an assay to identify a substance that
interferes with interaction of two cell surface molecules, one
specific to artery and the other specific to vein (e.g., binding of
a ligand to a receptor that recognizes it; interaction between
adhesion proteins; interaction between a cell surface protein and a
carbohydrate moiety on a cell surface), samples of cells expressing
one type of cell surface molecule (e.g., cells expressing an Eph
receptor, such as a a vein-derived cell line or other cells
genetically manipulated. to express the Eph receptor) are contacted
with either labeled ligand (e.g., an ephrin ligand, a soluble
portion thereof, or a soluble fusion protein such as a fusion of
the extracellular domain and the Fe domain of IgG) or labeled
ligand plus a test compound or group of test compounds. The amount
of labeled ligand which has bound to the cells is determined. A
lesser amount of label (where the label can be, for example, a
radioactive isotope, a fluorescent or colormetric label) in the
sample contacted with the test compound(s) is an indication that
the test compound(s) interferes with binding. The reciprocal assay
using cells expressing a ligand (e.g., an Ephrin ligand or a
soluble form thereof) can be used to test for a substance that
interferes with the binding of a receptor or soluble portion
thereof.
[0051] An assay to identify a substance which interferes with
interaction between artery-specific and vein-specific cell surface
protein can be performed with the component (e.g., cells, purified
protein, including fusion proteins and portions having binding
activity) which is not to be in competition with a test compound,
linked to a solid support. The solid support can he any suitable
solid phase or matrix, such as a bead, the wall of a plate or other
suitable surface (e.g., a well of a microtiter plate), column. pore
glass (CPG) or a pin that can be submerged into a solution, such as
in a well. Linkage of cells or purified protein to the solid
support can be either direct or through one or more linker
molecules.
[0052] Upon the isolation from a mammal of a gene expressing an
artery-specific or a vein-specific protein, the gene can be
incorporated into an expression system for production of a
recombinant protein or fusion protein, followed by isolation and
testing of the protein in vitro. The isolated or purified protein
can also be used in further structural studies that allow for the
design of agents which specifically bind to the protein and can act
as agonists or antagonists of the receptor or ligand activity of
the protein.
[0053] In one embodiment, an isolated or purified artery-specific
or vein-specific protein can be immobilized on a suitable affinity
matrix by standard techniques, such as chemical cross-linking, or
via an antibody raised against the isolated or purified protein,
and bound to a solid support. The matrix can be packed in a column
or other suitable container and is contacted with one or more
compounds (e.g., a mixture) to be tested under conditions suitable
for binding of the compound to the protein. For example, a solution
containing compounds can be made to flow through the matrix. The
matrix can be washed with a suitable wash buffer to remove unbound
compounds and non-specifically bound compounds. Compounds which
remain bound can be released by a suitable elution buffer. For
example, a change in the ionic strength or pH of the elution buffer
can lead to a release of compounds. Alternatively, the elution
buffer can comprise a release component or components designed to
disrupt binding of compounds (e.g., one or more ligands or
receptors, as appropriate, or analogs thereof which can disrupt
binding or competitively inhibit binding of test compound to the
protein).
[0054] Fusion proteins comprising all of, or a portion of, an
artery-specific or a vein specific protein linked to a second
moiety not occurring in that protein as found in nature can be
prepared for use in another embodiment of the method. Suitable
fusion proteins, for this purpose include those in which the second
moiety comprises an affinity ligand (e.g., an enzyme, antigen,
epitope). The fusion proteins can be produced by the insertion of a
gene specifically expressed in artery or vein cells or a portion
thereof into a suitable expression vector, which encodes an
affinity ligand. The expression vector can be introduced into a
suitable host cell for expression. Host cells are disrupted and the
cell material, containing fusion protein, can be bound to a
suitable affinity matrix by contacting the cell material with an
affinity matrix under conditions sufficient for binding of the
affinity ligand portion of the fusion protein to the affinity
matrix.
[0055] In one aspect of this embodiment, a fusion protein can be
immobilized on a suitable affinity matrix under conditions
sufficient to bind the affinity ligand portion of the fusion
protein to the matrix, and is contacted with one or more compounds
(e.g., a mixture) to be tested, under conditions suitable for
binding of compounds to the receptor or ligand protein portion of
the bound fusion protein. Next, the affinity matrix with bound
fusion protein can be washed with a suitable wash buffer to remove
unbound compounds and non-specifically bound compounds without
significantly disrupting binding of specifically bound compounds.
Compounds which remain bound can be released by contacting the
affinity matrix having fusion protein bound thereto with a suitable
elution buffer (a compound elution buffer). In this aspect,
compound elution buffer can be formulated to permit retention of
the fusion protein by the affinity matrix, but can be formulated to
interfere with binding of the compound(s) tested to the receptor or
ligand protein portion of the fusion protein. For example, a change
in the ionic strength or pH of the elution buffer can lead to
release of compounds, or the elution buffer can comprise a release
component or components designed to disrupt binding of compounds to
the receptor or ligand protein portion of the fusion protein (e.g.,
one or more ligands or receptors or analogs thereof which can
disrupt binding of compounds to the receptor or ligand protein
portion of the fusion protein).
[0056] Immobilization can be performed prior to, simultaneous with,
or after contacting the fusion protein with compound, as
appropriate. Various permutations of the method are possible,
depending upon factors such as the compounds tested, the affinity
matrix selected, and elution buffer formulation. For example, after
the wash step, fusion protein with compound bound thereto can he
eluted from the affinity matrix with a suitable elution buffer (a
matrix elution buffer). Where the fusion protein comprises a
cleavable linker, such as a thrombin cleavage site, cleavage from
the affinity ligand can release a portion of the fusion with
compound bound thereto. Bound compound can then be released from
the fusion protein or its cleavage product by an appropriate
method, such as extraction.
