U.S. patent application number 11/455506 was filed with the patent office on 2006-11-16 for extracellular matrix signalling molecules.
Invention is credited to Lester F. Lau.
Application Number | 20060257965 11/455506 |
Document ID | / |
Family ID | 21762734 |
Filed Date | 2006-11-16 |
United States Patent
Application |
20060257965 |
Kind Code |
A1 |
Lau; Lester F. |
November 16, 2006 |
Extracellular matrix signalling molecules
Abstract
Polynucleotides encoding mammalian ECM signalling molecules
affecting the cell adhesion, migration, and proliferation
activities characterizing such complex biological processes as
angiogenesis, chondrogenesis, and oncogenesis, are provided. The
polynucleotide compositions include DNAs and RNAs comprising part,
or all, of an ECM signalling molecule coding sequence, or
biological equivalents. Polypeptide compositions are also provided.
The polypeptide compositions comprise mammalian ECM signalling
molecules, peptide fragments, inhibitory peptides capable of
interacting with receptors for ECM signalling molecules, and
antibody products recognizing Cyr61. Also provided are methods for
producing mammalian ECM signalling molecules. Further provided are
methods for using mammalian ECM signalling molecules to screen for,
and/or modulate, disorders associated with angiogenesis,
chondrogenesis, and oncogenesis: ex vivo methods for using
mammalian ECM signalling molecules to prepare blood products are
also provided.
Inventors: |
Lau; Lester F.; (Chicago,
IL) |
Correspondence
Address: |
HOWREY LLP
C/O IP DOCKETING DEPARTMENT
2941 FAIRVIEW PARK DR, SUITE 200
FALLS CHURCH
VA
22042-2924
US
|
Family ID: |
21762734 |
Appl. No.: |
11/455506 |
Filed: |
June 19, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10053753 |
Jan 22, 2002 |
7064185 |
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11455506 |
Jun 19, 2006 |
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09142569 |
Apr 2, 1999 |
6413735 |
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PCT/US97/04193 |
Mar 14, 1997 |
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10053753 |
Jan 22, 2002 |
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60013958 |
Mar 15, 1996 |
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Current U.S.
Class: |
435/29 ; 435/189;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 38/00 20130101; C07K 2317/73 20130101; C12N 2799/026 20130101;
A61P 43/00 20180101; A61P 17/00 20180101; C07K 14/475 20130101;
C07K 16/18 20130101; A61P 11/00 20180101; A01K 2217/05 20130101;
A61P 9/00 20180101; C07K 2317/76 20130101; A61P 19/00 20180101;
A61P 17/02 20180101 |
Class at
Publication: |
435/029 ;
435/069.1; 435/189; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C12N 9/02 20060101 C12N009/02 |
Claims
1-64. (canceled)
65. A humanized monoclonal antibody that binds specifically to
native human Cyr61.
Description
[0001] This application claims the benefit of the filing date of
U.S. provisional patent application Ser. No. 60/013,958, filed Mar.
15, 1996.
FIELD OF THE INVENTION
[0002] The present invention is directed to materials and methods
involving extracellular matrix signalling molecules--polypeptides
involved in cellular responses to growth factors. More
particularly, the invention is directed to Cyr61-, Fisp12-, and
CTGF-related polynucleotides, polypeptides, compositions thereof,
methods of purifying these polypeptides, and methods of using these
polypeptides.
BACKGROUND OF THE INVENTION
[0003] The growth of mammalian cells is tightly regulated by
polypeptide growth factors. In the adult animal, most cells are
metabolically active but are quiescent with regard to cell
division. Under certain conditions, these cells can be stimulated
to reenter the cell cycle and divide. As quiescent cells reenter
the active growth and division phases of the cell cycle, a number
of specific genes, the immediate early genes, are rapidly
activated. Reentry to the active cell cycle is by necessity tightly
regulated, since a breakdown of this control can result in
uncontrolled growth, frequently recognized as cancer. Controlled
reentry of particular cells into the growth phase is essential for
such biological processes as angiogenesis (e.g., blood vessel
growth and repair), chondrogenesis (e.g., skeletal development and
prosthesis integration), oncogenesis (e.g., cancer cell metastasis
and tumor neovascularization), and other growth-requiring
processes.
[0004] Angiogenesis, the formation of new blood vessels from the
endothelial cells of preexisting blood vessels, is a complex
process which involves a changing profile of endothelial cell gene
expression, associated with cell migration, proliferation, and
differentiation. Angiogenesis begins with localized breakdown of
the basement membrane of the parent vessel. In vivo, basement
membranes (primarily composed of laminin, collagen type IV,
nidogen/entactin, and proteoglycan) support the endothelial cells
and provide a barrier separating these cells from the underlying
stroma. The basement membrane also affects a variety of biological
activities including cell adhesion, migration, and growth during
development and differentiation.
[0005] Following breakdown of the basement membrane, endothelial
cells migrate away from the parent vessel into the interstitial
extracellular matrix (ECM), at least partially due to
chemoattractant gradients. The migrating endothelial cells form a
capillary sprout, which elongates. This elongation is the result of
migration and proliferation of cells in the sprout. Cells located
in the leading capillary tip migrate toward the angiogenic
stimulus, but neither synthesize DNA nor divide. Meanwhile, behind
these leading tip cells, other endothelial cells undergo rapid
proliferation to ensure an adequate supply of endothelial cells for
formation of the new vessel. Capillary sprouts then branch at their
tips, the branches anastomose or join with one another to form a
lumen, the basement membrane is reconstituted, and a vascular
connection is established leading to blood flow.
[0006] Alterations in at least three endothelial cell functions
occur during angiogenesis: 1) modulations of interactions with the
ECM, which require alterations of cell-matrix contacts and the
production of matrix-degrading proteolytic enzymes; 2) an initial
increase and subsequent decrease in endothelial cell migration,
effecting cell translocation towards an angiogenic stimulus; and 3)
a transient increase in cell proliferation, providing cells for the
growing and elongating vessel, with a subsequent return to the
quiescent cell state once the vessel is formed. These three
functions are realized by adhesive, chemotactic, and mitogenic
interactions or responses, respectively. Therefore, control of
angiogenesis requires intervention in three distinct cellular
activities: 1) cell adhesion, 2) cell migration, and 3) cell
proliferation. Another biological process involving a similar
complex array of cellular activities is chondrogenesis.
[0007] Chondrogenesis is the cellular process responsible for
skeletal organization, including the development of bone and
cartilage. Chondrogenesis, like angiogenesis, involves the
controlled reentry of quiescent cells into the growth phase of the
cell cycle. The growth phase transition is associated with altered
cell adhesion characteristics, changed patterns of cell migration,
and transiently increased cell proliferation. Chondrogenesis
involves the initial development of chondrogenic capacity (i.e.,
the proto-differentiated state) by primitive undifferentiated
mesenchyme cells. This stage involves the production of
chondrocyte-specific markers without the ability to produce a
typical cartilage ECM. Subsequently, the cells develop the capacity
to produce a cartilage-specific ECM as they differentiate into
chondrocytes. Langille, Microscop. Res. & Tech. 28:455-469
(1994). Chondrocyte migration, adhesion, and proliferation then
contribute to the development of bony, and cartilaginous, skeleton.
Abnormal elaboration of the programmed development of cells
participating in the process of chondrogenesis results in skeletal
defects presenting problems that range from cosmetic concerns to
life-threatening disorders.
[0008] Like angiogeniesis and chondrogenesis, oncogenesis is
characterized by changes in cell adhesion, migration, and
proliferation. Metastasizing cancer cells exhibit altered adhesion
and migration properties. Establishment of tumorous masses requires
increased cell proliferation and the elaboration of the cellular
properties characteristic of angiogenesis during the
neovascularization of tumors.
[0009] Abnormal progression of angiogenesis or chondrogenesis, as
well as mere progression of oncogenesis, substantially impairs the
quality of life for afflicted individuals and adds to modern health
care costs. The features common to these complex biological
processes, comprising altered cell adhesion, migration, and
proliferation, suggest that agents capable of influencing all three
of these cellular activities would be effective in screening for,
and modulating, the aforementioned complex biological processes.
Although the art is aware of agents that influence individual
cellular activities. e.g., integrins and selectins (cell adhesion),
chemokines (cell migration), and a variety of growth factors or
cytokines (cell proliferation), until recently no agent has been
identified that exerts an influence over all three cellular
activities in humans.
[0010] Murine Cyr61 (CYsteine-Rich protein) is a protein expressed
in actively growing and dividing cells that may influence each of
these three cellular activities. RNase protection analyses have
shown that the gene encoding murine Cyr61, murine cyr61, is
transcribed in the developing mouse embryo. O'Brien et al., Cell
Growth & Diff 3:645-654 (1992). In situ hybridization analysis
showed that expression of cyr61 during mouse embryogenesis is
closely correlated with the differentiation of mesenchymal cells,
derived from ectoderm and mesoderm, into chondrocytes. In addition,
cyr61 is expressed in the vessel walls of the developing
circulatory system. These observations indicate that murine cyr61
is expressed during cell proliferation and differentiation, which
are characteristics of expression of genes involved in regulatory
cascades that control the cell growth cycle.
[0011] Further characterization of the Cyr61 polypeptide has been
hampered by an inability to purify useful quantities of the
protein. Efforts to purify Cyr61 in quantity by overexpression from
either eukaryotic or prokaryotic cells typically fail. Yang,
University of Illinois at Chicago, Ph.D. Thesis (1993). One problem
associated with attempting to obtain useful quantities of Cyr61 is
the reduction in mammalian growth rates induced by overexpression
of Cyr61. Another problem with Cyr61 purification is that the
cysteine-rich polypeptide, when expressed in bacterial cells using
recombinant DNA techniques, is often found in insoluble protein
masses. Nevertheless. Cyr61 has been characterized as a polypeptide
of 349 amino acids, containing 39 cysteine residues, a hydrophobic
putative N-terminal signal sequence, and potential N-linked
glycosylation sites (Asn.sub.28 and Asn.sub.225). U.S. Pat. No.
5,408,040 at column 3, lines 41-54, Grotendorst et al.,
incorporated herein by reference (the '040 patent).
[0012] Recently, proteins related to Cyr61 have been characterized.
For example, a human protein, Connective Tissue Growth Factor
(CTGF), has been identified. (See '040 patent). CTGF is expressed
in actively growing cells such as fibroblasts and endothelial cells
('040 patent, at column 5, lines 62-64), an expression pattern
shared by Cyr61. In terms of function, CTGF has been described as a
protein growth factor because its primary biological activity has
been alleged to be its mitogenicity ('040 patent, at column 2,
lines 25-27 and 53-55). In addition, CTGF reportedly exhibits
chemotactic activity. '040 patent, at column 2, lines 56-59. In
terms of structure, the polynucleotide sequence encoding CTGF, and
the amino acid sequence of CTGF, have been published. '040 patent,
SEQ ID NO:7 and SEQ ID NO:8, respectively.
[0013] Another apparently related protein is the mouse protein
Fisp12 (FIbroblast Secreted Protein). Fisp12 has been subjected to
amino acid sequence analysis, revealing a primary structure that is
rich in cysteines. Ryseck et al., Cell Growth & Diff. 2:225-233
(1991), incorporated herein by reference. The protein also
possesses a hydrophobic N-terminal sequence suggestive of the
signal sequence characteristic of secreted proteins.
[0014] Sequence analyses involving Cyr61, Fisp12, CTGF, and other
proteins, have contributed to the identification of a family of
cysteine-rich secreted proteins. Members of the family share
similar primary structures encoded by genes exhibiting similar
sequences. Each of the proteins in this emerging family is further
characterized by the presence of a hydrophobic N-terminal signal
sequence and 38 cysteine residues in the secreted forms of the
proteins. Members of the family identified to date include the
aforementioned Cyr61 (human and mouse), Fisp12 (mouse), and CTGF
(the human ortholog of Fisp12), as well as CEF10 (chicken), and Nov
(avian).
[0015] One of several applications for a purified protein able to
affect cell adhesion, migration, and proliferation properties
involves the development of stable, long term ex vivo hematopoietic
stem cell cultures. Patients subjected to high-dose chemotherapy
have suppressed hematopoiesis: expansion of stem cells, their
maturation into various hematopoietic lineages, and mobilization of
mature cells into circulating blood routinely take many weeks to
complete. For such patients, and others who need hematopoietic cell
transplantation, introduction into those patients of autologous
stem cells that have been manipulated and expanded in culture is
advantageous. Such hematopoietic stem cells (HSC) express the CD34
stem cell antigen, but do not express lineage commitment antigens.
These cells can eventually give rise to all blood cell lineages
(e.g., erythrocytes, lymphocytes, and myelocytes).
[0016] Hematopoietic progenitor cells that can initiate and sustain
long term cultures (i.e., long term culture system-initiating cells
or LTC-IC) represent a primitive population of stem cells. The
frequency of LTC-IC has been estimated at only 1-2 per 10.sup.4
cells in normal human marrow and only about 1 per 50-100 cells in a
highly purified CD34.sup.+ subpopulation. Thus, it would be useful
to have methods and systems for long term cell culture that
maintain and expand primitive, pluripotent human HSC to be used for
repopulation of the hematopoietic system in vivo.
[0017] Cell culture models of hematopoiesis have revealed a
multitude of cytokines that appear to play a role in the
hematopoietic process, including various colony stimulating
factors, interleukins, stem cell factor, and the c-kit ligand.
However, in ex vivo cultures, different combinations of these
cytokines favor expansion of different sets of committed
progenitors. For example, a factor in cord blood plasma enhanced
expansion of granulocyte-erythroid-macrophage-megakaryocyte colony
forming unit (CFU-GEMM) progenitors, but expansion in these
cultures favored the more mature subsets of cells. Therefore, it
has been difficult to establish a culture system that mimics in
vivo hematopoiesis.
[0018] An HSC culture system should maintain and expand a large
number of multi- or pluripotent stem cells capable of both long
term repopulation and eventual lineage commitment under appropriate
induction. However, in most ex vivo culture systems, the fraction
of the cell population comprised of LTC-IC decreases steadily with
continued culturing, often declining to 20% of their initial level
after several weeks, as the culture becomes populated by more
mature subsets of hematopoietic progenitor cells that are no longer
pluripotent. Moreover, the proliferative capacity exhibited by
individual LTC-IC may vary extensively. Thus, a need exists in the
art for HSC culture systems comprising biological agents that
maintain or promote the pluripotent potential of cells such as
LTC-IC cells. In addition to a role in developing ex vivo HSC
cultures, biological agents affecting cell adhesion, migration, and
proliferation are useful in a variety of other contexts.
[0019] Proteins that potentiate the activity of mitogens but have
no mitogenic activity themselves may play important roles as
signalling molecules in such processes as hematopoiesis. Moreover,
these signalling proteins could also serve as probes in the search
for additional mitogens, many of which have not been identified or
characterized. Several biological factors have been shown to
potentiate the mitogenic activity of other factors, without being
mitogenic themselves. Some of these potentiators are associated
with the cell surface and/or extracellular matrix. Included in this
group are a secreted basic Fibroblast Growth Factor-binding protein
(bFGF-binding protein), the basal lamina protein perlecan, and the
Human Immunodeficiency Virus-1 TAT protein, each protein being able
to promote bFGF-induced cell proliferation and angiogenesis. Also
included in this group of mitogen potentiators are thrombospondin,
capable of activating a latent form of Transforming Growth
Factor-.beta., and an unidentified secreted growth-potentiating
factor from vascular smooth muscle cells (Nakano et al., J. Biol.
Chem. 270:5702-5705 [1995]), the latter factor being required for
efficient activation of Epidermal Growth Factor- or
thrombin-induced DNA synthesis. Further, the B cell stimulatory
factor-1/interleukin-4, a T cell product with no demonstrable
mitogenic activity, is able to 1) enhance the proliferative
response of granulocyte-macrophage progenitors to
granulocyte-colony stimulating factor, 2) enhance the proliferative
response of erythroid progenitors to erythropoietin, and 3)
together with erythropoietin, induce colony formation by
multipotent progenitor cells. Similarly, interleukin-7 enhanced
stem cell factor-induced colony formation by primitive murine bone
marrow progenitors, although interleukin-7 had no proliferative
effect by itself. In addition, lymphocyte growth enhancing factor
(LGEF) was found to enhance mitogen-stimulated human peripheral
blood lymphocyte (PBL) or purified T cell proliferation in a
dose-dependent fashion. LGEF alone did not stimulate PBL or T cell
proliferation.