[0057] One or more compounds can be tested simultaneously according
to the method. Where a mixture of compounds is tested, the
compounds selected by the foregoing processes can be separated (as
appropriate) and identified by suitable methods (e.g., PCR,
sequencing, chromatography). Large combinatorial libraries of
compounds (e.g., organic compounds, peptides, nucleic acids)
produced by combinatorial chemical synthesis or other methods can
be tested (see e.g., Ohlmeyer, M. H. J. et al., Proc. Natl. Acad.
Sci. USA 90:10922-10926 (1993) and DeWitt, S. H. et al., Proc.
Natl. Acad. Sci. USA 90:6909-6913 (1993), relating to tagged
compounds; see also, Rutter, W. J. et al., U.S. Pat. No. 5,010,175;
Huebner, V. D. et al., U.S. Pat. No. 5,182,366; and Geysen, H. M.,
U.S. Pat. No. 4,833,092). Where compounds selected from a
combinatorial library by the present method carry unique tags,
identification of individual compounds by chromatographic methods
is possible. Where compounds do not carry tags, chromatographic
separation, followed by mass spectrophotometry to ascertain
structure, can be used to identify individual compounds selected by
the method, for example.
[0058] An in vivo assay useful to identify drugs which act
selectively on arteries or on veins is also available. It is
carried out using transgenic animals, such as those described
herein, which make it possible to visualize angiogenic processes.
For example, an EphrinB2 knockout mouse containing a marker, such
as a tau-lacZ insertion, that marks all arteries but not veins, can
be used for a variety of in vivo assays. Other marker genes that
can be used, for instance, are genes expressing alkaline
phosphatase, blue fluorescent protein or green fluorescent protein.
The mouse, or the targeted allele it contains, can be used to study
angiogenic processes, such as tumor angiogenesis and
ischemia-associated cardiac neovascularization, in arteries,
independent of veins. For example, tumor cells can be implanted in
the indicator mouse and arterial, vessel growth into the tumor can
be visualized by lacZ staining. Alternatively, mice bearing the
targeted allele can be crossed with a mouse model of another
condition, such as vascular degeneration or neovascularization, to
be visualized. The arterial-specific aspects of the process can be
visually monitored by lacZ staining An indicator of this type can
also be used to assess drags for their angiogenic or
anti-angiogenic effects.
[0059] A gene product produced specifically by arterial endothelial
cells (arteries) and not by other cell types allows for the
specific targeting of drugs, diagnostic agents, tagging labels,
histological stains or other substances specifically to arteries.
In an analogous manner, a gene product identified as produced
specifically by venous endothelial cells (veins) and not detectably
produced by other cell types allows for the specific targeting and
delivery of drugs, diagnostic agents, tagging labels, histological
stains or other substances specifically to veins. The following
description of targeting vehicles, targeted agents and methods is
presented using EphrinB2 as an illustration of a gene product
produced by arterial endothelial cells and not by vein cells and
EphB4 as an illustration of a gene product produced by venous
endothelial cells and not by artery cells. However, this
description applies equally well to other artery-specific and
vein-specific gene products that can be used to identify these
tissue types.
[0060] The differential expression of EphrinB2 in arteries and of
Eph4 in veins allows for the specific targeting of drugs,
diagnostic agents, imaging agents, or other substances to the cells
of arteries or of veins. A targeting vehicle can be used for the
delivery of such a substance. Targeting vehicles which bind
specifically to EphrinB2 or to Eph4 can be linked to a substance to
be delivered to the cells of arteries or veins, respectively. The
linkage can be via one or more covalent bonds, or by high affinity
non-covalent bonds. A targeting vehicle can be an antibody, for
instance, or other compound which binds either to EphrinB2 or to
EphB4 with high specificity. Another example is an aqueously
soluble polypeptide having the amino acid sequence of the
extracellular domain of EphB4, or a sufficient portion of the
extracellular domain (or a polypeptide having an amino acid
sequence conferring a similar enough conformation to allow specific
binding to EphrinB2), which can be used as a targeting vehicle for
delivery of substances to EphrinB2 in arteries. Similarly, a
soluble polypeptide having the amino acid sequence of the
extracellular domain of EphrinB2 or a sufficient antigenic portion
of the extracellular domain (or a polypeptide having an amino acid
sequence conferring a similar enough conformation to allow specific
binding to EphB4), can be used to target substances to EphB4 in
veins.
[0061] Targeting vehicles specific to an, artery-specific Ephrin
ligand (e.g., EphrinB2) or to a vein-specific Eph receptor (e.g.,
EphB4) have in vivo (e.g., therapeutic and diagnostic)
applications. For example, an antibody which specifically binds to
EphrinB2 can be conjugated to a drug to be targeted to arteries
(e.g., a therapeutic, such as an anti-plaque agent). Alternatively,
an antibody which specifically binds to EphB4 can be used to target
a drug to veins. A substance (e.g., a radioactive substance) which
can be detected (e.g., a label) in vivo can also be linked to a
targeting vehicle which specifically binds to an artery-specific
Ephrin ligand (e.g., EphrinB2) and the conjugate can be used as a
labeling agent to identify arteries. Similarly, a detectable label
can be linked to a targeting vehicle which specifically binds a
vein-specific Eph receptor (e.g., EphB4) to identify veins.
[0062] Targeting vehicles specific to EphrinB2 or to EphB4 find
further applications in vitro. For example, an EphB4-specific
targeting vehicle, such as an antibody (a polyclonal preparation or
monoclonal) which specifically binds to EphB4, can be linked to a
substance which can be used as a stain for a tissue sample (e.g.,
horseradish peroxidase) to provide a method for the identification
of veins in a sample. Likewise, an antibody which specifically
binds to EphrinB2 or to the extracellular domain of EphrinB2 can be
used in the identification of arteries. For instance, in a biopsied
tissue sample, as from a tumor, or from an arteriovenous
malformation in a child or adult, antibody to EphrinB2 or to the
extracellular domain of EphrinB2 can be used to identify artery
tissue and to distinguish it from vein tissue.
[0063] To treat malformed, painful or cosmetically undesirable
veins, an agent which acts against them (e.g, antiangiogenic
factors) can be linked to an EphB4-specific vehicle for local
administration to the veins. For example, anti-angiogenic factors
can be injected into varicose veins.
[0064] Targeted agents directed to either an artery-specific Ephrin
family ligand (e.g., EphrinB2) or a vein-specific Eph family
receptor (e.g., EphB4) can also be used when it is desired to
produce an effect on both arteries and veins. For example, limited
amounts of targeted agents comprising an anti-angiogenic drug and a
targeting vehicle to either EphrinB2, EphB4, or both, can be
administered locally to sites of angiogenesis, such as sites of
tumor formation or sites of undesirable neovascularization where it
is desired to inhibit the growth of blood vessels, or to areas in
which increased vascularization is desired to enhance growth or
establishment of blood vessels.
[0065] Substances that act as agonists or antagonists of an
artery-specific Ephrin family ligand (e.g., EphrinB2) or a
vein-specific Eph family receptor (e.g., EphB4) can be used as
angiogenic or anti-angiogenic agents. Drugs that target these
molecules will selectively influence arterial and venous
angiogenesis. For example, monoclonal antibodies to EphrinB2 or
EphB4 can serve as artery- or vein-specific angiogenic or
anti-angiogenic agents. Drugs that interfere with EphrinB2 function
(for instance, blocking antibodies) can be used in anti-angiogenic
methods of therapy. As can be concluded from the phenotype shown by
the EphrinB2.sup.tlacZ/EphrinB2.sup.tlacZ mutant mice, antagonists
of EphrinB2 or antagonists of EphB4 will inhibit angiogenesis.
Agents which, are agonists of both EphrinB2 and EphB4 will promote
angiogenesis.
[0066] In another example, soluble agonists Which comprise the
extracellular domain of an Ephrin family ligand or the
extracellular domain of an Eph family receptor fused to the Fc
domain of human IgG can be produced. For example, an EphB4 or an
EphrinB2 hybrid protein in which the extracellular domain of the
membrane protein is fused to the Fe domain of human IgG can be used
(Wang, H. U. and D. J. Anderson, Neuron 18:383-396 (1997)). See,
for examples of methods Stein, E. et al., Genes and Dev. 12:667-678
(1998), regarding experiments on responses of cells to clustered
Ephrin-B1/Fc fusion proteins. Clustering of these hybrid molecules
with anti-human Fc antibodies generates soluble agonists:
Ephrin-derived "ligand-bodies" for Eph receptors, and conversely,
Eph-derived "receptor bodies" for Ephrins. Non-clustered forms of
these hybrid molecules can be used as antagonists.
[0067] A further application of isolated arterial endothelial cells
and isolated venous endothelial cells is the genetic alteration of
the isolated cells and the administration of these cells,
preferably intravenously, to the host mammal from which the cells
were isolated, or into another compatible host, where the cells can
be incorporated into a blood vessel of the appropriate type. In
this way, the effects of a genetic defect which is manifested in
arteries or in veins can he ameliorated. It has been demonstrated
that circulating endothelial cell progenitors can migrate to sites
of neovascularization and be incorporated into blood vessels
(Asahara et al., Science 275:964-967 (1997)).
[0068] The introduction of a gene (an endogenous gene that has been
altered, or a gene originally isolated from a different organism,
for example) into cells can be accomplished by any of several known
techniques, for example, by vector mediated gene transfer, as by
amphotropic retroviruses, calcium phosphate, or liposome fusion,
for example.
[0069] A gene intended to have an effect on arteries or veins in a
host mammal can he delivered to isolated artery cells or isolated
vein cells by the use of viral vectors comprising one or more
nucleic acid sequences encoding the gene of interest. Generally,
the nucleic acid sequence has been incorporated into the genuine of
the viral vector. In vitro, the viral vector containing the nucleic
acid sequences encoding the gene can be contacted with a cell and
infection can occur. The cell can then be used experimentally to
study, for example, the effect of the gene on growth of artery or
vein cells in vitro or the cells can be implanted into a patient
for therapeutic use. The cells to be altered by introduction or
substitution of a gene can be present in a biological sample
obtained from the patient and used in the treatment of disease, or
can be obtained from cell culture and used. to dissect
developmental pathways of arteries and veins in in vivo and in
vitro systems.
[0070] After contact with the viral vector comprising a nucleic
acid sequence encoding the gene of interest, the treated artery or
vein cells can be returned or readministered to a patient according
to methods known to those practiced in the art. Such a treatment
procedure is sometimes referred to as ex vivo treatment. Ex vivo
gene therapy has been described, for example, in Kasid, et al.,
Proc. Natl. Acad. Sci. USA 87:473 (1990); Rosenberg, et al., New
Engl. J. Med. 323:570 (1990); Williams, et al., Nature 3 10476
(1984); Dick, et al., Cell 42.71 (1985); Keller, et al., Nature
318:149 (1985) and Anderson, et al., U.S. Pat. No. 5,399,346
(1994).