[0020] Therefore, a need continues to exist for biological agents
capable of exerting a concerted and coordinated influence on one or
more of the particularized functions collectively characterizing
such complex biological processes as angiogenesis, chondrogenesis,
and oncogenesis. In addition, a need persists in the art for agents
contributing to the reproduction of these in vivo processes in an
en vitro environment, e.g., the development of HSC cultures.
Further, there continues to be a need for tools to search for the
remaining biological components of these complex processes, e.g.,
mitogen probes, the absence of which impedes efforts to
advantageously modulate and thereby control such processes.
SUMMARY OF THE INVENTION
[0021] The present invention provides extracellular matrix (ECM)
signalling molecule-related materials and methods. In particular,
the present invention is directed to polynucleotides encoding ECM
signalling molecules and fragments or analogs thereof, ECM
signalling molecule-related polypeptides and fragments, analogs,
and derivatives thereof, methods of producing ECM signalling
molecules, and methods of using ECM signalling molecules.
[0022] One aspect of the present invention relates to a purified
and isolated polypeptide comprising an ECM signalling molecule. The
polypeptides according to the invention retain at least one
biological activity of an ECM signalling molecule, such as the
ability to stimulate cell adhesion, cell migration, or cell
proliferation; the ability to modulate angiogenesis,
chondrogenesis, or oncogenesis; immunogenicity or the ability to
elicit an immune response; and the ability to bind to polypeptides
having specific binding sites for ECM signalling molecules,
including antibodies and integrins. The polypeptides may be native
or recombinant molecules. Further, the invention comprehends
full-length ECM signalling molecules, and fragments thereof. In
addition, the polypeptides of the invention may be underivatized,
or derivatized in conformity with a native or non-native
derivatization pattern. The invention further extends to
polypeptides having a native or naturally occurring amino acid
sequence, and variants (i.e., polypeptides having different amino
acid sequences), analogs (i.e., polypeptides having a non-standard
amino acid or other structural variation from the conventional set
of amino acids) and homologs (i.e., polypeptides sharing a common
evolutionary ancestor with another polypeptide) thereof.
Polypeptides that are covalently linked to other compounds, such as
polyethylene glycol, or other proteins or peptides, i.e. fusion
proteins, are contemplated by the invention.
[0023] Exemplary ECM signaling molecules include mammalian Cyr61,
Fisp12, and CTGF polypeptides. Beyond ECM signalling molecules, the
invention includes polypeptides that specifically bind an ECM
signalling molecule of the invention, such as the aforementioned
antibody products. A wide variety of antibody products fall within
the scope of the invention, including polyclonal and monoclonal
antibodies, antibody fragments, chimeric antibodies, CDR-grafted
antibodies, "humanized" antibodies, and other antibody forms known
in the art. Other molecules such as peptides, carbohydrates or
lipids designed to bind to an active site of the ECM molecules
thereby inhibiting their activities are also contemplated by the
invention. However molecules such as peptides that enhance or
potentiate the activities of ECM molecule are also within the scope
of the invention. The invention further extends to a pharmaceutical
composition comprising a biologically effective amount of a
polypeptide and a pharmaceutically acceptable adjuvant, diluent or
carrier, according to the invention. A "biologically effective
amount" of the biomaterial is an amount that is sufficient to
result in a detectable response in the biological sample when
compared to a control lacking the biomaterial.
[0024] Another aspect of the invention relates to a purified and
isolated polynucleotide comprising a sequence that encodes a
polypeptide of the invention. A polynucleotide according to the
invention may be DNA or RNA, single- or double-stranded, and may be
may purified and isolated from a native source, or produced using
synthetic or recombinant techniques known in the art. The invention
also extends to polynucleotides encoding fragments, analogs (i.e.,
polynucleotides having a non-standard nucleotide), homologs (i.e.,
polynucleotides having a common evolutionary ancestor with another
polynucleotide), variants (i.e., polynucleotides differing in
nucleotide sequence), and derivatives (i.e., polynucleotides
differing in a structural manner that does not involve the primary
nucleotide sequence) of ECM molecules. Vectors comprising a
polynucleotide according to the invention are also contemplated. In
addition, the invention comprehends host cells transformed or
transfected with a polynucleotide or vector of the invention.
[0025] Other aspects of the invention relate to methods for making
or using the polypeptides and/or polynucleotides of the invention.
A method for making a polypeptide according to the invention
comprises expressing a polynucleotide encoding a polypeptide
according to the present invention in a suitable host cell and
purifying the polypeptide. Other methods for making a polypeptide
of the invention use techniques that are known in the art, such as
the isolation and purification of native polypeptides or the use of
synthetic techniques for polypeptide production. In particular, a
method of purifying an ECM signalling molecule such as human Cyr61
comprises the steps of identifying a source containing human Cyr61,
exposing the source to a human Cyr61-specific biomolecule that
binds Cyr61 such as an anti-human Cyr61 antibody, and eluting the
human Cyr61 from the antibody or other biomolecule, thereby
purifying the human Cyr61.
[0026] Another aspect of the invention is a method of screening for
a modulator of angiogenesis comprising the steps of: (a) contacting
a first biological sample capable of undergoing angiogenesis with a
biologically effective (i.e., angiogenically effective) amount of
an ECM signalling molecule-related biomaterial and a suspected
modulator (inhibitor or potentiator); (b) separately contacting a
second biological sample with a biologically effective amount of an
ECM signalling molecule-related biomaterial, thereby providing a
control; (c) measuring the level of angiogenesis resulting from
step (a) and from step (b); and (d) comparing the levels of
angiogenesis measured in step (c), whereby a modulator of
angiogenesis is identified by its ability to alter the level of
angiogenesis when compared to the control of step (b). The
modulator may be either a potentiator or inhibitor of angiogenesis
and the ECM signalling molecule-related biomaterial includes, but
is not limited to, Cyr61, and fragments, variants, homologs,
analogs, derivatives, and antibodies thereof.
[0027] The invention also extends to a method of screening for a
modulator of angiogenesis comprising the steps of: (a) preparing a
first implant comprising Cyr61 and a second implant comprising
Cyr61 and a suspected modulator of Cyr61 angiogenesis; (b)
implanting the first implant in a first cornea of a test animal and
the second implant in a second cornea of the test animal; (c)
measuring the development of blood vessels in the first and second
corneas; and (d) comparing the levels of blood vessel development
measured in step (c), whereby a modulator of angiogenesis is
identified by its ability to alter the level of blood vessel
development in the first cornea when compared to the blood vessel
development in the second cornea.
[0028] Another aspect of the invention relates to a method of
screening for a modulator of chondrogenesis comprising the steps
of: (a) contacting a first biological sample capable of undergoing
chondrogenesis with a biologically effective (e.g. chondrogenically
effective) amount of an ECM signalling molecule-related biomaterial
and a suspected modulator; (b) separately contacting a second
biological sample capable of undergoing chondrogenesis with a
biologically effective amount of an ECM signalling molecule-related
biomaterial, thereby providing a control; (c) measuring the level
of chondrogenesis resulting from step (a) and from step (b); and
(d) comparing the levels of chondrogenesis measured in step (c),
whereby a modulator of chondrogenesis is identified by its ability
to alter the level of chondrogenesis when compared to the control
of step (b). The modulator may be either a promoter or an inhibitor
of chondrogenesis; the ECM signalling molecules include those
defined above and compounds such as mannose-6-phosphate, heparin,
and tenascin.
[0029] The invention also relates to an in vitro method of
screening for a modulator of oncogenesis comprising the steps of:
(a) inducing a first tumor and a second tumor; (b) administering a
biologically effective amount of an ECM signalling molecule-related
biomaterial and a suspected modulator to the first tumor; (c)
separately administering a biologically effective amount of an ECM
signalling molecule-related biomaterial to the second tumor,
thereby providing a control; (d) measuring the level of oncogenesis
resulting from step (b) and from step (c); and (e) comparing the
levels of oncogenesis measured in step (d), whereby a modulator of
oncogenesis is identified by its ability to alter the level of
oncogenesis when compared to the control of step (c). Modulators of
oncogenesis contemplated by the invention include inhibitors of
oncogenesis. Tumors may be induced by a variety of techniques
including, but not limited to, the administration of chemicals,
e.g., carcinogens, and the implantation of cancer cells. A related
aspect of the invention is a method for treating a solid tumor
comprising the step of delivering a therapeutically effective
amount of a Cyr61 inhibitor to an individual, thereby inhibiting
the neovascularization of the tumor. Inhibitors include, but are
not limited to, inhibitor peptides such as peptides having the
"RGD" motif, and cytotoxins, which may be free or attached to
molecules such as Cyr61.
[0030] Yet another aspect of the invention is directed to a method
of screening for a modulator of cell adhesion comprising the steps
of: (a) preparing a surface compatible with cell adherence; (b)
separately placing first and second biological samples capable of
undergoing cell adhesion on the surface; (c) contacting a first
biological sample with a suspected modulator and a biologically
effective amount of an ECM signalling molecule-related biomaterial
selected from the group consisting of a human Cyr61, a human Cyr61
fragment, a human Cyr61 analog, and a human Cyr61 derivative; (d)
separately contacting a second biological sample with a
biologically effective amount of an ECM signalling molecule-related
biomaterial selected from the group consisting of a human Cyr61, a
human Cyr61 fragment, a human Cyr61 analog, and a human Cyr61
derivative, thereby providing a control; (e) measuring the level of
cell adhesion resulting from step (c) and from step (d); and (f)
comparing the levels of cell adhesion measured in step (e), whereby
a modulator of cell adhesion is identified by its ability to alter
the level of cell adhesion when compared to the control of step
(d).
[0031] The invention also extends to a method of screening for a
modulator of cell migration comprising the steps of: (a) forming a
gel matrix comprising Cyr61 and a suspected modulator of cell
migration: (b) preparing a control gel matrix comprising Cyr61; (c)
seeding endothelial cells capable of undergoing cell migration onto
the gel matrix of step (a) and the control gel matrix of step (b);
(d) incubating the endothelial cells; (e) measuring the levels of
cell migration by inspecting the interior of the gel matrix and the
control gel matrix for cells; (f) comparing the levels of cell
migration measured in step (e), whereby a modulator of cell
migration is identified by its ability to alter the level of cell
migration in the gel matrix when compared to the level of cell
migration in the control gel matrix. The endothelial cells include,
but are not limited to, human cells, e.g., human microvascular
endothelial cells. The matrix may be formed from gelling materials
such as Matrigel, collagen, or fibrin, or combinations thereof.
[0032] Another aspect of the invention is directed to an in vitro
method of screening for cell migration comprising the steps of: (a)
forming a first gelatinized filter and a second gelatinized filter,
each filter having two sides; (b) contacting a first side of each
the filter with endothelial cells, thereby adhering the cells to
each the filter; (c) applying an ECM signalling molecule and a
suspected modulator of cell migration to a second side of the first
gelatinized filter and an ECM signalling molecule to a second side
of the second gelatinized filter; (d) incubating each the filter;
(e) detecting cells on the second side of each the filter; and (f)
comparing the presence of cells on the second side of the first
gelatinized filter with the presence of cells on the second side of
the second gelatinized filter, whereby a modulator of cell
migration is identified by its ability to alter the level of cell
migration measured on the first gelatinized filter when compared to
the cell migration measured on the second gelatinized filter. The
endothelial cells are defined above. The ECM signalling molecules
extend to human Cyr61 and each of the filters may be placed in
apparatus such as a Boyden chamber, including modified Boyden
chambers.
[0033] The invention also embraces an in vivo method of screening
for a modulator of cell migration comprising the steps of: (a)
removing a first central portion of a first biocompatible sponge
and a second central portion of a second biocompatible sponge; (b)
applying an ECM signalling molecule and a suspected modulator to
the first central portion and an ECM signalling molecule to the
second central portion; (c) reassociating the first central portion
with said first biocompatible sponge and said second central
portion with the second biocompatible sponge; (d) attaching a first
filter to a first side of the first biocompatible sponge and a
second filter to a second side of the first biocompatible sponge;
(e) attaching a third filter to a first side of the second
biocompatible sponge and a fourth filter to a second side of the
second biocompatible sponge; (f) implanting each of the
biocompatible sponges, each biocompatible sponge comprising the
central portion and the filters, in a test animal; (e) removing
each the sponge following a period of incubation; (f) measuring the
cells found within each of the biocompatible sponges; and (g)
comparing the presence of cells in the first biocompatible sponge
with the presence of cells in the second biocompatible sponge,
whereby a modulator of cell migration is identified by its ability
to alter the level of cell migration measured using the first
biocompatible sponge when compared to the cell migration measured
using the second biocompatible sponge. ECM signalling molecules
include, but are not limited to human Cyr61; the ECM signalling
molecule may also be associated with Hydron. In addition, the in
vivo method of screening for a modulator of cell migration may
include the step of providing a radiolabel to the test animal and
detecting the radiolabel in one or more of the sponges.
[0034] Another aspect of the invention relates to a method for
modulating hemostasis comprising the step of administering an ECM
signalling molecule in a pharmaceutically acceptable adjuvant,
diluent or carrier. Also, the invention extends to a method of
inducing wound healing in a tissue comprising the step of
contacting a wounded tissue with a biologically effective amount of
an ECM signalling molecule, thereby promoting wound healing. The
ECM signalling molecule may be provided in the form of an ECM
signalling molecule polypeptide or an ECM signalling molecule
nucleic acid, e.g., using a gene therapy technique. For example,
the nucleic acid may comprise an expression control sequence
operably linked to an ECM signalling molecule which is then
introduced into the cells of a wounded tissue. The expression of
the coding sequence is controlled, e.g., by using a tissue-specific
promoter such as the K14 promoter operative in skin tissue to
effect the controlled induction of wound healing. The nucleic acid
may include a vector such as a Herpesvirus, an Adenovirus, an
Adeno-associated Virus, a Cytomegalovirus, a Baculovirus, a
retrovirus, and a Vaccinia Virus. Suitable wounded tissues for
treatment by this method include, but are not limited to, skin
tissue and lung epithelium. A related method comprises
administering a biologically effective amount of an ECM signalling
molecule, e.g. Cyr61, to an animal to promote organ regeneration.
The impaired organ may be the result of trauma, e.g. surgery, or
disease. Another method of the invention relates to improving the
vascularization of grafts, e.g., skin grafts. Another method of the
invention is directed to a process for promoting bone implantation,
including bone grafts. The method for promoting bone implantation
comprises the step of contacting a bone implant or receptive site
with a biologically effective (i.e., chondrogenically effective)
amount of an ECM signalling molecule. The contacting step may be
effected by applying the ECM signalling molecule to a biocompatible
wrap such as a biodegradable gauze and contacting the wrap with a
bone implant, thereby promoting bone implantation. The bone
implants comprise natural bones and fragments thereof, as well as
inanimate natural and synthetic materials that are biocompatible,
such as prostheses. In addition to direct application of an ECM
signalling molecule to a bone, prosthesis, or receptive site, the
invention contemplates the use of matrix materials for controlled
release of the ECM signalling molecule, in addition to such
application materials as gauzes.
[0035] Yet another aspect of the invention relates to a method of
screening for a modulator of cell proliferation comprising the
steps of: (a) contacting a first biological sample capable of
undergoing cell proliferation with a suspected modulator and a
biologically effective (i.e., mitogenically effective) amount of an
ECM signalling molecule-related biomaterial selected from the group
consisting of a human Cyr61, a human Cyr61 fragment, a human Cyr61
analog, and a human Cyr61 derivative; (b) separately contacting a
second biological sample capable of undergoing cell proliferation
with a biologically effective amount of an ECM signalling
molecule-related biomaterial selected from the group consisting of
a human Cyr61, a human Cyr61 fragment, a human Cyr61 analog, and a
human Cyr61 derivative, thereby providing a control; (c) incubating
the first and second biological samples; (d) measuring the level of
cell proliferation resulting from step (c); and (e) comparing the
levels of cell proliferation measured in step (d), whereby a
modulator of cell proliferation is identified by its ability to
alter the level of cell adhesion when compared to the control of
step (b).