[0071] Generally, viral vectors which can be used therapeutically
and experimentally are known in the art. Examples include the
vectors described by Srivastava, A., U.S. Pat. No. 5,252,479
(1993); Anderson, W. F., et al., U.S. Pat. No. 5,399,346 (1994);
Ausubel et al., "Current Protocols in Molecular Biology", John
Wiley & Sons, Inc, (1998). Suitable viral vectors for the
delivery of nucleic acids to cells include, for example,
replication defective retrovirus, adenovirus, parvovirus (e.g.,
adeno-associated viruses), and coronavirus. Examples of
retroviruses include avian leukosis-sarcoma, mammalian C-type,
B-type viruses, lentiviruses (Coffin, J. M., "Retroviridae: The
Viruses and Their Replication", In: Fundamental Virology, Third
Edition, B. N. Fields, et al., eds., Lippincott-Raven Publishers,
Philadelphia, Pa., (1996)). The mechanism of infectivity depends
upon the viral vector and target cell. For example, adenoviral
infectivity of HeLa cells occurs by binding to a viral surface
receptor, followed by receptor-mediated endocytosis and
extrachromasomal replication (Horwitz, M. S., "Adenoviruses" In:
Fundamental Virology, Third Edition, B. N. Fields, et al., eds.,
Lippincott-Raven Publishers, Philadelphia, Pa., (1996)).
[0072] The present invention is illustrated. by the following
examples, which are not intended to be limiting in any way.
EXAMPLES
Experimental Procedures
[0073] The following experimental procedures were used in the
examples which follow.
[0074] Targeted disruption of the EphrinB2 gene. A 200 base pair
probe starting from the ATG of the mouse EphrinB2 gene (Bennett, B.
D., et al., Proc. Natl. Acad. Sci. USA 92:1866-1870 (1.995)) was
used to screen a 129SVJ genomic library (Stratagene). Analysis of
several overlapping clones revealed that the first exon, including
the signal sequence, ends at 131 base pairs after the ATG. Further
phage analysis and library screens revealed that the rest of the
EphrinB2 gene was located at least 7 kb downstream from the first
exon. To construct a targeting vector (FIG. 1B), a 3 kb Xbal-NcoI
fragment whose 3' end terminated at the ATG was used as the 5' arm.
A 5.3 kb Tau-lacZ coding sequence (Mombaerts. P., et al., Cell
87:675-686 (1996)) was fused in frame after the ATG. The PGKneo
gene (Ma, Q., et al., Neuron 20:469-482 (1998)) was used to replace
a 2.8 kb intronic sequence 3' to the first exon. Finally, a 3.2 kb
downstream EcoRI-EcoRI fragment was used as the 3' arm. Normal (6
kb) and targeted (9 kb) loci are distinguished by HindIII digestion
when probed with a 1 kb genomic' fragment. Electroporation,
selection and blastocyst-injection of AB-1 ES cells were performed
essentially as described Ma, Q., et al. (Neuron 20:469-482 (1998)),
with the exception that FLAU-selection was omitted. ES cell
targeting efficiency via G418 selection was 1 out of 18 clones.
Germline transmission of the targeted EphrinB2 locus (FIG. 1C) in
heterzygous males was confirmed by Southern blotting of tail DNA of
adult mice, using a 1 kb HindIII-XbaI probe. Subsequent genotyping
was done by genomic PCR. Primers for Neo are
5'-AAGATGGATTGCACGCAGGTTCTC-3' (SEQ ID NO.: 1) and
5'-CCTGATGCTCTICGTCCAGATCAT-3' (SEQ ID NO.: 2). Primers for the
replaced intronic fragment are 5'-AGGACGGAGGACGTTGCCACTAAC-3' (SEQ
ID NO.: 3) and 5' -ACCACCAGTTCCGACGCGAAGGGA-3' (SEQ ID NO.: 4).
[0075] LacZ, PECAM-1, and histological staining. Embryos and yolk
sacs were removed between E7.5 and E10.0, fixed in cold 4%
paraformaldehyde/PBS for 10 minutes, rinsed twice with PBS, and
stained for 1 hour to overnight at 37.degree. C. in X-gal buffer
(1.3 mg/ml potassium ferrocyanide, 1 mg/ml potassium ferricyanide,
0.2% Triton X-100, 1 mM MgCl.sub.2, and 1 mg/ml X-gal
[5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside] in PBS, pH
7.2). LacZ stained embryos were post-fixed and photographed, or
sectioned on a cryostat after embedding in 15% sucrose and 7.5%
gelatin in PBS. Procedures for whole mount or section staining with
anti-PECAM-1 antibody (clone MEC 13.3, Pharmingen) were done
essentially as described Ma et al. (Neuron 20:469-482 (1998); Fong
et al., Nature 376:66-70 (1995)). Horseradish peroxidase-conjugated
secondary antibodies were used for all PECAM-1 stainings.
LacZ-stained yolk sacs were Sectioned in gelatin and then subjected
to hematoxylin counter-staining by standard procedures.
[0076] In situ hybridization. In situ hybridization, on frozen
sections was performed as previously described (Birren et al.
Development 119:597-610 (1993)). Whole-mount in situ hybridization
followed a protocol by Wilkinson, D. G., (Whole-mount in situ
hybridization of vertebrate embryos. pp. 75-83 In: In Situ
Hybridization: A Practical Approach (ed. D. G. Wilkinson) IRL
Press, Oxford :75-83 (1992). Bluescript vectors (Stratagene)
containing cDNAs for EphB2/Nuk and EphB4/Myk-1 were generated as
described Wang, H. U. and Anderson, D. J. (Neuron 18:383-396
(1997)).
Example 1
Targeted Mutagenesis of EphrinB2 in Mice
[0077] Targeted disruption of the EphrinB2 gene was achieved by
homologous recombination in embryonic stem cells. The targeting
strategy involved deleting the signal sequence and fusing a
tau-lacZ indicator gene in frame with the initiation codon. The
expression pattern of .beta.-galactosidase in heterozygous
(EphrinB2.sup.tlacZ/+) embryos was indistinguishable from that
previously reported for the endogenous gene. (Bennett, B. D. et al.
Proc. Natl. Acad. Sci. USA 92: 1866-1870 (1995); Bergemann, A. D.
et al. Mol. Cell. Bio. 1995:4921-4929 (1995); Wang, H. U. and
Anderson, D. J. Neuron 18:383-396 (1 997)). While prominent
expression was detected in the hindbrain and somites, lower levels
were observed in the aorta and heart as early as E8.25. Expression
in the yolk sac was first detected at E8.5. Heterozygous animals
appeared phenotypically normal. In homozygous embryos, growth
retardation was evident at E10 and lethality occurred with 100%
penetrance around E11. No expression of endogenous EphrinB2 mRNA
was detected by in situ hybridization, indicating that the mutation
is a null. Somite polarity, hindbrain segmentation, and the
metameric patterning of neural crest migration (in which EphrinB2
and related ligands have previously been implicated Xu, W. et al.
Development 121:4005-4016 (1995); Wang, H. U. and Anderson, D. J.
Neuron 18:383-396 (1997); Krull, C. E. et al, Curr. Biol. 7:571-580
(1997); Smith, A. et al. Curr. Biol. 7:561-570 (1997)) appeared
grossly normal in homozygous mutant embryos.
Example 2
Reciprocal Expression Pattern of EphrinB2 and EphB4 in Arteries and
Veins
[0078] The enlarged heart observed in dying mutant embryos prompted
examination of the expression of EphrinB2.sup.tlacZ in the vascular
system in detail. Expression was consistently observed in arteries
but not veins. In the yolk sac, for example, the posterior vessels
connected to the vitelline artery, but not the vitelline vein,
expressed the gene, as detected by lacZ staining In the trunk,
labeling was detected in the dorsal aorta, vitelline artery,
umbilical artery and its allantoic vascular plexus, but not the
umbilical, anterior and common cardinal veins (the umbilical vein
is labeled with anti-PECAM-1 antibody). In the head, labeling was
seen in branches of the internal carotid artery, but not in those
of the anterior cardinal vein. In situ hybridization with EphrinB2
cDNA probes confirmed that the selective expression of tau-lacZ in
arteries correctly reflected the pattern of expression of the
endogenous gene. Examination of the expression of the four EphB
family genes, as well as Eph A4/Sek1, which are receptors for
EphrinB2 Gale, N. W. et al., Neuron 17:9-19 (1996) revealed
complementary expression of EphB4 in veins but not arteries,
including the vitelline vein and its branches in anterior portion
of the yolk sac.
Example 3
Vasculogenesis Occurs Normally in EphrinB2 Mutant Embryos
[0079] The formation of the major vessels in the trunk was
unaffected by the lack of EphrinB2, as examined by lacZ and PECAM-1
double staining of 9 somite embryos. Expression of EphrinB2-lacZ
was seen in the dorsal aorta and vitelline artery, but not the
umbilical and posterior cardinal veins. The dorsal aorta, vitelline
artery, posterior cardinal and umbilical veins, for example,
formed, although some dilation and wrinkling of the vessel wall was
observed. Similarly the intersomitic vessels originating from the
dorsal aorta fowled at this stage. Between E8.5 and E9.0, the
primitive endocardium appeared only mildly perturbed in mutants,
while a pronounced disorganization was apparent at E10. Red blood
cells developed and circulated. normally up to E9.5 in both the
mutant yolk sac and embryo proper.
Example 4
Extensive Intercalation of Yolk Sac Arteries and Veins Revealed by
EphrinB2 Expression
[0080] In the yolk sac, the vitelline artery and its capillary
network occupy the posterior region, and the vitelline vein and its
capillaries the anterior region. At E8.5, a stage at which the
primary capillary plexus has formed but remodeling has not yet
occurred, asymmetric expression of EphrinB2-taulacZ in heterozygous
embryos was evident at the interface between the anterior and
posterior regions. Apparently homotypic remodeling of
.beta.-galactosidase.sup.+ arterial capillaries into larger,
branched trunks clearly segregated from. venous vessels was evident
between E9.0 and E9.5. At this stage, expression of the receptor
EphB4 was clearly visible on the vitelline veins but not arteries.
Thus arterial and venous endothelial capillaries are already
molecularly distinct following vasculogenesis and prior to
angiogenesis.
[0081] While textbook diagrams (Carlson, B. M. Patten's Foundations
of Embryology (1981)) of the yolk sac capillary plexus depict a
non-overlapping boundary between the arterial and venous capillary
beds, expression of EphrinB2-taulacZ allowed detection of a
previously-unrecognized extensive intercalation between arteries
and veins across the entire anterior-posterior extent of the yolk
sac; this was observed in the heterozygote, but not in the
homozygote. Double-labeling for PECAM and .beta.-galactosidase,
revealed that the interface between the arteries and veins occurs
between microvessel extensions that bridge larger vessels
interdigitating en passant.