[0036] Also comprehended by the invention is a method for expanding
a population of undifferentiated hematopoietic stem cells in
culture, comprising the steps of: (a) obtaining hematopoietic stem
cells from a donor; and (b) culturing said cells under suitable
nutrient conditions in the presence of a biologically effective
(i.e., hematopoietically effective) amount of Cyr61.
[0037] Another method according to the invention is a method of
screening for a mitogen comprising the steps of: (a) plating cells
capable of undergoing cell proliferation; (b) contacting a first
portion of the cells with a solution comprising Cyr61 and a
suspected mitogen; (c) contacting a second portion of the cells
with a solution comprising Cyr61, thereby providing a control; (c)
incubating the cells; (d) detecting the growth of the first portion
of cells and the second portion of the cells; and (e) comparing
growth of the first and second portions of cells, whereby a mitogen
is identified by its ability to induce greater growth in the first
portion of cells when compared to the growth of the second portion
of cells. The cells include, but are not limited to, endothelial
cells and fibroblast cells. Further, the method may involve
contacting the cells with a nucleic acid label, e.g.,
[.sup.3H]-thymidine, and detecting the presence of the label in the
cells. Another method relates to improving tissue grafting,
comprising administering to an animal a quantity of Cyr61 effective
in improving the rate of neovascularization of a graft.
[0038] Numerous additional aspects and advantages of the present
invention will be apparent upon consideration of the following
drawing and detailed description.
BRIEF DESCRIPTION OF THE DRAWING
[0039] FIG. 1 presents the comparative amino acid sequences of
members of the cysteine-rich protein family of growth-regulating
proteins.
DETAILED DESCRIPTION OF THE INVENTION
[0040] In the mouse, the Cyr61 protein has been found to influence
cell adhesion, migration, and proliferation. The cyr61 gene, which
encodes Cyr61, is an immediate-early gene that is transcriptionally
activated by serum growth factors in mouse fibroblasts. Lau et al.,
EMBO J. 4:3145-3151 (1985), incorporated herein by reference; Lau
et al., Proc. Natl. Acad. Sci. (USA) 84:1182-1186 (1987),
incorporated herein by reference. The murine cyr61 cDNA coding
sequence is set forth in SEQ ID NO:1. (The human cyr61 cDNA coding
sequence is provided in SEQ ID NO:3). The amino acid sequence of
murine Cyr61 is set out in SEQ ID NO:2. (The human Cyr61 amino acid
sequence is presented in SEQ ID NO:4). Cyr61 is a 41 kDa
polypeptide exhibiting 39 cysteine residues, approximately 10% of
the 379 amino acids constituting the unprocessed protein. Yang et
al., Cell Growth & Diff. 2:351-357 (1991), incorporated herein
by reference. Investigations have revealed that murine Cyr61 binds
heparin and is secreted. Yang et al. Consistent with the observed
secretion of Cyr61 is the identification of an N-terminal signal
sequence in nascent Cyr61, deduced from inspection of the murine
cyr61 cDNA sequence. Yang et al. Additionally, Cyr61 is not found
in the conditioned medium of cultured cells expressing cyr61, but
is found associated with the extracellular matrix (ECM) and the
cell surface. Yang et al. Structurally similar cysteine-rich
mammalian proteins have been characterized.
[0041] Fisp12, a cysteine-rich murine protein, exhibits structural
similarity to Cyr61. The cDNA sequence encoding Fisp12 is set forth
in SEQ ID NO:5; the amino acid sequence of Fisp12 is presented in
SEQ ID NO:6. Murine Fisp12, like Cyr61, influences cell adhesion,
proliferation and migration. The human ortholog of Fisp12 is
Connective Tissue Growth Factor (CTGF), a protein similar in
structure and function to Cyr61. Fisp12, and CTGF, are
distinguishable from Cyr61, however. For example, a greater
proportion of secreted Fisp12 is found in the culture medium than
is the case with Cyr61; a correspondingly lower proportion of
Fisp12 is localized in the area of expressing cells (cell surface
and nearby extracellular matrix) than is found with Cyr61.
Additional similarities and distinctions among the proteins
comprising the ECM signalling molecules of the invention will
become apparent in the recitations hereinbelow.
[0042] The present invention has multiple aspects, illustrated by
the following examples. Example 1 describes the cloning of
polynucleotides encoding members of the cysteine-rich protein
family of ECM signalling molecules; Example 2 describes sequence
analyses; Example 3 describes RNA analyses; Example 4 describes the
production of transgenic animals; Example 5 describes the
expression of Cyr61 polypeptides; Example 6 describes the
expression of Fisp12 polypeptides; Example 7 sets out methods of
polypeptide purification; Example 8 provides a characterization of
the polypeptides of the invention; Example 9 discloses a heparin
binding assay for the polypeptide members of the cysteine-rich
protein family; Example 10 is directed to receptors for the
polypeptides; Example 11 describes anti-ECM signalling molecule
antibodies; Example 12 is directed to inhibitory peptides; Example
13 describes cell adhesion and polypeptide-based methods for
influencing the process of cell adhesion; Example 14 describes
polypeptide-influenced migration of fibroblasts; Example 15
describes the migration of endothelial cells and in vitro assays
for migration; Example 16 describes an in vitro assay for
inhibitors of endothelial cell migration; Example 17 describes an
in vivo assay for endothelial cell migration; Example 18 describes
mitogen potentiation by the polypeptides of the invention; Example
19 describes an in vivo cornea assay for angiogenic factors and
modulators; Example 20 is directed to methods for influencing blood
clotting using the polypeptides of the invention; Example 21
discloses the use of the polypeptides for ex vivo hematopoietic
stem cell cultures; Example 22 addresses organ regeneration;
Example 23 describes chondrogenesis and the expression of
extracellular matrix signalling molecules in mesenchyme cells;
Example 24 describes the promotion of cell adhesion in the process
of chondrogenesis using the polypeptides of the invention; Example
25 describes chondrogenesis and the influence of the polypeptides
of the invention on cell aggregation; Example 26 describes the
promotion of cell proliferation by polypeptides of the invention in
the process of chondrogenesis; Example 27 addresses methods for
using the polypeptides of the invention to affect chondrogenesis;
and Example 28 provides genetic approaches to the use of
polynucleotides of the invention. These examples are intended to be
illustrative of the present invention and should not be construed
to limit the scope of the invention.
EXAMPLE 1
Polynucleotide Cloning
[0043] A human cyr61 cDNA was isolated from a human placental cDNA
library by probing with the murine cyr61 cDNA sequence using
techniques that are standard in the art. See Sambrook et al.,
incorporated herein by reference. Isolation of the complete murine
cyr61 cDNA from a BALB/c 3T3 (ATCC CRL-1658) cDNA library has been
described. O'Brien et al., Mol. Cell. Biol. 10:3569-3577 (1990),
incorporated herein by reference. The nucleotide and deduced amino
acid sequences of murine cyr61 are available from the GenBank
database under accession number M32490. The nucleotide sequence of
murine cyr61 is presented in SEQ ID NO:1; the murine Cyr61 amino
acid sequence is presented in SEQ ID NO:2.
[0044] The human cDNA library was constructed using .lamda.gt11
(Promega Corp., Madison. WI) as a vector which was transfected into
E. coli and plated on LB agar. A murine cDNA expression construct
cloned in pGEM-2 (O'Brien et al., [1990]), containing the entire
murine cyr61 coding sequence [nucleotides 56-1560, using the
numbering of O'Brien et al., (1990); see SEQ ID NO:1] was used as a
probe. The mouse cDNA probe was radiolabeled by techniques standard
in the art. Sambrook et al. Plaque screenings using the mouse probe
were performed using standard techniques. Sambrook et al.
[0045] More particularly, agar plates containing the human cDNA
library described above were exposed to nitrocellulose filters
(BA85, 82 mm. Schleicher & Schuell, Keene, N.H.) were placed on
each plate. After plaque adsorption (approximately 20 minutes), the
filters were removed and air dried for approximately 30 minutes.
Subsequently, each filter was sequentially submerged for 30-60
seconds in 0.2M NaOH, 1.5M NaCl (100 ml); 2.times.SSC, 0.4M
Tris-HCl, pH 7.4 (100 ml); and 0.2.times.SSC (100 ml). Filters were
then dried at room temperature for approximately 1 hour and
subjected to 80.degree. C. under vacuum for 2 hours. Filters were
probed with radiolabeled murine cyr61 cDNA.
[0046] Alternatively, human cyr61 cDNA clones were identified with
probes generated by RT-PCR. In particular, the probe for screening
the human placental cDNA library was a PCR fragment generated with
degenerate primers by RT-PCR of total RNA from logarithmically
growing WI38 cells. The primers were derived from the sequences
corresponding to the most conserved region of the open reading
frame of the mouse cyr61 cDNA. One primer, designated H61-5
[5'-GGGAATICTG(TC)GG(GATC)TG(TC)TG(TC)AA(GA)GT(GC)TG-3'], contains
a degenerate sequence which, with the exception of the "GGGAATTC"
sequence at the 5' end which was used to introduce an EcoRI site,
is derived from nucleotides 327-346 (sense strand) of the mouse
cyr61 sequence set forth in SEQ ID NO: 1. The degeneracies appear
in positions corresponding to the third position of codons in SEQ
ID NO: 1. The second primer used for PCR amplification of a human
cyr61 sequence was designated H61-3
[5'-CCGGATCC(GA)CA(GA)TT(GA)TA(GA)TT(GA)CA-3'], which, with the
exception of the 5' sequence "CCGGATCC" used to introduce a BamHI
site, corresponds to the anti-sense strand complementary to
nucleotides 1236-1250 of the mouse cyr61 sequence set forth in SEQ
ID NO: 1. The degeneracies occur in positions complementary to the
third positions of codons in mouse cyr61 as set forth in SEQ ID NO:
1. The amplified cyr61 cDNA was cloned into the pBlueScript SK+
vector (Stratagene, La Jolla, Calif.) and sequenced with a
Sequenase II kit (U.S. Biochemicals, Cleveland, Ohio).
[0047] Serial screenings of the human placental cDNA library led to
the isolation of a clone containing a human cyr61 cDNA. The human
cyr61 cDNA is approximately 1,500 bp in length. The human cDNA is
contained on an EcoRI fragment cloned into the EcoRI site in
pGEM-2. As shown in SEQ ID NO:3, the human cDNA sequence includes
the entire coding region for human Cyr61, along with 120 bp of 5'
flanking sequence, and about 150 bp of 3' flanking sequence.
[0048] The polynucleotides of the invention may be wholly or
partially synthetic. DNA or RNA, and single- or double-stranded.
Because polynucleotides of the invention encode ECM signalling
molecule polypeptides which may be fragments of an ECM signalling
molecule protein, the polynucleotides may encode a partial sequence
of an ECM signalling molecule. Polynucleotide sequences of the
invention are useful for the production of ECM signalling molecules
by recombinant methods and as hybridization probes for
polynucleotides encoding ECM signalling molecules.
[0049] DNA polynucleotides according to the invention include
genomic DNAs, cDNAs, and oligonucleotides comprising a coding
sequence of an ECM signalling molecule, or a fragment or analog of
an ECM signalling molecule, as described above, that retains at
least one of the biological activities of an ECM signalling
molecule such as the ability to promote cell adhesion, cell
migration, or cell proliferation in such biological processes as
angiogenesis, chondrogenesis, and oncogenesis, or the ability to
elicit an antibody recognizing an ECM signalling molecule.
[0050] Other polynucleotides according to the invention differ in
sequence from sequences contained within native ECM signalling
molecule polynucleotides (i.e., by the addition, deletion,
insertion, or substitution of nucleotides) provided the
polynucleotides encode a protein that retains at least one of the
biological activities of an ECM signalling molecule. A
polynucleotide sequence of the invention may differ from a native
ECM signalling molecule polynucleotide sequence by silent mutations
that do not alter the sequence of amino acids encoded therein.
Additionally, polynucleotides of the invention may specify an ECM
signalling molecule that differs in amino acid sequence from native
ECM signalling molecule sequences or subsequences, as described
above. For example, polynucleotides encoding polypeptides that
differ in amino acid sequence from native ECM signalling molecules
by conservative replacement of one or more amino acid residues, are
contemplated by the invention. The invention also extends to
polynucleotides that hybridize under standard stringent conditions
to polynucleotides encoding an ECM signalling molecule of the
invention, or that would hybridize but for the degeneracy of the
genetic code. Exemplary stringent hybridization conditions involve
hybridization at 42.degree. C. in 50% formamide, 5.times.SSC, 20 mM
Na.PO.sub.4, pH 6.8 and washing in 0.2.times.SSC at 55.degree. C.
It is understood by those of skill in the art that variation in
these conditions occurs based on the length and GC nucleotide
content of the sequences to be hybridized. Formulas standard in the
art are appropriate for determining exact hybridization conditions.
See Sambrook et al., Molecular Cloning: A Laboratory Manual (Second
ed., Cold Spring Harbor Laboratory Press 1989) .sctn..sctn.
9.47-9.51.
[0051] ECM signalling molecule polynucleotides comprising RNA are
also within the scope of the present invention. A preferred RNA
polynucleotide according to the invention is an mRNA of human
cyr61. Other RNA polynucleotides of the invention include RNAs that
differ from a native ECM signalling molecule mRNA by the insertion,
deletion, addition, or substitution of nucleotides (see above),
with the proviso that they encode a polypeptide retaining a
biological activity associated with an ECM signalling molecule.
Still other RNAs of the invention include anti-sense RNAs (i.e.,
RNAs comprising an RNA sequence that is complementary to an ECM
signalling molecule mRNA).
[0052] Accordingly, in another embodiment a set of DNA fragments
collectively spanning the human cyr61 cDNA were cloned in pGEM-2
and M13 derivatives using methods well known in the all to
facilitate nucleotide sequence analyses. The pGEM-2 clones provided
substrates for the enzymatic generation of serial deletions using
techniques known in the art. This collection of clones,
collectively containing a series of DNA fragments spanning various
parts of the cyr61 cDNA coding region, are useful in the methods of
the invention. The resulting series of nested pGEM-2 clones, in
turn, provided substrates for nucleotide sequence analyses using
the enzymatic chain terminating technique. The fragments are also
useful as nucleic acid probes and for preparing Cyr61 deletion or
truncation analogs. For example, the cyr61 cDNA clones may be used
to isolate cyr61 clones from human genomic libraries that are
commercially available. (Clontech Laboratories, Inc. Palo Alto,
CA). Genomic clones, in turn, may be used to map the cyr61 locus in
the human genome, a locus that may be associated with a known
disease locus.
[0053] Other embodiments involve the polynucleotides of the
invention contained in a variety of vectors, including plasmid,
viral (e.g., prokaryotic and eukaryotic viral vectors derived from
Lambda phage, Herpesviruses, Adenovirus, Adeno-associated viruses,
Cytomegalovirus, Vaccinia Virus, the M13-fl-fd family of viruses,
retroviruses, Baculovirus, and others), phagemid, cosmid, and YAC
(i.e., Yeast Artificial Chromosome) vectors.
[0054] Yet other embodiments involve the polynucleotides of the
invention contained within heterologous polynucleotide
environments. Polynucleotides of the invention have been inserted
into heterologous genomes, thereby creating transgenes, and
transgenic animals, according to the invention. In particular, two
types of gene fusions containing partial murine cyr61 gene
sequences have been used to generate transgenic mice, (See below).
One type of fused gene recombined the coding sequence of cyr61 with
one of three different promoters: 1) the K14 keratin promoter, 2)
the .beta.-actin promoter, or 3) the phosphoglycerokinase promoter.
Adra et al. Gene 60:65-74 (1987). These fusion constructs were
generated using standard techniques, as described below in the
context of a phosphoglycerokinase promoter (pgk-1)-cyr61 fusion. An
XhoI-ScaI genomic DNA fragment containing the entire cyr61 coding
region and all introns, but lacking the transcription initiation
site and polyadenylation signal, was cloned into plasmid
pgk/.beta.-gal, replacing the lacZ coding sequence. The resulting
construct placed cyr61 under the control of the strong pgk-1
promoter which is active in all cells.