Example 5
Disrupted Angiogenesis in the Yolk Sac of
EphrinB2.sup.tlacZ/EphrinB2.sup.tlacZ Embryos
[0082] Defects in yolk sac angiogenesis was were apparent by E9.0
and obvious at E9.5. There was an apparent block to remodeling at
the capillary plexus stage, for both arterial vessels as revealed
by .beta.-galactosidase staining and venous vessels in the anterior
region of the sac as revealed by PECAM staining. Thus, disruption
of the EphrinB2 ligand gene caused both a non-autonomous defect in
EphB4 receptor-expressing venous cells, and an autonomous defect in
the arteries themselves,
[0083] This defect was accompanied by a failure of intercalating
bi-directional growth of arteries and veins across the
antero-posterior extent of the yolk sac, so that an interface
between EphrinB2-expressing and non-expressing zones at the
midpoint of the sac was apparent. (However, small patches of lacZ
expression were occasionally visible within the anterior venous
plexus, suggesting that some arterial endothelial cells may have
become incorporated into venous capillaries.) These observations
imply a close relationship between the remodeling of the capillary
plexus into larger vessels and the intercalating growth of these
vessels. The large .beta.-galactosidase vitelline arteries as well
as vitelline veins present at the point of entry to the yolk sac of
the embryo-derived vasculature appeared unperturbed in the mutant,
however. This is consistent with the observation that the mutation
does not affect formation of the primary trunk vasculature. It also
argues that the yolk sac phenotype is due to a disruption of
intrinsic angiogenesis and is not secondary to a failure of
ingrowth of embryo-derived vessels.
[0084] Histological staining (hematoxylin) of sectioned yolk sacs
revealed an accumulation of elongated. support cells (mesenchymal
cells or pericytes) in close association with the endothelial
vessels at E10 and E10.5. In mutant yolk sacs, these support cells
appeared more rounded, suggesting a defect in their
differentiation. Moreover, in contrast to heterozygous yolk sacs,
where vessels of different diameters began to appear at E9.5 and
vessel diameter increased through E10.5, capillary diameter
appeared relatively uniform and did not increase with age in the
mutants. At E10.5, arteries appear dilated, as if fusion of vessels
occurred without encapsulation by support cells. The mutant
capillaries also failed to delaminate from the basal endodermal
layer.
Example 6
Absence of Internal Carotid Arterial Branches and Defective
Angiogenesis of Venous Capillaries in the Head of Mutant
Embryos
[0085] Similar to the yolk sac phenotype, the capillary bed of the
head appeared dilated in the mutant, and apparently arrested at the
primary plexus stage. Staining for .beta.-galactosidase revealed
that the anterior-most branches of the internal carotid artery
failed to develop in the mutant. Unlike the case in the yolk sac,
therefore, the malformed capillary beds must be entirely of venous
origin. However the anterior branches of the anterior cardinal vein
formed although they were slightly dilated. Taken together, these
data indicate that in the head, venous angiogenesis is blocked if
the normal interaction with arterial capillaries is prevented. The
angiogenic defects observed in the head and yolk sac are unlikely
to be secondary consequences of heart defects (see below), since
they are observed starting at E9.0 and the embryonic blood
circulation appears normal until E9.5.
Example 7
EphrinB2-Dependent Signaling Between Endocardial Cells is Required
for Myocardial Trabeculae Formation
[0086] Examination of ligand and receptor expression in wild-type
hearts revealed expression in the atrium of both EphrinB2 (as
detected by lacZ staining) and EphB4 (as detected by in situ
hybridization). Expression of both ligand and receptor was also
detected in the ventricle in the endocardial cells lining the
trabecular extensions of the myocardium. Double-labeling
experiments suggested that the ligand and receptor are expressed by
distinct but partially overlapping cell populations, although the
resolution of the method does not permit us to distinguish whether
this overlap reflects co-expression by the same cells, or a close
association of different cells. In any case, expression of EphrinB2
and EphB4 does not define complementary arterial (ventricular) and
venous (atrial) compartments of the heart, unlike the extra-cardiac
vasculature.
[0087] Heart defects commenced at E9.5 and were apparent in mutant
embryos at El 0 both morphologically and by wholemount PECAM-1
staining. Sections revealed an absence of myocardial trabecular
extensions, although strands of EphrinB2-expressing endocardial
cells were still visible. Thus, mutation of the ligand-encoding
gene caused a non-autonomous defect in myocardial cells, similar to
the effect of a mutation in the neuregulin-1 gene. (Meyer, D. and
Birchmeier, C. Nature 378:386-390 (1995)) Paradoxically, however,
in this case the EphB4 receptor is expressed not on myocardial
cells, as is the case for the neuregulin-1 receptors erbB2 and
erbB4 (Lee et al., Nature 378:394-398 (1995); Gassmann, et al.,
Nature 378:390-394 (1995), but rather on endocardial cells.
Expression of any of the other receptors for Ephrin B family
ligands (Eph B1, B2, B3 and A4) was detected in this tissue. This
suggests that in the heart, ligand-receptor interactions among
endothelial cells may in turn affect interactions with smooth
muscle cells.
Example 8
EphrinB2 is Required for Vascularization of the Neural Tube
[0088] In EphrinB2.sup.tlacZ/EphrinB2.sup.tlacZ embryos capillary
ingrowth into the neural tube failed to occur. Instead,
EphrinB2-expressing endothelial cells remained associated with the
exterior surface of the developing spinal cord. Comparison
.beta.-galactosidase to pan-endothelial PECAM-1 and EphB4
expression provided no evidence of a separate, venous capillary
network expressing EphB4 in the CNS at this early stage (E9-E10).