[0055] The second type of gene fusion recombined the cyr61
expression control sequences (i.e., promoter) with the E. coli
.beta.-galactosidase coding sequence. The cyr61-lacZ fusion gene
was constructed using the following approach. A DNA fragment
spanning nucleotides -2065 to +65 relative to the transcription
initiation nucleotide was used to replace the pgk-1 promoter (Adra
et al., Gene 60:65-74 [1987]) in plasmid pgk/.beta.-gal by
blunt-end cloning. In addition, the polyadenylation signal from the
bovine growth hormone gene was cloned into the plasmid containing
the fusion gene. The resulting construct, plasmid 2/lacZ, has the
E. coli lacZ gene under the transcriptional control of a 2 kb DNA
fragment containing the cyr61 promoter. The related plasmid
1.4/lacZ was derived from plasmid 2lacZ by removing about 600 bp of
cyr61 DNA found upstream of an AfII site. Also, plasmid 2M/lacZ
resembles plasmid 2/lacZ, except for a C-to-T transition in the
CArG Box, created by PCR. These constructs were excised from the
vectors by NotI digestion, purified using GeneClean [Bio101, Inc.,
La Jolla, Calif.), and used to generate transgenic mice (see
below).
[0056] A cDNA fragment encoding mouse fisp12 has also been cloned
using standard techniques. Ryseck et al., Cell Growth & Diff.
2:225-233 (1991), incorporated herein by reference. The cloning was
accomplished by ligating an XhoII fragment containing the fisp12
cDNA coding region into BamHI-cleaved pBlueBacIII, a baculovirus
expression vector (Invitrogen Corp., San Diego, Calif.).
Recombinant baculovirus clones were obtained as described in
Summers et al., TX Ag. Exp. Sta., Bulletin 1555 (1987).
[0057] The human ortholog of fisp12, the gene encoding CTGF, was
cloned by screening a fusion cDNA library with
anti-Platelet-Derived Growth Factor (anti-PDGF) antibodies, as
described in U.S. Pat. No. 5,408,040, column 12, line 16, to column
13, line 29, incorporated herein by reference. The screening
strategy exploited the immunological cross-reactivity of CTGF and
PDGF.
[0058] The cloned copies of the cyr61, fisp12, and ctgf cDNAs
provide a ready source for polynucleotide probes to facilitate the
isolation of genomic coding regions, as well as allelic variants of
the genomic DNAs or cDNAs. In addition, the existing cDNA clones,
or clones isolated by probing as described above, may be used to
generate transgenic organisms. For example, transgenic mice
harboring cyr61 have been generated using standard techniques, as
described in the next Example.
[0059] A clone, hCyr61cDNA, containing the human cyr61 cDNA
sequence set forth in SEQ ID NO: 3, and a bacterial strain
transformed with that clone, Escherichia coli DH5.alpha.
(hCyr61cDNA), were deposited with the American Type Culture
Collection 12301 Parklawn Drive, Rockville, Md. 20852 USA, on Mar.
14, 1997.
EXAMPLE 2
Sequence Analyses
[0060] The nucleotide sequence of murine cyr61 has been described,
O'Brien et al. (1990); Latinkic et al., Nucl. Acids Res.
19:3261-3267 (1991), and is set out herein as SEQ ID NO:1.
[0061] The deduced amino acid sequence of murine Cyr61 has been
reported, O'Brien et al. (1990), and is set forth in SEQ ID
NO:2.
[0062] The nucleotide sequence of the human cyr61 cDNA was
determined using the method of Sanger, as described in Sambrook et
al. Sequencing templates were generated by constructing a series of
nested deletions from a pGEM-2 human cyr61 cDNA clone, as described
in Example 1 above. The human cyr61 cDNA sequence is set forth in
SEQ ID NO:3. The amino acid sequence of human Cyr61 was deduced
from the human cyr6 cDNA sequence and is set forth in SEQ ID
NO:4.
[0063] A comparison of the mouse and human Cyr61 sequences,
presented in SEQ ID NO:2 and SEQ ID NO:4, respectively, reveals 91%
similarity. Both sequences exhibit an N-terminal signal sequence
indicative of a processed and secreted protein; both proteins also
contain 38 cysteine residues, distributed throughout both proteins
but notably absent from the central regions of both murine and
human Cyr61. Notably, the region of greatest sequence divergence
between the mouse and human Cyr61 coding regions is this central
region free of cysteine residues. However, the 5' untranslated
regions of the mouse and human cyr61 cDNAs are even more divergent
(67% similarity). In contrast, the 3' untranslated regions are the
most similar regions (91% similarity). In overall length, the
encoded murine Cyr61 has 379 amino acids; human Cyr61 has 381 amino
acids.
[0064] A fisp12 cDNA sequence has also been determined and is set
out in SEQ ID NO:5. The amino acid sequence of Fisp12 has been
deduced from the fisp12 cDNA sequence and is set forth in SEQ ID
NO:6. A comparison of the amino acid sequences of murine Cyr61 and
Fisp12 reveals that the two proteins are 65% identical. The
structural similarity of Cyr61 and Fisp12 is consistent with the
similar functional properties of the two proteins, described
below.
[0065] A partial cDNA sequence of CTGF, containing the complete
CTGF coding region, has also been determined. The CTGF cDNA
sequence was obtained using M13 clones as templates for enzymatic
sequencing reactions, as described. '040 patent, at column 12, line
68 to column 13, line 14. Additional cloning coupled with
double-stranded enzymatic sequencing reactions, elucidated the
entire sequence of the cDNA encoding CTGF. U.S. Pat. No. 5,408,040,
column 14, line 44, to column 15, line 8, incorporated herein by
reference. The nucleotide sequence of the cDNA encoding CTGF is
presented herein in SEQ ID NO:7. The deduced amino acid sequence of
the cDNA encoding CTGF is presented in SEQ ID NO:8.
EXAMPLE 3
RNA Analyses
[0066] Polynucleotide probes are useful diagnostic tools for
angiogenic, and other, disorders correlated with Cyr61 expression
because properly designed probes can reveal the location, and
level, of cyr61 gene expression at the transcriptional level. The
expression of cyr61, in turn, indicates whether or not genes
controlling the process of angiogenesis are being expressed at
typical, or expected, levels.
[0067] Using these tools, the mouse cyr61 mRNA expression pattern
was determined using an RNase protection technique. O'Brien et al.,
(1992). In particular, a 289 nucleotide antisense riboprobe was
used that would protect 246 nucleotides of the murine cyr61 mRNA
(nucleotides 67 to 313 using the numbering of O'Brien et al.) The
assays showed levels of cyr61 mRNA in PSA-1 cells (10 .mu.g of
total RNA) from either the undifferentiated state or stages 1, 2,
and 3 of differentiation (PSA-1 cells undergo three stages of
cellular differentiation corresponding to mouse embryonic cells of
the following gestational ages, in days: 4.5-6.5 [PSA-1 stage 1];
6.5-8.5 [PSA-1 stage 2]; 8.5-10.5 [PSA-1 stage 3]). A comparison of
the protection of whole embryonic and placental total RNAs (20
.mu.g each) showed that cyr61 is expressed in embryonic tissues at
times that are coincident with the processes of cell
differentiation and proliferation.
[0068] Expression characteristics of human cyr61 were determined by
Northern analyses, using techniques that are standard in the art.
Sambrook et al. RNA was isolated from the human diploid
fibroblastic cell line WI38 (ATCC CCL-75). In addition, RNA was
isolated from rat cells (REF52), hamster cells (CHO), and monkey
cells (BSC40). Each of the cell lines was grown to confluence in
MEM-10 (Eagle's Minimal Essential Medium with Earle's salts
[GIBCO-BRL. Inc.], 2 mM glutamine, and 10% fetal calf serum [fcs])
and maintained in MEM-0.5 (a 0.5% serum medium) for two days.
Cultures were then stimulated with 20% fcs, in the presence or
absence of cycloheximide, by techniques known in the art. Lau et
al. (1985; 1987). Ten microgram aliquots of RNA isolated from these
cell lines were then fractionated by formaldehyde-agarose gel
electrophoresis, transferred and immobilized on nitrocellulose
filters, and exposed to a full-length [.sup.32P]-radiolabeled
murine cyr61 cDNA probe under hybridization conditions of high
stringency. Human cyr61 RNA expression was similar to murine cyr61
expression. Both mouse and human cyr61 expression yielded
approximately 2 kilobase RNAs. Additionally, both mouse and human
expression of Cyr61 were stimulated by serum and were resistant to
cycloheximide.
[0069] The distribution of human cyr61 mRNA was also examined using
multiple tissue Northern blots (Clontech). The blots were
hybridized in an ExpressHyb Solution (Clontech) according to the
manufacturer's instructions. The results showed that cyr61 mRNA is
abundant in the human heart, lung, pancreas, and placenta; is
present at low levels in skeletal muscle, kidney and brain; and is
not detectable in liver. These results are consistent with the
expression of cyr61 in mouse tissues.
[0070] In addition, total cellular RNA was isolated from human skin
fibroblasts (HSFs) that were either quiescent, growing
exponentially, stimulated by serum, or exposed to cycloheximide.
HUVE cells (ATCC CRL 1730) were maintained in Ham's F12 medium
supplemented with 10% fbs (Intergene), 100 .mu.g/ml heparin (Gibco
BRL) and 30 .mu.g/ml endothelial cell growth supplement
(Collaborative Biomedical Products). Human skin fibroblasts (HSF,
ATCC CRL-1475) and WI38 fibroblasts (ATCC CCL-75) were grown in
Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10%
fbs. Quiescent HSFs were prepared by growth in DMEM supplemented
with 10% fbs to confluence followed by changing the medium to DMEM
containing 0.1% fbs, for 2 days. Serum stimulation was carried out
by changing the medium to 20% fbs for 1 hour. Where indicated,
cycloheximide was added to 10 .mu.g/ml simultaneously with serum
for 3 hours.
[0071] RNAs from the aforementioned cells were isolated using a
guanidinium isothiocyanate protocol. Chomczynski et al., Anal.
Biochem. 162:156-159 (1987). RNA samples were analyzed by
electrophoretic separation in formaldehyde-agarose gels followed by
transfer to nylon filters. Blots were hybridized with random-primed
probes generated using either cyr61 or GAPDH as a template. Adams
et al., Nature 355:632-634 (1992). The results indicated that human
cyr61 mRNA is not detectably present in quiescent human skin
fibroblasts, is abundant in logarithmically growing and serum
stimulated HSFs, and is superinduced by cycloheximide.
[0072] The analysis of RNA encoding CTGF also involved techniques
that are standard in the art. In particular, investigation of RNA
encoding CTGF involved the isolation of total cellular RNA and
Northern analyses, performed as described in U.S. Pat. No.
5,408,040, column 11, line 59, to column 12, line 14, and column
13, lines 10-29, incorporated herein by reference. A 2.4 kb RNA was
identified. The expression of CTGF was high in the placenta, lung,
heart, kidney, skeletal muscle and pancreas. However, CTGF
expression was low in the liver and brain.
EXAMPLE 4
Transgenic Animals
[0073] The construction of transgenic mice bearing integrated
copies of recombinant cyr61 sequences was accomplished using linear
DNA fragments containing a fusion gene. The cyr61 coding sequence
was independently fused to the .beta.-actin, K14, and pgk
promoters, described above. Expression of cyr61 was driven by these
promoters in the transgenic animals. The fusion gene was produced
by appropriate restriction endonuclease digestions, using standard
techniques. The fusion gene fragments were injected into
single-cell zygotes of Swiss Webster mice. The injected zygotes
were then implanted into pseudopregnant females. Several litters of
mice were produced in this manner. Newborns exhibiting unusual
phenotypes were subjected to additional analyses. For example,
neonatal transgenic mice expressing cyr61 under the pgk promoter
exhibited skeletal deformities, including curly tails, immobile
joints, and twisted limbs, resulting in locomotive difficulties.
These mice typically were runted and died within seven days of
birth. Transgenic mice expressing cyr61 under the .beta.-actin
promoter showed no obvious phenotype except that the mice were
smaller. When mice bearing the transgene were back-crossed to the
in-bred strain C57BL/6, the progeny mice became progressively more
runted with continued back-crossing. After three to four such
back-crosses, essentially no progeny survive to reproduce.
Transgenic mice expressing cyr61 under the K14 promoter exhibited a
form of fibrotic dermatitis. The pathology involved excessive
surface scratching, sometimes resulting in bleeding. Transgenic
organisms having knockout mutations of cyr61 can also be created
using these standard techniques. Hogan et al., Manipulating the
Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory
Press 1994), and are useful as models of disease states.
EXAMPLE 5
Cyr61 Expression
[0074] Native Cyr61 is expressed in embryonic tissues and is
induced in a variety of wounded tissues. See below; see also,
O'Brien et al. (1992). The tissue distribution of Cyr61 was
examined with rabbit anti-Cyr61 polyclonal antibodies elicited
using a conventional immunological technique (Harlow et al., 1987)
and affinity-purified. Using affinity-purified anti-Cyr61
polyclonal antibodies according to the invention, cyr61 expression
was found in a variety of tissues, including smooth muscle,
cardiomyocytes, and endothelia of the cardiovascular system; brain,
spinal cord, ganglia and neurons, and retina of the nervous system;
cartilage and bone of the skeletal system; epidermis, hair, oral
epithelia, and cornea of the skin; bronchioles and blood vessels of
the lung; and placental tissues. In addition to expression studies
directed towards native cyr61 (mRNA and protein), studies using
cyr61 transgenes, as described above, have contributed to our
understanding of Cyr61 expression. The use of transgene fusions
comprising the expression control sequences of cyr61 and the coding
sequence of lacZ (encoding .beta.-galactosidase) has provided a
convenient calorimetric assay for protein expression.
[0075] The colorimetric assay involves the use of
5-Bromo-4-Chloro-3-Indolyl-.beta.-D-Galactopyranoside (i.e., X-Gal)
as a substrate for .beta.-galactosidase, the gene product of lacZ.
Enzymatic cleavage of X-Gal by .beta.-galactosidase produces an
intensely colored indigo dye useful in histochemical staining. In
practice, embryonic and adult tissues subjected to analysis were
dissected and fixed in 2% formaldehyde, 0.2% glutaraldehyde, 0.02%
Nonidet P-40, and 0.01 sodium deoxycholate, in standard
phosphate-buffered saline (PBS). Fixation times varied from 15-120
minutes, depending on the size and density of organ or embryo
samples being subjected to analysis. Subsequently, samples were
rinsed in PBS and stained overnight at 37.degree. C. in a PBS
solution containing 5 mM potassium ferrocyanide, 5 mM potassium
ferricyanide, 2 mM MgCl.sub.2, 0.02% Nonidet P-40, 0.01% sodium
deoxycholate and 1 mg/ml of X-Gal (40 mg/ml in dimethylsulfoxide
[DMSO]). Samples were then rinsed in PBS, post-fixed in 4%
paraformaldehyde for 1-2 hours, and stored in 70% ethanol at
4.degree. C. until subjected to microscopic examination. Mice
containing the cyr61-lacZ transgene were used to map the expression
profile of cyr61. The results are presented in Table I for
embryonic tissues at day 12.5. TABLE-US-00001 TABLE I Transgenic
Blood Nervous Mouse Line Vessels Skeleton System Epidermis .sup.
1.S.sup.1 .sup. +.sup.2 - + + 2.S + + + + 3.S + +/- + + 4.T + - -
NA 5.T + - - NA 6.T + +/- - NA 7.T + +/- - NA 8.T + +/- + NA
.sup.1Transgenic lines, S--stable (established) transgenic lines;
T--transient lines .sup.2+/- Expression pattern only partially
reproduced.
[0076] The results indicate that Cyr61 is expressed in a variety of
embryonic cell types. Additional information has been gleaned from
the ectopic expression of Cyr61 resulting from another type of
transgene fusion comprising a heterologous expression control
sequence coupled to the coding sequence of cyr61. The control
sequences, the K14 keratin promoter, the .beta.-actin promoter, and
the phosphoglycerokinase promoter, directed the expression of Cyr61
in a pattern that differed from its native expression.
[0077] Transgenic mice ectopically expressing Cyr61 were routinely
smaller than wild type mice and exhibited a reduction in average
life span. Moreover, these transgenic mice had abnormal hearts
(i.e., thickened chamber walls with a corresponding reduction in
internal capacity) and abnormal skeletons characterized by curved
spines, joints swollen to the point of immobility, and curly tails.
Therefore, ectopic expression of Cyr61 interferes with angiogenesis
(blood vessel development and heart development) and chondrogenesis
(skeletal development). In addition, transgenic mice carrying
knockout mutations of cyr61 may be developed and tested as models
of disease states associated with a lack of Cyr61 activity.
[0078] A strategy for the expression of recombinant cyr61 was
designed using a Baculovirus expression vector in Sf9 cells.
Expression systems involving Baculovirus expression vectors and Sf9
cells are described in Current Protocols in Molecular Biology
.sctn..sctn. 16.9.1-16.12.6 (Ausubel et al., eds., 1987). One
embodiment of the present invention implemented the expression
strategy by cloning the murine cyr61 cDNA into pBlueBac2, a
transfer vector. The recombinant clone, along with target AcMNPV
(i.e., Autographia california nuclear polyhedrosis virus, or
Baculovirus) DNA, were delivered into Sf9 cells by
liposome-mediated transfection, using the MaxBac Kit (Invitrogen,
Inc., San Diego, Calif.) according to the manufacturer's
instructions. Recombinant virus was plaque-purified and amplified
by 3 passages through Sf9 cells via infection.
[0079] Conditioned medium of Sf9 insect cells infected with a
baculovirus construct driving the synthesis of murine Cyr61 was
used as a source for purification of Cyr61 (see below). The
purified recombinant Cyr61 retains certain characteristics of the
endogenous protein, e.g., the heparin-binding activity of Cyr61
(described below) from 3T3 fibroblast cells and had a structure
similar to the endogenous protein as revealed by independent
peptide profiles produced by partial proteolysis using either
chymotrypsin or trypsin (sequencing grade: Boehringer-Mannheim.
Inc. Indianapolis, Ind.).
[0080] Human cyr61 was also expressed using the baculovirus system.
A SmaI-HindIII fragment (corresponding to nucleotides 100-1649 of
SEQ ID NO: 3) of cyr61 cDNA spanning the entire human cyr61 open
reading frame was subcloned into a pBlueBac3 baculovirus expression
vector (Invitrogen). Recombinant baculovirus clones were obtained,
plaque purified and amplified through three passages of Sf9
infection, using conventional techniques. Infection of Sf9 cells
and human Cyr61 (hCyr61) purification was performed using standard
techniques, with some modifications. Sf9 cells were maintained in
serum-free Sf900-II medium (Sigma). Sf9 cells were seeded, at
2-3.times.10.sup.6 cells per 150 mm dish, in monolayer cultures and
were infected with 5 plaque forming units (PFU) of recombinant
virus per cell. The conditioned medium was collected at 8 and 96
hours post-infection, cleared by centrifugation (5000.times.g, 5
minutes) and adjusted to 50 mM MES [2-(N-Morpholino)ethanesulfonic
acid], pH 6.0. 1 mM PMSF (phenylmethylsulfonyl fluoride), and 1 mM
EDTA. The medium was mixed with Sepharose S beads equilibrated with
loading buffer (50 mM MES, pH 6.0, 1 mM PMSF, 1 mM EDTA. 150 mM
NaCl) at a ratio of 5 ml Sepharose S beads per 500 ml of
conditioned medium and the proteins were allowed to bind to the
Sepharose S at 4.degree. C. (o/n) with gentle stirring. Sepharose S
beads were collected by sedimentation without stirring for 20
minutes and applied to the column. The column was washed with 6
volumes of 0.3 M NaCl in loading buffer and recombinant human Cyr61
was eluted from the column with a step gradient of NaCl (0.4-0.8 M)
in loading buffer. This procedure resulted in 3-4 milligrams of
purified Cyr61 protein from 500 ml of conditioned medium, and the
purified Cyr61 was over 90% pure as judged by Coomassie Blue
staining of SDS-gels.
[0081] In another embodiment, the complete human cyr61 cDNA is
cloned into a cytomegalovirus vector such as pBK-CMV (Stratagene,
LaJolla, Calif.) using the Polymerase Chain Reaction (Hayasyhi, in
PCR: The Polymerase Chain Reaction 3-13 [Mullis et al. eds.,
Birkhauser 1994]) and Taq Polymerase with editing function,
followed by conventional cloning techniques to insert the PCR
fragment into a vector. The expression vector is then introduced
into HUE cells by liposome-mediated transfection. Recipient clones
containing the vector-borne neo gene are selected using G418.
Selected clones are expanded and Cyr61 expression is identified by
Reverse Transcription-Polymerase Chain Reaction (i.e., RT-PCR;
Chelly et al., in PCR: The Polymerase Chain Reaction 97-109 [Mullis
et al. eds., Birkhauser 1994]) or Enzyme-Linked Immunosorbent
Assays (i.e., ELISA; Stites et al., in Basic and Clinical
Immunology 243 [Stites et al. eds., Appleton & Lange 1991])
assays.
[0082] In other embodiments of the invention, Cyr61 protein is
expressed in bacterial cells or other expression systems (e.g.,
yeast) using the cyr61 cDNA coding region linked to promoters that
are operative in the cell type being used. Using one of these
approaches, Cyr61 protein may be obtained in a form that can be
administered directly to patients, e.g., by intravenous routes, to
treat angiogenic, chondrogenic, or oncogenic disorders. One of
skill in the art would recognize that other administration routes
are also available, e.g., topical or local application,
liposome-mediated delivery techniques, or subcutaneous,
intradermal, intraperitoneal, or intramuscular injection.
EXAMPLE 6
Fisp12 Expression
[0083] The expression of Fisp12, and a comparison of the expression
characteristics of Cyr61 and Fisp12, were investigated using
immunohistochemical techniques. For these immunohistochemical
analyses, tissue samples (see below) were initially subjected to
methyl-Carnoy's fixative (60% methanol, 30% chloroform and 10%
glacial acetic acid) for 2-4 hours. They were then dehydrated,
cleared and infiltrated in Paraplast X-tra wax at 55-56 C for
minimal duration. 7 .mu.m thick sections were collected on
poly-L-lysine-coated slides (Sigma), mounted and dewaxed. They were
then treated with 0.03% solution of H.sub.2O.sub.2 in methanol for
30 min. to inactivate endogenous peroxidase activity. After
rehydration, sections were put in Tris-buffered saline (TBS: 10 mM
Tris, pH 7.6 and 140 mM NaCl) for 15 minutes. At that point,
sections were blotted to remove excess TBS with paper towels and
blocked with 3% normal goat serum in TES for 10 minutes in a humid
chamber. Excess buffer was then drained and primary antibodies
applied. Affinity purified anti-Cyr61 antibodies were diluted 1:50
in 3% normal goat serum-TBS solution. Dilution for
affinity-purified anti-Fisp12 antibody was 1:25. Routine control
was 3% normal goat serum-TBS, or irrelevant antibody (for example,
monoclonal anti-smooth muscle cell .alpha.-actin). Specificity of
staining was confirmed by incubation of anti-Cyr61 or anti-Fisp12
antibodies with an excess of the corresponding antigen on ice for
at least two hours prior to applying to sections. Complete
competition was observed. By contrast, cross-competition
(incubation of anti-Cyr61 antibodies with Fisp12 antigen and vice
versa) did not occur.
[0084] Primary antibodies were left on sections overnight at 4 C.
They were then washed with TES twice, and subjected to 30 minutes
incubation with secondary antibodies at room temperature. Secondary
antibodies used were goat anti-rabbit horseradish peroxidase
conjugates from Boehringer-Mannheim, Inc., Indianapolis, Ind. (used
at 1:400 dilution). Sections were washed twice in TBS and
chromogenic horseradish peroxidase substrate was applied for 5
minutes (1 mg/ml of diaminobenzidine in 50 mM Tris-HCl, pH 7.2 and
0.03% H.sub.2O.sub.2). Sections were then counterstained in
Ehrlich's haematoxylin or in Alcian blue, dehydrated and mounted in
Permount.
[0085] Mouse embryos between the neural fold (E8.5) and late
organogenesis (E18.5) stages of development were sectioned and
subjected to immunostaining with antigen-affinity-purified rabbit
anti-Cyr61 and anti-Fisp12 antibodies. As various organs developed
during embryogenesis, the presence of Cyr61 and Fisp12 was
determined. Cyr61 and Fisp12 were co-localized in a number of
tissues and organs. A notable example is the placenta, where both
proteins were readily detectable. In particular, both Cyr61 and
Fisp12 were found in and around the trophoblastic giant cells,
corroborating the previous detection of cyr61 mRNA in these cells
by in situ hybridization (O'Brien and Lau, 1992). Both Cyr61 and
Fisp12 signals in immunohistochemical staining were blocked by
either the corresponding Cyr61 or Fisp12 antigen but not by each
other, nor by irrelevant proteins, demonstrating specificity. In
general, Cyr61 and Fisp12 proteins could be detected both
intracellularly and extracellularly.
[0086] In addition to the placenta, both Cyr61 and Fisp12 were
detected in the cardiovascular system, including the smooth muscle,
the cardiomyocytes, and the endothelia. Both proteins were also
found in the bronchioles and the blood vessels in the lung. Low
levels of anti-Cyr61 and anti-Fisp12 staining could be detected
transiently in the skeletal muscle. This staining is associated
with connective tissue sheets, rather than myocytes; in this
instance the staining pattern was clearly extracellular.
[0087] A more complex pattern of distribution was found in the
epidermis and the epithelia. Both Cyr61 and Fisp12 staining could
be detected in the early, single-cell layer of embryonic epidermis,
as well as in later, multilayered differentiating epidermis. Fisp12
in epidermis declined to an undetectable level by the end of
gestation and remained as such through adulthood, whereas Cyr61 was
readily detectable in the epidermis. In the neonate, a strong
staining for Fisp12 was seen in the oral epithelia where Cyr61
staining was much weaker, while Cyr61 was found in the upper
jawbone where Fisp12 was not observed. The anti-Fisp12 signal in
the oral epithelia gradually increased and remained intense into
adulthood. In the tongue, both Cyr61 and Fisp12 were seen in the
keratinized epithelia, although the Fisp12 staining pattern, but
not that of Cyr61, excludes the filiform papillae.
[0088] Aside from the aforementioned sites of localization, Cyr61
and Fisp12 were also uniquely localized in several organ systems.
For example, Cyr61, but not Fisp12, was present in skeletal and
nervous systems. As expected from in situ hybridization results
(O'Brien and Lau, 1992), Cyr61 protein was readily detected in the
sclerotomal masses of the somites, and in cartilage and bone at
later stages of development. In contrast, Fisp12 was not detectable
in the skeletal system. Since correlation with chondrocytic
differentiation is one of the most striking features of cyr61
expression (O'Brien and Lau, 1992), the absence of Fisp12 in the
skeletal system may underscore an important difference in the
biological roles of Cyr61 and Fisp12. In the E14.5 embryo, Cyr61
could be detected in the ventral spinal cord, dorsal ganglia, axial
muscle and sclerotome-derived cartilaginous vertebrae. Fisp12,
however, was not detected in these tissues.
[0089] By contrast, Fisp12 was uniquely present in various
secretory tissues. Beginning at E16.5, Fisp12 could be detected in
the pancreas, kidneys, and salivary glands. In the pancreas, Fisp12
was strictly localized to the periphery of the islets of
Langerhans. In the kidney, strong Fisp12 staining was seen in the
collecting tubules and Henle's loops, regions where Cyr61 was not
found. In the mucous-type submandibular salivary gland only
collecting ducts stained for Fisp12, whereas in the mixed
mucous-serous submandibular gland, both serous acini and collecting
ducts stained. The signal in acini was peripheral, raising the
possibility that Fisp12 is capsule-associated. In simple holocrine
sebaceous glands a strong acellular Fisp12 signal was detected.
[0090] In summary, Cyr61 and Fisp12 have been co-localized in the
placenta, the cardiovascular system, the lung and the skin. Neither
protein was detected in the digestive system or the endocrine
glands. Unique localization of Cyr61 can be detected in the
skeletal and central nervous system, and Fisp12 is found in
secretory tissues where Cyr61 is not.
[0091] An issue closely related to protein expression concerns the
metabolic fate of the expressed proteins. Members of the
cysteine-rich protein family have been localized. As discussed
above, secreted Cyr61 is found in the ECM and on the cell surface
but not in the culture medium (Yang and Lau, 1991), yet secreted
Fisp12 was readily detected in the culture medium (Ryseck et al.,
1991). To address the question of whether Fisp12 is also
ECM-associated, the fate of both Cyr61 and Fisp12 was followed
using pulse-chase experiments. Serum-stimulated, sub-confluent NIH
3T3 fibroblasts were metabolically pulse-labeled for 1 hour and
chased in cold medium for various times. Samples were
fractionated-into cellular, ECM, and medium fractions followed by
immunoprecipitation to detect Cyr61 and Fisp12. Both proteins have
a similar short half-life of approximately 30 minutes in the
cellular fraction, which includes both newly synthesized
intracellular proteins as well as secreted proteins associated with
the cell surface (Yang and Lau, 1991). It should be noted that
since Cyr61 is quantitatively secreted after synthesis and only a
minor fraction is stably associated with the ECM, the bulk of
secreted Cyr61 is cell-surface associated (Yang and Lau, 1991).
[0092] A fraction of Cyr61 was chased into the ECM where it
remained stable for several hours. Newly synthesized Fisp12 was
also chased into the ECM, where its half-life was only about 1
hour. A larger fraction of Fisp12 was chased to the conditioned
medium, where no Cyr61 was detectable. Fisp12 in the conditioned
medium also had a short half-life of about 2 hours. Thus, whereas
Cyr61 is strongly associated with the ECM, Fisp12 is associated
with the ECM more transiently. This result suggests that Fisp12
might be able to act at a site distant from its site of synthesis
and secretion, whereas Cyr61 may act more locally.
[0093] Since many ECM proteins associate with the matrix via
interaction with heparan sulfate proteoglycans, the affinity with
which a protein binds heparin might be a factor in its interaction
with the ECM. The results of heparin binding assays, described
below, are consistent with this hypothesis.
EXAMPLE 7
Protein Purification
[0094] Serum-stimulated NIH 3T3 fibroblast cells were lysed to
provide a source of native murine Cyr61. Yang et al. Similarly,
human fibroblasts are a source of native human Cyr61.
[0095] Recombinant murine Cyr61 was purified from Sf9 cells
harboring the recombinant Baculovirus vector, described above,
containing the complete cyr61 coding sequence. Although murine
Cyr61 in Sf9 cell lysates formed insoluble aggregates as was the
case with bacterial cell extracts, approximately 10% of the Cyr61
synthesized was secreted into the medium in a soluble form. The
soluble, secreted form of Cyr61 was therefore subjected to
purification.
[0096] Initially, subconfluent Sf9 cells in monolayer cultures were
generated in supplemented Grace's medium (GIBCO-BRL, Inc., Grand
Island, N.Y.). Grace, Nature 195:788 (1962). The Sf9 cells were
then infected with 10 plaque-forming-units/cell of the recombinant
Baculovirus vector, incubated for 16 hours, and fed with serum-free
Grace's medium. These cells were expanded in serum-free Grace's
Medium. The conditioned medium was collected 48 hours
post-infection, although Cyr61 expression could be detected in the
medium 24 hours after infection. Subsequently, the conditioned
medium was cleared by centrifugation at 5000.times.g for 5 minutes,
chilled to 4.degree. C. adjusted to 50 mM MES, pH 6.0, 2 mM EDTA
(Ethylenediamine tetraacetic acid), 1 mM PMSF (Phenylmethylsulfonyl
fluoride) and applied to a Sepharose S column (Sigma Chemical Co.,
St. Louis, Mo.) at 4.degree. C. (5 ml void volume per 500 ml
medium). The column was washed with a buffer (50 mM MES, pH 6.0, 2
mM EDTA, 0.5 mM PMSF) containing 150 NaCl, and bound proteins were
eluted with a linear gradient of NaCl (0.2-1.0 M) in the same
buffer. The pooled fractions of Cyr61 eluted at 0.6-0.7 M NaCl as a
distinct broad peak. The column fractions were 90% pure, as
deter-mined by 10% SDS-PAGE followed by Coomassie Blue staining or
Western analysis, using techniques that are standard in the art.