Rather, expression of a different EphrinB2 receptor, Eph B2, was
seen in the neural tube as previously reported Henkemeyer, et al.,
Oncogene 9:1001-1014 (1994), where no gross morphological or
patterning defects were detectable. In this case, therefore, the
mutation does not appear to cause a non-autonomous phenotype in
receptor-expressing cells, rather only an autonomous effect on
ligand-expressing cells.
Example 9
EphrinB2 is Artery-Specific in Adult Tissues
[0089] To determine whether ephrin-B2 is expressed in adult tissues
in an artery-specific mariner, we performed histochemical staining
for .beta.-galactosidase on ephrin-B2.sup.taulacZ/+heterozygous
mice. Antibody staining for PECAM-1, pan-endothelial marker, was
performed on the same or on adjacent sections to reveal
non-arterial vessel (i.e., veins). Ephrin-B2 is expressed
throughout the adult mouse in an artery-specific manner, in tissues
including the heart, leg muscle, kidney, liver and fat. Expression
was detected in vessels of all diameters, including large arteries,
arterioles and the smallest-diameter capillaries. It had been
previously assumed that capillaries, by definition have neither
arterial nor venous identity. These results show that this is not
the case, and that arterial identity extends into the capillary
beds.
[0090] Sections through adult arteries were double labeled by
histochemical staining fox .beta.-galactosidase (lacZ) to reveal
ephrin-B2-taulacZ expression, and with antibody to PECAM-1 as a
pan-endothelial marker. LacZ (blue from X-gal) staining revealed
ephrinB2 expression, in dorsal aorta but not in inferior vena cava,
in femoral artery next to the leg bone, but not in femoral vein,
and in coronary epicardial artery, but not in coronary vein.
[0091] Similar staining of other sections revealed the presence of
EphrinB2 in kidney arteriole, liver arteriole and small muscle
arteriole, as well as arterial capillary. Kidney venule, hepatic
vein, and muscle veins were lacZ-negative but PECAM-1 positive.
[0092] Gut fat was stained for lacZ (.beta.-galactosidase) and
labeled with PECAM-1 to reveal venous vessels as well as arterial
vessels. EphrinB2 is expressed by arterioles and arterial
capillaries and in arteriole but not in PECAM-1 positive venule. A
further section showed that EphrinB2 is expressed by arterial
capillaries surrounded by PECAM-1 positive non-arterial
capillaries.
Example 10
Ephrin-B2 is Expressed During Tumor Angiogenesis
[0093] It had been assumed that tumor vessels sprout from the
post-capillary venules. To address the question of whether EphrinB2
is expressed during tumor angiogenesis, Lewis Lung Carcinomas were
implanted subcutaneously in the dorsal region of
ephrin-B2.sup.taulacZ/+heterozygous females. After one week, the
tumors were removed and processed for .beta.-galactosidase
histochemistry in whole mounts. The results indicate clearly that
ephrin-B2 is in fact expressed by tumor vessels. This was confirmed
by double-staining of sections cut through such tumors, for
.beta.-galactosidase and PECAM-1.
[0094] EphrinB2 expression by tumor capillaries. Lewis Lung
Carcinoma (LLC) cells were implanted subcutaneously in the dorsal
region of EphrinB2-taulacZ heterozygous females. After one week,
tumors were removed for staining. EphrinB2 positive arterial
capillaries were observed in peripheral tumor tissue. Double
labeling with PECAM-1 colocalized the EphrinB2 lacZ (staining blue)
and PECAM-1 (staining brown) signals in arterial capillaries, and
reveals non-arterial capillaries labeled by PECAM-1 only.
Example 11
Screen for Clones Containing Artery-Specific Genes
[0095] A summary of the screening procedures used is provided in
Appendix I. Briefly, endothelial cells were isolated from
dissociated embryonic yolk sac, vitelline arteries or vitelline
veins using positive selection with antibody to PECAM-1 (magnetic
bead or FACS). cDNA was synthesized. from lysates of either single
cells or small numbers (ca. 200) of cells, and amplified by PCR. To
confirm that the cDNAs were from arterial or venous endothelial
cells, the cDNA synthesized from each cell prep was Southern
blotted and hybridized in quadruplicate with a series of probes,
including tubulin (ubiquitously expressed) the pan-endothelial
probes Flk1 and Flt1, and the arterial-specific probe ephrin-B2.
cDNAs that contained Flk1, Flt1 and ephrin-B2 sequences were
considered arterial, while those that contained the pan-endothelial
markers but not ephrin-B2 were considered venous. Thus the use of
ephrin-B2 probes in this procedure was essential to confirm the
arterial vs. venous nature of the cDNAs synthesized.
[0096] To isolate additional arterial-specific genes from these
cDNAs, they were cloned into a phase lambda vector to generate cDNA
libraries. Plaques from these libraries were then screened in
duplicate filter lifts with arterial- or venous-specific cDNA
probes made from either single cells or pools of cells. Plaques
exhibiting differential hybridization to the arterial vs. venous
probes were isolated, and the inserts were amplified using T3-T7
primers, and re-analyzed by cDNA Southern blotting. Two different
pairs of arterial-venous endothelial cell cDNA probes (vitelline
artery-vein cells and single yolk sac arterial and venous
endothelial. cells) were used in the Southern blot. Most of the
clones were strongly expressed in arterial cells, and expressed
weakly or not detectably by venous cells. The initial screen was
designed to isolate additional arterial-specific genes. Twelve
candidate arterial-specific clones were isolated using the
single-cell probes, while one clone was isolated using the pooled
probe. The arterial-specific expression of these genes in vivo can
be confirmed by in situ hybridization experiments. Methods such as
these can be applied to arterial endothelial cells or vein-specific
cells.