Yang et al.; see also, Sambrook et al., supra. For Western
analysis, blots were probed with affinity-purified anti-Cyr61
antibodies as described in Yang et al., supra. After antibody
probing, Western blots were stained with ECL.TM. (i.e., Enhanced
ChemiLuminescence) detection reagents (Amersham Corp., Arlington
Heights, Ill.). Fractions containing Cyr61 were pooled, adjusted to
pH 7.5 with Tris-HCl, pH 7.5, and glycerol was added to 10% prior
to storage of the aliquots at -70.degree. C. Protein concentration
was determined by the modified Lowry method using the BioRad
protein assay kit (BioRad Laboratories, Inc., Hercules, Calif.).
This purification procedure was repeated at least five times with
similar results. The typical yield was 3-4 mg of 90% pure Cyr61
protein from 500 ml of conditioned medium.
[0097] Fisp12 was purified using a modification of the Cyr61
purification scheme (Kireeva et al., Experimental Cell Research, in
press). Serum-free conditioned media (500 ml) of Sf9 cells infected
at 10 pfu per cell were collected 48 hours post-infection and
loaded onto a 5-ml Sepharose S (Sigma Chemical Co., St. Louis, Mo.)
column. After extensive washing at 0.2 M and 0.4 M NaCl, bound
proteins were recovered by step elution with 50 mM MES (pH 6.0)
containing 0.5 M NaCl. Fractions containing Fisp12 of greater than
80% purity were pooled, NaCl adjusted to 0.15 M and the protein was
concentrated 3-5 fold on a 0.5 ml Sepharose S column with elution
of the protein at 0.6 M NaCl.
[0098] This purification scheme allowed the isolation of 1.5 mg of
recombinant Fisp12 protein of at least 80% purity from 500 ml of
serum-free conditioned media.
[0099] CTGF was purified by affinity chromatography using anti-PDGF
cross-reactivity between CTGF and PDGF, as described in U.S. Pat.
No. 5,408,040, column 7, line 15, to column 9, line 63,
incorporated herein by reference.
EXAMPLE 8
Polypeptide Characterization
[0100] The murine Cyr61 protein has a M.sub.r of 41,000 and is 379
amino acids long including the N-terminal secretory signal. There
is 91% amino acid sequence identity with the 381 amino acid
sequence of the human protein. Those regions of the mouse and human
proteins contributing to the similarity of the two proteins would
be expected to participate in the biological activities shared by
the two polypeptides and disclosed herein. However, the mouse and
human proteins do diverge significantly in the central portion of
the proteins, where each protein is devoid of cysteines. See,
O'Brien et al., Cell Growth & Diff. 3:645-654 (1992). A
cysteine-free region in the murine Cyr61 amino acid sequence is
found between amino acid residues 164 to 226 (SEQ ID NO:2). A
corresponding cysteine-free region is found in the human Cyr6]amino
acid sequence between amino acid residues 163 to 229 (SEQ ID NO:
4). More particularly, the mouse and human Cyr61 proteins are most
divergent between Cyr61 amino acids 170-185 and 210-225. Other
members of the ECM signalling molecule family of cysteine-rich
proteins, e.g., Fisp12 (SEQ ID NO:6) and CTGF (SEQ ID NO:8),
exhibit similar structures suggestive of secreted proteins having
sequences dominated by cysteine residues.
[0101] Because murine Cyr61 contains 38 cysteines in the 355 amino
acid secreted portion, the contribution of disulfide bond formation
to Cyr61 tertiary structure was investigated. Exposure of Cyr61 to
10 mM dithiothreitol (DTT) for 16 hours did not affect the ability
of Cyr61 to mediate cell attachment (see below). However, Cyr61 was
inactivated by heating at 75.degree. C. for 5 minutes, by
incubation in 100 mM HCl, or upon extensive digestion with
chymotrypsin. These results indicate that murine Cyr61 is a heat-
and acid-labile protein whose active conformation is not sensitive
to reducing agents. The aforementioned structural similarities of
murine and human Cyr61 polypeptides suggests that human Cyr61 may
also be sensitive to heat or acid, but insensitive to reducing
agents. In addition, Cyr61 is neither phosphorylated nor
glycosylated.
[0102] To determine if the purified recombinant murine Cyr61
described above was the same as native murine Cyr61, two additional
characteristics of mouse Cyr61 were determined. First, two
independent protein fingerprints of recombinant and native murine
Cyr61 were obtained. Purified recombinant murine Cyr61 and a lysate
of serum-stimulated 3T3 cells, known to contain native murine
Cyr61, were subjected to limited proteolysis with either trypsin or
chymotrypsin, and their digestion products were compared. Partial
tryptic digests of both the recombinant protein and cell lysate
resulted in two Cyr61 fragments of approximately 21 and 19 kDa.
Similarly, fingerprinting of both preparations by partial
chymotrypsin digestion produced stable 23 kDa fragments from
recombinant murine Cyr61 and native murine Cyr61.
[0103] Another criterion used to assess the properties of
recombinant Cyr61 was its ability to bind heparin, described below.
Purified recombinant murine Cyr61 bound quantitatively to
heparin-sepharose at 0.15 M NaCl and was eluted at 0.8-1.0 M NaCl.
This heparin binding capacity is similar to native murine Cyr61
obtained from serum-stimulated mouse fibroblasts. Because of the
similarities of the murine and human Cyr61 proteins, recombinant
human Cyr61 should exhibit properties similar to the native human
Cyr61, as was the case for the murine polypeptides.
[0104] The polypeptides of the invention also extend to fragments,
analogs, and derivatives of the aforementioned full-length ECM
signalling molecules such as human and mouse Cyr61. The invention
contemplates peptide fragments of ECM signalling molecules that
retain at least one biological activity of an ECM signalling
molecule, as described above. Candidate fragments for retaining at
least one biological activity of an ECM signalling molecule include
fragments that have an amino acid sequence corresponding to a
conserved region of the known ECM signalling molecules. For
example, fragments retaining one or more of the conserved cysteine
residues of ECM signalling molecules would be likely candidates for
ECM signalling molecule fragments that retain at least one
biological activity. Beyond the naturally occurring amino acid
sequences of ECM signalling molecule fragments, the polypeptides of
the invention include analogs of the amino acid sequences or
subsequences of native ECM signalling molecules.
[0105] ECM signalling molecule analogs are polypeptides that differ
in amino acid sequence from native ECM signalling molecules but
retain at least one biological activity of a native ECM signalling
molecule, as described above. These analogs may differ in amino
acid sequence from native ECM signalling molecules. e.g., by the
insertion, deletion, or conservative substitution of amino acids. A
conservative substitution of an amino acid, i.e., replacing an
amino acid with a different amino acid of similar properties (e.g.,
hydrophilicity, degree and distribution of charged regions) is
recognized in the art as typically involving a minor change. These
minor changes can be identified, in part, by considering the
hydropathic index of amino acids, as understood in the art. Kyte et
al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an
amino acid is based on a consideration of its hydrophobicity and
charge, and include the following values: alanine (+1.8), arginine
(-4.5), asparagine (-3.5), aspartate (-3.5), cysteine/cystine
(+2.5), glycine (-0.4), glutamate (-3.5), glutamine (-3.5),
histidine (-3.2), isoleucine (+4.5), leucine (+3.8), lysine (-3.9),
methionine (+1.9), phenylalanine (+2.8), proline (-1.6), serine
(-0.8), threonine (-0.7), tryptophan (-0.9), tyrosine (-1.3), and
valine (+4.2). It is known in the art that amino acids of similar
hydropathic indexes can be substituted and still retain protein
function. Preferably, amino acids having hydropathic indexes of
.+-.2 are substituted.
[0106] The hydrophilicity of amino acids can also be used to reveal
substitutions that would result in proteins retaining biological
function. A consideration of the hydrophilicity of amino acids in
the context of a polypeptide permits calculation of the greatest
local average hydrophilicity of that polypeptide, a useful measure
that has been reported to correlate well with antigenicity and
immunogenicity. U.S. Pat. No. 4,554,101, incorporated herein by
reference. Hydrophilicity values for each of the common amino
acids, as reported in U.S. Pat. No. 4,554,101, are: alanine (-0.5),
arginine (+3.0), asparagine (+0.2), aspartate (+3.0.+-.1), cysteine
(-1.0), glycine (0), glutamate (+3.0.+-.1), glutamine (+0.2),
histidine (-0.5), isoleucine (-1.8), leucine (-1.8), lysine (+3.0),
methionine (-1.3), phenylalanine (-2.5), proline (-0.5.+-.1),
serine (+0.3), threonine (-0.4), tryptophan (-3.4), tyrosine
(-2.3), and valine (-1.5). Substitution of amino acids having
similar hydrophilicity values can result in proteins retaining
biological activity, for example immunogenicity, as is understood
in the art. Preferably, substitutions are performed with amino
acids having hydrophilicity values within .+-.2 of each other. Both
the hyrophobicity index and the hydrophilicity value of amino acids
are influenced by the particular side chain of that amino acid.
Consistent with that observation, amino acid substitutions that are
compatible with biological function are understood to depend on the
relative similarity of the amino acids, and particularly the side
chains of those amino acids, as revealed by the hydrophobicity,
hydrophilicity, charge, size, and other properties.
[0107] Additionally, computerized algorithms are available to
assist in predicting amino acid sequence domains likely to be
accessible to an aqueous solvent. These domains are known in the
art to frequently be disposed towards the exterior of a protein,
thereby potentially contributing to binding determinants, including
antigenic determinants. Having the DNA sequence in hand, the
preparation of such analogs is accomplished by methods well known
in the art (e.g., site-directed) mutagenesis and other
techniques.
[0108] Derivatives of ECM signalling molecules are also
contemplated by the invention. ECM signalling molecule derivatives
are proteins or peptides that differ from native ECM signalling
molecules in ways other than primary structure (i.e., amino acid
sequence). By way of illustration, ECM signalling molecule
derivatives may differ from native ECM signalling molecules by
being glycosylated, one form of post-translational modification.
For example, polypeptides may exhibit glycosylation patterns due to
expression in heterologous systems. If these polypeptides retain at
least one biological activity of a native ECM signalling molecule,
then these polypeptides are ECM signalling molecule derivatives
according to the invention. Other ECM signalling molecule
derivatives include, but are not limited to, fusion proteins having
a covalently modified N- or C-terminus, PEGylated polypeptides,
polypeptides associated with lipid moieties, alkylated
polypeptides, polypeptides linked via an amino acid side-chain
functional group to other polypeptides or chemicals, and additional
modifications as would be understood in the art. In addition, the
invention contemplates ECM signalling molecule-related polypeptides
that bind to an ECM signalling molecule receptor, as described
below.
[0109] The various polypeptides of the present invention, as
described above, may be provided as discrete polypeptides or be
linked, e.g., by covalent bonds, to other compounds. For example,
immunogenic carriers such as Keyhole Limpet Hemocyanin may be bound
to a ECM signalling molecule of the invention.
EXAMPLE 9
Heparin Binding Assay
[0110] The heparin binding assay for native murine Cyr61, described
in Yang et al., was modified for the purified recombinant murine
protein. Initially, recombinant purified Cyr61 was suspended in
RIPA (Radioimmuno-precipitation assay) buffer (150 mM NaCl, 1.0%
NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0, 1 mM
phenylmethylsulfonyl fluoride). Next, 200 .mu.l of a 50% (v/v)
slurry of heparin-Sepharose CL 6B beads (Pharmacia-LKB
Biotechnology, Inc., Piscataway, N.J.) was added to 100 .mu.l of
the recombinant Cyr61 solution and incubated for 1 hour. Under
these conditions, human Cyr61 was quantitatively bound to
heparin-agarose. Application of a salt concentration gradient in
RIPA buffer resulted in the elution of recombinant murine Cyr61 at
0.8-1.0 M NaCl. The elution profile of the recombinant protein was
similar to the elution profile for native murine Cyr61.
[0111] One might expect that Fisp12 would bind heparin with lower
affinity than Cyr61, as it does not interact with the ECM as
strongly as Cyr61. To examine this possibility, metabolically
labeled [.sup.35S-cysteine; 100 .mu.Ci per 100 mm dish: ICN] cell
lysates were incubated with heparin agarose beads which were
subsequently washed to remove unbound proteins. Bound proteins were
eluted in increasing salt concentrations. Fisp12 from cell lysates
was retained on heparin agarose but was eluted by 0.2 to 0.6 M NaCl
with peak elution at 0.4 M NaCl. This is in contrast to Cyr61,
which was eluted at significantly higher concentrations of NaCl.
This difference in heparin binding is consistent with the differing
affinities of Cyr61 and Fisp12 for the ECM, suggesting that binding
to heparan sulfate proteoglycans may be a primary mechanism by
which both proteins associate with the ECM.
EXAMPLE 10
Receptors
[0112] Human Cyr61, like murine Cyr61, was localized to the cell
surface and ECM. The localization of Cyr61 to the cell surface
implicated a cell surface receptor binding Cyr61. Consistent with
that implication, the biological effects of Cyr61 are mediated by
the .alpha..sub.v.beta..sub.3 integrin, or vitronectin receptor.
The .alpha..sub.v.beta..sub.3 integrin, in association with other
integrins, forms protein clusters providing focal points for
cytoskeletal attachment. Cyr61 induces the formation of protein
clusters, including the protein clusters containing the
.alpha..sub.v.beta..sub.3 integrin. In addition, using an in vitro
assay, the biological effects of Cyr61, including Cyr61-induced
cell adhesion and mitogenesis, were abolished by the addition of
either one of two monoclonal antibodies-LM609 (Cheresh, Proc. Natl.
Acad. Sci. [USA] 84:6471-6475 [1987]) or anti-VnR 1 (Chen et al.,
Blood 86:2606-2615 [1995])--directed to the
.alpha..sub.v.beta..sub.3 integrin. This data led to the
identification of the .alpha..sub.v.beta..sub.3 integrin as the
Cyr61 receptor.
[0113] Cyr61 induction of HUVE cell adhesion, described in Example
13 below, led to an investigation of the divalent cation-sensitive
cell surface receptors expressed by HUVE cells. The cell adhesion
properties of Cyr61 were used to identify the receptor, which is a
divalent cation-sensitive cell surface receptor. The ability of
Cyr61 to mediate cell adhesion, coupled with the strict requirement
for divalent cations in the process, indicated that Cyr61 interacts
with one of the divalent cation-dependent cell adhesion molecules
from the integrin, selectin, or cadherin families. Ruoslahti et
al., Exp. Cell Res. 227:1-11 (1996). Using well-characterized
approaches to receptor identification, a series of inhibition
studies were conducted. Inhibitors, or blocking agents, of various
degrees of specificity (EDTA, similar to the EGTA described above;
inhibitory peptides bearing variants of the RGD (single letter
amino acid code) integrin recognition motif, such as RGDS, SGDR,
and RGDSPK (Ruoslahti, et al., Science 238:491-497 [1987],
Ruoslahti, E., Ann. Rev. of Cell and Dev. Biol. 12:698-715 [1996]);
and known, specific anti-receptor antibodies) were used to identify
a Cyr61 receptor. That receptor was the .alpha..sub.v.beta..sub.3
integrin, also known to function as the vitronectin receptor.
Confirmation of that identification was obtained by showing that
antibody LM609, a specific anti-.alpha..sub.v.beta..sub.3 integrin
antibody, could block the effect of Cyr61 on cell adhesion.
Integrins form a large family of heterodimeric adhesion receptors,
with a broad ligand specificity range, involved in cell-cell and
cell-matrix interactions. Beyond their requirement for divalent
cations and their involvement in cell-matrix adhesion events
[Hynes, R. O., Cell 69:11-25 (1992)], integrins also are involved
in cell migration. [Damsky et al., Curr. Opin. Cell Biol. 4:772-781
(1992): Doerr et al., J. Biol. Chem. 271:2443-447 (1996)] and
proliferation [Juliano et al., J. Cell Biol., 120:577-585 (1993);
Plopper et al., Mol. Biol. Cell 6:1349-1365 (1995); and Clark et
al., Science 268:233-239 (1995)], two additional processes
associated with Cyr61 activity. The .alpha..sup.v.beta..sub.3
integrin was found to be essential for Cyr61-mediated cell
adhesion.
[0114] Characterization of CTGF binding to cells has been reported
to occur through a cell surface receptor that also interacts with
PDGF-BB (the BB isoform of PDGF), as recited in U.S. Pat. No.
5,408,040, column 11, line 10, to column 12, line 14, incorporated
herein by reference. The identification of the foregoing receptors
permits the the design and production of molecules and which bind
to the respective receptors to inhibit the activities of ECM
molecules.
EXAMPLE 11
Anti-ECM Signalling Molecule Antibodies
[0115] Antibodies, optionally attached to a label or to a toxin as
described below, are also contemplated by the present invention.
The availability of the human cyr61 cDNA sequence and the Cyr61
deduced protein sequence facilitate the implementation of methods
designed to elicit anti-Cyr61 antibodies using a number of
techniques that are standard in the art. Harlow et al.
[0116] In one embodiment, polyclonal antibodies directed against
Cyr61 are generated. The generation of anti-Cyr61 antibodies
specific for human Cyr61, for example, is optimized by designing
appropriate antigens. The human Cyr61 protein is 381 amino acids
long, including the N-terminal secretory signal. As described
above, human Cyr61 exhibits a 91% amino acid sequence identity with
the 379 amino acid sequence of the mouse protein. However, the
mouse and human proteins diverge most significantly in the central
portion of the proteins, where they are devoid of cysteines (see
above). These sequence differences are exploited to elicit
antibodies specific to the human Cyr61 by using as an antigen a
peptide having a sequence derived from one of the divergent regions
in the human protein, although antibodies directed to a conserved
region are also contemplated by the invention.
[0117] In another embodiment of the present invention, monoclonal
antibodies are elicited using intact recombinant human Cyr61
although a fragment may be used. Female BALB/c mice are inoculated
intraperitoneally with a mixture of 0.25 ml recombinant human Cyr61
(5-50 micrograms), bacterially produced or produced in eukaryotic
cells, and 0.25 ml complete Freund's adjuvant. Fourteen days later
the injections are repeated with the substitution of incomplete
Freund's adjuvant for complete Freund's adjuvant. After an
additional two weeks, another injection of human Cyr61 in
incomplete Freund's adjuvant is administered. About two weeks after
the third injection, tail bleeds are performed and serum samples
are screened for human anti-Cyr61 antibodies by immunoprecipitation
with radiolabeled recombinant human Cyr61. About two months after
the initial injection, mice whose sera yield the highest antibody
titers are given booster injections of Cyr61 (5-50 micrograms in
incomplete Freund's adjuvant, 0.1 ml intravenously and 0.1 ml
intraperitoneally). Three days after the booster injection, the
mice are sacrificed. Splenocytes are then isolated from each mouse
using standard techniques, and the cells are washed and
individually fused with a myeloma cell line, e.g., the X63Ag8.653
cell line (Harlow, et al.), using polyethylene glycol, by
techniques that are known in the art. Other suitable cell lines for
fusion with splenocytes are described in Harlow et al., at page
144, Table 6.2, incorporated herein by reference. Fused cells are
removed from the PEG solution, diluted into a counter-selective
medium (e.g., Hypoxanthine-Aminopterin-Thymidine or HAT medium) to
kill unfused myeloma cells, and inoculated into multi-well tissue
culture dishes.
[0118] About 1-2 weeks later, samples of the tissue culture
supernatants are removed from wells containing growing hybridomas,
and tested for the presence of anti-Cyr61 antibodies by binding to
recombinant human Cyr61 bound to nitrocellulose and screening with
labeled anti-immunoglobulin antibody in a standard antibody-capture
assay. Cells from positive wells are grown and single cells are
cloned on feeder layers of splenocytes. The cloned cell lines are
stored frozen. Monoclonal antibodies are collected and purified
using standard techniques, e.g., hydroxylapatite chromatography. In
an alternative, Cyr61 peptides used as antigens, may be attached to
immunogenic carriers such as keyhole limpet hemocyanin carrier
protein, to elicit monoclonal anti-Cyr61 antibodies.
[0119] Another embodiment involves the generation of antibody
products against a fusion protein containing part, or all, of human
Cyr61, including enough of the protein sequence to exhibit a useful
epitope in a fusion protein. The fusion of the large subunit of
anthranilate synthase (i.e., TrpE) to marine Cyr61, and the fusion
of glutathione S-transferase (i.e., GST) to murine Cyr61, have been
used to successfully raise antibodies against murine Cyr61. Yang et
al. In addition, a wide variety of polypeptides, well known to
those of skill in the art, may be used in the formation of Cyr61
fusion polypeptides according to the invention.
[0120] More particularly, Yang reported a TrpE-Cyr61 fusion
polypeptide that was expressed from a recombinant clone constructed
by cloning a fragment of the murine cyr61 cDNA containing
nucleotide 456 through nucleotide 951 (encoding Cyr61 amino acids
93-379) into the SacI site of the pATH1 vector. Dieckman et al., J.
Biol. Chem. 260:1513-1520 (1985). The recombinant construct was
transformed into a bacterial host, e.g., E. coli K12, and
expression of the fusion protein was induced by addition of 25
.mu.g/ml indoleacrylic acid to growing cultures. Subsequently,
cells were lysed and total cell lysate was fractionated by
electrophoresis on a 7.5% polyacrylamide gel. The fusion protein of
predicted size was the only band induced by indoleacrylic acid;
that band was eluted from the gel and used as an antigen to
immunize New Zealand White rabbits (Langshaw Farms) using
techniques that are standard in the art. Harlow et al. In addition
to polyclonal antibodies, the invention comprehends monoclonal
antibodies directed to such fusion proteins.
[0121] In other embodiments of the invention, recombinant antibody
products are used. For example, chimeric antibody products,
"humanized" antibody products, and CDR-grafted antibody products
are within the scope of the invention. Kashmiri et al., Hybridoma
14:461-473 (1995), incorporated herein by reference. Also
contemplated by the invention are antibody fragments. The antibody
products include the aforementioned types of antibody products used
as isolated antibodies or as antibodies attached to labels. Labels
can be signal-generating enzymes, antigens, other antibodies,
lectins, carbohydrates, biotin, avidin, radioisotopes, toxins,
heavy metals, and other compositions known in the art; attachment
techniques are also well known in the art.
[0122] Anti-Cyr61 antibodies are useful in diagnosing the risk of
thrombosis, as explained more fully in Example 20 below. In
addition, anti-Cyr61 antibodies are used in therapies designed to
prevent or relieve undesirable clotting attributable to abnormal
levels of Cyr61. Further, antibodies according to the invention can
be attached to toxins such as ricin using techniques well known in
the art. These antibody products according to the invention are
useful in delivering specifically-targeted cytotoxins to cells
expressing Cyr61, e.g., cells participating in the
neovascularization of solid tumors. These antibodies are delivered
by a variety of administrative routes, in pharmaceutical
compositions comprising carriers or diluents, as would be
understood by one of skill in the art.
[0123] Antibodies specifically recognizing Fisp12 have also been
elicited using a fusion protein. The antigen used to raise
anti-Fisp12 antibodies linked glutathione-S-transferase (GST) to
the central portion of Fisp12 (GST-Fisp12), where there is no
sequence similarity to Cyr61 (O'Brien and Lau, 1992). A construct
containing cDNA encoding amino acids 165 to 200 of Fisp12 was fused
to the glutathione-S-transferase (GST) coding sequence. This was
done by using polymerase chain reaction (PCR) to direct synthesis
of a fragment of DNA encompassing that fragment of fisp12 flanked
by a 5' BamHI restriction site and a 3' EcoRI restriction site. The
5' primer has the sequence 5'-GGGGATCTGTGACGAGCCCAAGGAC-3' (SEQ ID
NO: 9) and the 3' primer has the sequence
5'-GGGAATTCGACCAGGCAGTTGGCTCG-3' (SEQ ID NO: 10). For
Cyr61-specific antiserum, a construct fusing the central portion of
Cyr61 (amino acids 163 to 229), which contains no sequence
similarity to Fisp12, to GST was made in the same manner using the
5' primer 5'-GGGGATCCTGTGATGAAGACAGCATT-3' (SEQ ID NO: 11) and the
3' primer 5'-GGGAATTCAACGATGCATTTCTGGCC-3' (SEQ ID NO: 12). These
were directionally cloned into pGEX2T vector (Pharmacia-LKB, Inc.)
and the clones confirmed by sequence analysis. The GST-fusion
protein was isolated on glutathione sepharose 4B (Pharmacia-LKB,
Inc.) according to manufacturer's instructions, and used to
immunize New Zealand white rabbits. For affinity purifications,
antisera were first passed through a GST-protein affinity column to
remove antibodies raised against GST, then through a GST-Fisp12 or
GST-Cyr61 protein affinity column to isolate anti-Fisp12 or
anti-Cyr61 antibodies (Harlow et al., 1988).
[0124] These antibodies immunoprecipitated the correct size Fisp12
protein product synthesized in vitro directed by fisp12 mRNA. The
antibodies are specific for the Fisp12 polypeptide and show no
cross-reactivity with Cyr61.
[0125] Polyclonal antibodies recognizing CTGF are also known. U.S.
Pat. No. 5,408,040, column 7, line 41, to column 9, line 63,
incorporated by reference hereinabove, reveals an immunological
cross-reactivity between PDGF and CTGF, as described above.
EXAMPLE 12
Inhibitor Peptides
[0126] Another embodiment of the present invention involves the use
of inhibitory peptides in therapeutic strategies designed to
inhibit the activity of the Cyr61 protein. One approach is to
synthesize an inhibitory peptide based on the protein sequence of
Cyr61. For example, a peptide comprising an amino acid sequence
that is conserved between murine Cyr61 (SEQ ID NO:2) and human
Cyr61 (SEQ ID NO:4) competes with native Cyr61 for its binding
sites. This competition thereby inhibits the action of native
Cyr61. For example, administration of an inhibitory peptide by
well-known routes inhibits the capacity of Cyr61 to influence the
cascade of events resulting in blood clots, the vascularization of
tumors, or the abnormal vascularization of the eye (e.g., eye
disorders characterized by vascularization of the retina or the
vitreous humor), etc. In particular, an inhibitory peptide prevents
Cyr61 from inhibiting the action of Tissue Factor Pathway
Inhibitor, or TFPI, as described below.
[0127] In an embodiment of the invention, inhibitory peptides were
designed to compete with Cyr61. These inhibitory peptides, like the
antibodies of the preceding Example, exemplify modulators of Cyr61
activity, as described in the context of a variety of assays for
Cyr61 activity that are disclosed herein. The peptide design was
guided by sequence comparisons among murine Cyr61, Fisp12, and Nov
(an avian proto-oncogene). The amino acid sequences of several
members of this family are compared in FIG. 1. These types of
sequence comparisons provide a basis for a rational design for a
variety of inhibitory peptides. Some of these designed peptides,
for example peptides spanning amino acids 48-68 (SEQ ID NO:13),
115-135 (SEQ ID NO:14), 227-250 (SEQ ID NO:15), 245-270 (SEQ ID
NO:16), and 310-330 (SEQ ID NO:17) of SEQ ID NO:2, have been
synthesized. A comparison of the murine Cyr61 amino acid sequence
and the human Cyr61 amino acid sequence reveals that similar
domains from the human protein may be used in the design of
peptides inhibiting human Cyr61. In addition, sequence comparisons
may involve the human Cyr61 amino acid sequence; comparisons may
also include the human homolog of Fisp12. Connective Tissue Growth
Factor, also identified as a member of this protein family. O'Brien
et al. (1992).
[0128] Inhibitory peptides may also be designed to compete with
other ECM signalling molecules, e.g., Fisp12 or CTGF, for binding
to their respective receptors. The design of inhibiting peptides is
facilitated by the similarity in amino acid sequences among the ECM
signalling molecules. In addition, inhibitory peptide design may be
guided by one or more of the methods known in the art for
identifying amino acid sequences likely to comprise functional
domains (e.g., hydrophilic amino acid sequences as external/surface
protein domains; sequences compatible with .alpha.-helical
formation as membrane-spanning domains). These methods have been
implemented in the form of commercially available software, known
to those of ordinary skill in the art. See e.g., the
Intelligenetics Suite of Analytical Programs for Biomolecules.
Intelligenetics, Inc., Mountain View, Calif. Using these
approaches, inhibitory peptides interfering with the biological
activity of an ECM signalling molecule such as Cyr61, Fisp 12 or
CTGF, may be designed. With the design of the amino acid sequence
of an inhibitory peptide, production of that peptide may be
realized by a variety of well-known techniques including, but not
limited to, recombinant production and chemical synthesis.
Exemplary peptides that have been shown to specifically inhibit at
least one biological activity of Cyr61 include peptides exhibiting
the "RGD" motif, or motif variants such as "RGDS," "RGDSPK," "GDR,"
or "SGDR." (Ruoslahti, et al., Science 238:491-497 [1987],
Ruoslahti, E., Ann. Rev. of Cell and Dev. Biol. 12:698-715 [1996])
as described in Example 10 above.
EXAMPLE 13
Cell Adhesion
[0129] Another embodiment of the invention is directed to the use
of Cyr61 to mediate cellular attachment to the extracellular
matrix. Induction of cellular adhesion was investigated using
murine Cyr61, fibronectin, and bovine serum albumin (BSA).
Immunological 96-well plates (Falcon brand) were coated with 50
.mu.l of 0.1% BSA in PBS at 4.degree. C. in the presence of 0-30
.mu.g/ml concentrations of murine Cyr61 or fibronectin. After two
hours exposure to the coating solution, non-diluted immune or
pre-immune antisera (30 .mu.l/well), or affinity-purified
anti-Cyr61 antibodies were added. For some wells, the coating
mixture was adjusted to 10 mM DTT or 100 mM HCl. After 16 hours
incubation, the coating solution was removed and the well surface
was blocked with 1% BSA in phosphate-buffered saline (PBS) for 1
hour at room temperature. HUVE cells were plated in Ham's complete
F12K medium [GIBCO-BRL. Inc.; Ham, Proc. Natl. Acad. Sci. (USA)
53:786 (1965)] at 5.times.10.sup.3-10.sup.4 cells/well.
Cycloheximide was added to 100 .mu.g/ml immediately before plating
and monensin was added to 1 .mu.M 14 hours before plating. After a
2-hour incubation at 37.degree. C., the wells were washed with PBS
and attached cells were fixed and stained with methylene blue. The
attachment efficiency was determined by quantitative dye extraction
and measurement of the extract absorbance at 650 nm. Oliver et al.,
J. Cell. Sci. 92:513-518 (1989).
[0130] HUVE cells attached poorly to dishes treated with BSA alone,
but adhered well to dishes coated with fibronectin. Murine
Cyr61-coated surfaces also supported HUVE cell attachment in a
dose-dependent manner, similar to fibronectin. For example, at 1
.mu.g/ml. Cyr61 and fibronectin yielded A.sub.650 values of 0.1. An
A.sub.650 value of 0.5 corresponded to the attachment of
6.times.10.sup.3 cells. At the other end of the tested
concentration range, 30 .mu.g/ml, Cyr61 yielded an A.sub.650 of
0.8; fibronectin yielded an A.sub.650 of 0.9. Cyr61 also promoted
the attachment of NIH 3T3 cells, though less effectively than
fibronectin. Cyr61-mediated cell attachment can be observed as
early as 30 minutes after plating, as visualized by light
microscopy.
[0131] The adhesion of HUVE cells on murine Cyr61-coated surfaces
was specifically inhibited by anti-Cyr61 antiserum and by
affinity-purified anti-Cyr61 antibodies, but not by pre-immune
serum. In contrast, attachment of cells to fibronectin-coated
dishes was not affected by either the anti-Cyr61 antiserum or
affinity-purified anti-Cyr61 antibodies. These results show that
enhancement of cell adhesion is a specific activity of the Cyr61
protein. Furthermore, the Cyr61-mediated cell attachment was
insensitive to cycloheximide or monensin treatments indicating that
Cyr61 does not act by inducing de novo synthesis of ECM components,
stimulation of fibronectin, or collagen secretion. Rather, the data
support the direct action of Cyr61 on cells in effecting adhesion.