[0097] These data show that it will be possible to isolate novel
arterial- or venous-specific genes from single endothelial cells.
Such vessel type-restricted genes may provide insights into the
physiological differences between arterial, and venous endothelial
cells. Methods such as those described herein can also identify
genes involved in the etiology of arterial and venous specific
diseases, such as arterial hypertension, atherosclerosis, deep
venous thrombosis, and certain types of venous malformations. They
can also provide candidate genes for human genetic disorders of the
circulatory system. The gene products of such genes can then serve
as novel drug targets.
Appendix I: Single Cell PCR, 3' cDNA Library Construction And
Differential Screening Procedure to Isolate Novel Arterial- or
Venous-Specific Genes [0098] 1. Dissection of vitelline arteries
and vitelline veins from E12.5 to E14.5 yolk sacs, based on
morphological criteria. [0099] 2. Dissociation of yolk sacs in
collagenase solution (5 mg/ml) at 37.degree. C. for 45'. [0100] 3.
Isolate single or small groups of endothelial cells (ECs) by one of
the following methods. [0101] a) Magnetic bead-based separation
using PECAM-1 as primary antibody. [0102] b) FACS purification
using PECAM-1-FITC primary antibody. [0103] c) GFP fluorescence
from tie2-GFP transgenic mouse for endothelial cell identification,
followed by microcapillary mouth-pipeting. [0104] 4. Lyse the
single cells in PCR tubes at 65.degree. C. for 1'. [0105] 5. Keep
1-2' at room temperature to allow the oligo-T to anneal to RNA.
[0106] 6. Reverse transcription using AMV and MMLV enzyme mixture,
at 37.degree. C. for 15'. [0107] 7. Poly-A tailing with terminal
transferase and dATP, at 37.degree. C. for 15'. [0108] 8. PCR
reaction setup: [0109] 100 .mu.l reaction, 5-fold normal level of
dNTP mix, high [0110] concentration of Taq. [0111] Using a single
PCR primer with 24 (T)s at 3' end for symmetrical amplification.
[0112] 9. cDNA. Southern blotting with endothelial specific and
arterial/venous specific 3' probes on the amplified cDNAs for each
cell prep. [0113] 10. Choose a few good cells that give strong
signals for the appropriate probes. [0114] Isolate cDNAs from 500
bp to 2 kb on agarose gel. [0115] 11. Precipitate and quantify
cDNA. [0116] 12. Ligate into lambda ZAPII (Stratagene; LaJolla,
Calif.) phage arms for cDNA libraries. [0117] 13. Plate the library
at very low density: 1000 pfu/plate. Take duplicate filter lifts.
[0118] 14. Screen duplicate filters with probes made from vitelline
artery cells and vitelline vein cells. [0119] 15. Pick
differentially expressed phage clones. Reverse (cDNA) Southern blot
confirmation of phage inserts by probing with probes made from
vitelline artery cells and vitelline vein cells. [0120] 16. In situ
hybridization to examine the expression patterns of cDNA
fragments.
Example 12
Generation of Monoclonal Antibodies Against the Extracellular
Domain of Ephrin-B2
[0121] While polyclonal rabbit antibodies to fragments of ephrin-B2
expressed in bacteria had previously been reported, such antibodies
are typically not reactive with native forms of the protein on the
cell surface, and therefore are not useful for many applications
including cell sorting, functional inhibition and drug-targeting.
To generate antibodies with more desirable binding properties, we
expressed the extracellular domain of ephrin-B2 as a
glycophosphatidylinositol (GPI)-linked form on Chinese Hamster
Ovary (CHO) cells. Hamsters were immunized with these cells,
hybridomas prepared by fusion with mouse myeloma cells, and
supernatants screened on COS cells expressing ephrin-B2-GPI.
[0122] Supernatant from clone #6E3 bound well to ephrinB2 on live
COS cells. The ephrin-B2-GPI COS cell lines are a pooled population
of G418-selected cells, with 30% of the cells being positive for
ephrinB2. Control (untransfected) COS cells were negative when
stained with the same antibody.
Example 13
Some Anti-Ephrin-B2 Antibodies Block the EphB2-EphrinB2 Binding
Interaction
[0123] In cases where the antibodies also block function (i.e.,
inhibit binding of ephrin-B2 to its receptors), they can be used as
potential anti-angiogenic agents. To demonstrate an assay to
identify such function-inhibiting antibodies, we screened 12
hamster anti-EphrinB2 hybridoma supernatants for their ability to
reduce the binding of GPI-ephrin-B2 (expressed on COS cells) to a
soluble EphB2-Fc fusion protein. Binding was detected by
.sup.125I-labeled goat anti-human Fc antibody. Pre-incubation of
cells with various supernatants revealed that the majority of the
antibodies have no blocking effects on subsequent receptor-ligand
binding (control as 100%), even though these supernatants all
contained antibodies that bound to ephrin-B2-GPI. One of the
antibodies produced a 40% reduction in EphB2-Fc binding to the
ephrin-B2-GPI COS cells (FIG. 2).
Equivalents
[0124] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims. Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the claims.
Sequence CWU 1
1
4124DNAArtificial SequencePrimer 1aagatggatt gcacgcaggt tctc
24224DNAArtificial SequencePrimer 2cctgatgctc ttcgtccaga tcat
24324DNAArtificial SequencePrimer 3aggacggagg acgttgccac taac
24424DNAArtificial SequencePrimer 4accaccagtt ccgacgcgaa ggga
24
* * * * *
References