The Cyr61-mediated attachment of HUVE cells was completely
abolished by the presence of EGTA; however, attachment was restored
by the addition of CaCl.sub.2 or MgSO.sub.4 to the medium. These
results indicate that the interaction between Cyr61 and its cell
surface receptor requires divalent cations, consistent with the
observations leading to the identification of the
.alpha..sub.v.beta..sub.3 integrin as the Cyr61 receptor described
in Example 10, above.
[0132] The ability of Cyr61 to promote cell adhesion, and the
ability of molecules such as anti-Cyr61 antibodies to inhibit that
process is exploited in an assay for modulators of cell adhesion.
The assay involves a comparison of cell adhesion to surfaces, e.g.,
plastic tissue culture wells, that are coated with Cyr61 and a
suspected modulator of cell adhesion. As a control, a similar
surface is coated with Cyr61 alone. Following contact with suitable
cells, the cells adhering to the surfaces are measured. A relative
increase in cell adhesion in the presence of the suspected
modulator, relative to the level of cell adherence to a
Cyr61-coated surface, identifies a promoter of cell adhesion. A
relative decrease in cell adhesion in the presence of the suspected
modulator identifies an inhibitor of cell adhesion.
[0133] The identification of a Cyr61 receptor led to the
development of a rapid and specific ligand-receptor assay (i.e.,
integrin binding assay) for Cyr61. Monoclonal antibody LM609
(anti-.alpha..sub.v.beta..sub.3) has been described. Cheresh, 1987.
Monoclonal antibody JBS5 (anti-fibronectin antibody) was purchased
from Chemicon. Anti-human and anti-bovine vitronectin antisera were
from Gibco BRL. HRP-conjugated goat anti-rabbit antibody was from
KPL. RGDSPK peptide was from Gibco BRL; RGDS and SDGR peptides were
from American Peptide Company. The peptides for functional assays
were dissolved in PBS at 10 mg/ml and the pH was adjusted to
7.5-8.0 with NaOH. Human plasma vitronectin was from Collaborative
Biomedical Products.
[0134] .alpha..sub.v.beta..sub.3 integrin purification from HUVE
cell lysates was done as described in Pytela et al., Meth.
Enzymol., 144:475-489 (1987). Briefly, 10.sup.8 cells were lysed in
1 ml of PBS containing 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 0.5 mM
PMSF and 100 mM octylglucoside. The lysate was passed four times
through a 0.5 ml column containing RGDSPK Sepharose (prepared from
the cyanogen bromide activated Sepharose CL 4B as described in Lam,
S.C.-T., J. Biol. Chem., 267:5649-5655 (1992). The column was
washed with 10 ml of the lysis buffer and the bound protein was
eluted with 2 ml of the same buffer containing 1 mM RGDS peptide at
room temperature. The .alpha..sub.v.beta..sub.3 integrin was
dialyzed against PBS containing 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 5
mM octyl-glucoside and 0.1 mM PMSF with three changes of the
dialysis buffer to remove the RGDS peptide. The protein was stored
in aliquots at -70.degree. C. The purity of the integrin was
determined by SDS-PAGE under non-reducing conditions, followed by
silver staining. Western blotting with anti-CD47 antibody showed
that this .alpha..sub.v.beta..sub.3 integrin preparation does not
contain any integrin-associated proteins.
[0135] The integrin binding assay was developed in accordance with
the disclosures in Brooks et al., Cell 85:683-693 (1996), and Lam,
S.C.-T. (1992). Approximately 50 ng of the integrin in a total
volume of 50 .mu.l were added per well of 96-well immunological
Pro-Bind plates (Falcon) and incubated overnight at 4.degree. C.
Non-specific sites were blocked with 20 mg/ml BSA in the same
buffer and washed four times in that buffer. Treated plates were
incubated with 1 .mu.g/ml Cyr61 or 0.1 .mu.g/ml vitronectin for 3
hours at room temperature. EDTA (5 mM), RGDS peptide (0.5 mM) and
blocking antibodies were either preincubated with the immobilized
integrin for 1 hour before the addition of the protein ligand or
added along with the ligand. The final dilution of the LM609
ascites fluid was 1:200. Bound proteins were detected by specific
polyclonal antisera (anti-Cyr61 antiserum was diluted 1:500 and
anti-vitronectin antiserum was diluted 1:1000 in PBS containing 1
mM CaCl.sub.2, 1 mM MgCl.sub.2, and 5 mg/ml BSA) followed by a
secondary antibody-horseradish peroxidase conjugate (1:20000 in the
same buffer). Plates were rinsed four times with PBS containing 1
mM CaCl.sub.2 and 1 mM MgCl.sub.2 after each incubation.
Horseradish peroxidase (HRP) was detected with an HRP immunoassay
kit (Bio-Rad Laboratories). The colorimetric reaction was developed
for 15-30 minutes at room temperature, stopped by the addition of
H.sub.2SO.sub.4, and the absorbance at 450 nm was measured. Those
of ordinary skill in the art will understand that a variety of
detection techniques could be employed in place of the
enzyme-linked immunological approach exemplified. For example,
other labels such as radiolabels, fluorescent compounds and the
like could be bound, e.g., covalently, to an antibody or other
agent recognizing the peptide of interest such as Cyr61.
[0136] The results of integrin binding assays showed that
vitronectin and Cyr61 bound to the immobilized integrin. Further,
both Cyr61 and vitronectin binding to .alpha..sub.v.beta..sub.3
were saturable. The concentration of Cyr61 at which saturation was
reached was significantly higher than the concentration of
vitronectin required for saturation. This difference may reflect a
lower affinity of .alpha..sub.v.beta..sub.3 for Cyr61 compared to
vitronectin, which is in agreement with the results of cell
adhesion assays, which show that HUVE cells adhere to vitronectin
and, more weakly, to Cyr61, in a concentration-dependent manner
(see below). The specificity of the interaction was addressed by
blocking the ligand binding site of the integrin using any one of
several techniques, including divalent cation deprivation, RGDS
peptide competition, and LM609 antibody inhibition. The interaction
of both proteins (Cyr61 and vitronectin) with
.alpha..sub.v.beta..sub.3 was inhibited by EDTA, the RGDS peptide,
and the LM609 antibody. These properties of the Cyr61 interaction
with .alpha..sub.v.beta..sub.3 were also in agreement with the
results of the cell adhesion assay and indicated that HUVE cell
adhesion to Cyr61 was mediated by the direct interaction of Cyr61
with the .alpha..sub.v.beta..sub.3 integrin.
[0137] In addition, Cyr61 induces focal adhesion, i.e., cell
surface foci for cytoskeletal attachments. Focal adhesion is
effected by cell surface protein complexes or clusters. These
protein clusters are complex, including a variety of receptors from
the integrin family, and a variety of protein kinases. The
induction of focal adhesion by Cyr61 is reflected in the capacity
of Cyr61 to induce particular members of these cell surface protein
clusters. For example, Cyr61 induces the phosphorylation of Focal
Adhesion Kinase, a 125 kDa polypeptide, and Paxillin, another
protein known to be involved in the focal adhesion cell surface
protein complexes. Moreover, indirect immunofluorescence studies
have shown that Cyr61 is bound to a receptor (see above) in focal
adhesive plaques. The plaques, in turn, are characteristic of focal
adhesion protein complexes. Focal Adhesion Kinase, Paxillin, and
.alpha..sub.v.beta..sub.3 Integrin are co-localized to the focal
adhesion plaques produced by focal adhesion complex formation
induced by Cyr61. These focal adhesion protein complexes bind Cyr61
at the cell surface; the complexes also attach internally to the
cytoskeleton. Therefore, murine Cyr61, and human Cyr61 (see below),
are, in part, adhesion molecules, a characteristic distinguishing
Cyr61 from conventional growth factors. Those of skill in the art
will also recognize that the .alpha..sub.v.beta..sub.3 integrin can
be used, in conjunction with Cyr61, to screen for modulators of
Cyr61 binding to its receptor. In one embodiment, the integrin is
immobilized and exposed to either (a) Cyr61 and a suspected
modulator of receptor binding; or (b) Cyr61 alone. Subsequently,
bound Cyr61 is detected, e.g., by anti-Cyr61 antibody that is
labeled using techniques known in the art, such as radiolabelling,
fluorescent labelling, or the use of enzymes catalyzing
colorimetric reactions. A promoter of Cyr61 binding to its receptor
would increase binding of Cyr61 (and an inhibitor would decrease
Cyr61), relative to the binding by Cyr61 alone.
[0138] In another embodiment of the invention, the effect of murine
Cyr61 on cell morphogenesis was assessed by a cell spreading assay.
Polystyrene Petri dishes were coated with 2 ml of a 10 .mu.g/ml
solution of Cyr61 or fibronectin in PBS with 0.1% BSA and treated
as described above. A third plate was treated with BSA and served
as a control. Each dish received 7.times.10.sup.6 cells and was
incubated for 2 hours. Cell spreading was analyzed by microscopy at
100-fold magnification. The results indicate that murine Cyr61
induces HUVE cell spreading to approximately the same extent as
fibronectin. The efficient attachment (see above) and spreading of
cells on marine Cyr61-coated substrates indicated that Cyr61 may
interact with a signal-transducing cell surface receptor, leading
to a cascade of cytoskeletal rearrangements and possible formation
of focal contacts. Consequently, Cyr61 and Cyr61-related
polypeptides may prove useful in controlling cell adhesion, e.g.,
the cell adhesion events that accompany metastasizing cancer cells,
organ repair and regeneration, or chondrocyte colonization of
prosthetic implants, discussed below.
[0139] In contrast to mouse Cyr61 which mediated both HUVE cell
attachment and migration, hCyr61 was found to mediate cell adhesion
but not spreading of HUVE cells. Immunological plates (96-well
ProBind assay plates, Falcon) were coated with 0.1-30 .mu.g/ml
hCyr61, fibronectin (Gibco BRL) or vitronectin (Gibco BRL) in
phosphate-buffered saline (PBS) containing 0.1% protease-free BSA
(Sigma) for 16 hrs at 4.degree. C. The wells were blocked with 1%
BSA in PBS for 1 hr at room temperature and washed with PBS. HUVE
cells were harvested with 0.02% EDTA in PBS, washed twice with
serum-free F12 medium and resuspended in serum-free F12. In some
experiments, fbs was added to 5-10%. Also, in experiments involving
vitronectin-coated plates, endogenous vitronectin was removed from
fbs by immunoaffinity chromatography using bovine polyclonal
anti-vitronectin antibodies (Gibco). Norris et al., J. Cell Sci.
95:255-262 (1990). Cells were plated at 10.sup.4 cells/well. After
2 hours, cells were fixed with 4% paraformaldehyde, stained with
methylene blue and quantified as described. Oliver et al., J. Cell
Sci. 92:513-518 (1989).
[0140] Under serum-free conditions, hCyr61 mediated cell attachment
but not spreading of HUVE cells. Attachment of HUVE cells to
hCyr61-coated plates was enhanced by inclusion of serum in the
culture medium. In the presence of serum. HUVE cells attached and
spread on hCyr61 in a manner similar to that seen on fibronectin.
Human Cyr61 supported HUVE cell adhesion in a dose-dependent manner
both under high-serum (10%) and low-serum (0.5%) conditions.
However, in the presence of 10% fbs, the maximal proportion of the
cells attaching at a lower concentration of hCyr61, and the
proportion of the cells attached, was higher. Human Cyr61 was also
found to cooperate with vitronectin in promoting HUVE cell adhesion
and spreading. Two major cell-adhesive proteins found in mammalian
sera are fibronectin and vitronectin, also known as "serum
spreading factor." For review, see Felding-Habermann et al., Curr.
Opin. Cell Biol. 5:864-868 (1993). Cell attachment, spreading and
growth on tissue-culture plastic depended upon vitronectin, rather
than fibronectin, in serum for the following reasons: (1)
considerable depletion of fibronectin in the batches of fbs due to
"clotting" at 4.degree. C.; and (2) inability of fibronectin to
efficiently coat the plastic in the presence of an excess amount of
other serum proteins. In contrast, vitronectin coated the plastic
surfaces efficiently under the same conditions.
[0141] The ability of HUVE cells to adhere to hCyr61-coated plates
in the presence of mock-immunodepleted fbs and serum immunodepleted
with anti-bovine vitronectin antibodies were compared. HUVE cells
adhered to hCyr61-coated surfaces significantly better in the
presence of soluble vitronectin or mock-immunodepleted fbs than
they did in the presence of serum-free medium or medium
supplemented with vitronectin-immunodepleted fbs. The addition of
vitronectin (30 .mu.g/ml) to vitronectin-immunodepleted serum
restored the ability of HUVE cells to adhere and spread on
hCyr61-coated plates to the same level observed when whole serum
was used in the cell attachment assay. Furthermore, soluble
vitronectin alone, at a concentration equal to its level in 10%
serum (30 .mu.g/ml), restored the level of cell adhesion and
spreading to the level found in the presence of 10% serum. Thus,
vitronectin is a necessary and sufficient serum component
contributing to HUVE cell adhesion and spreading on hCyr61-coated
plastic surfaces. Control studies showed that the effect of
vitronectin was not due to its preferential retention on the
plastic dish surfaces in the presence of hCYR61.
[0142] Additionally, HUVE cell attachment and spreading in the
presence of an increasing quantity of vitronectin was examined. The
solutions for coating the dishes contained increasing amounts of
vitronectin (0-10 .mu.g/ml) with a fixed amount of hCyr61 (10
.mu.g/ml). The results indicated that more cells adhered to plates
coated with the two proteins than would have been expected by
adding the individual adhesive capacities of vitronectin and
hCyr61. This non-additive increase of adhesion in the presence of
vitronectin and hCyr61 was not due to higher amounts of vitronectin
absorbed on the plastic. ELISA assay with anti-human vitronectin
antibodies showed that the amount of vitronectin adsorbed to
plastic dishes exposed to the vitronectin/hCyr61 mixture did not
exceed that of vitronectin alone by more than 20%. This difference
is insufficient to explain the observed difference in cell adhesion
(3-5 fold in different experiments). In addition, a higher
proportion of HUVE cells also adhered to the mixture of proteins
when the coating solution contained diluted vitronectin (2.5
.mu.g/ml) than were found to adhere to dishes coated with higher
concentrations of pure vitronectin (10 .mu.g/ml) or pure hCyr61 (10
.mu.g/ml). Thus, vitronectin and hCyr61 functionally cooperate and
exert a synergistic effect on HUVE cell adhesion.
[0143] The capacity of Fisp12 to affect cell adhesion was also
investigated. Fisp12 cell attachment assays were performed
essentially as described (Oliver et al., 1989), 96-well
immunological plates were coated for 16 hours at 4.degree. C. with
20 .mu.g/ml Cyr61, Fisp12 or fibronectin (Gibco BRL) in PBS
containing 0.1 mg/ml BSA and blocked with 10 mg/ml BSA for 1 hour
at room temperature. HUVE cells were plated at 10.sup.4 cells/well
in F12K media with 10% FBS (Hyclone Laboratories, Inc., Logan,
Utah), NIH 3T3 fibroblasts were plated at 3.times.10.sup.4
cells/well and Mv1Lu cells were plated at 5.times.10.sup.4
cells/well in minimal essential medium (MEM) with 10% FBS. After 1
hour incubation cells were fixed, stained with methylene blue and
quantified as described (Oliver et al., 1989). Cell spreading was
examined on cells plated on 100 mm polystyrene petri dishes coated
with 2.5 ml of a 20 .mu.g/ml solution of Cyr61, Fisp12 or
fibronectin. 10.sup.7 cells were plated on each dish and cell
spreading was analyzed 90 min. after plating by microscopy at
100.times. magnification.
[0144] The results indicated that Fisp12, as well as Cyr61, when
coated on plastic dishes, promoted the attachment of three
different cell types: HUVE cells, NIH 3T3 fibroblasts, and mink
lung epithelial (Mv1Lu) cells. These cells attached poorly to
uncoated plastic dishes or plastic dishes coated with bovine serum
albumin, but attached significantly better to dishes coated with
either fibronectin, Cyr61, or Fisp12. The ability of either Cyr61
or Fisp12 to mediate cell attachment is comparable to that of
fibronectin for all three cell types. While the ability of Cyr61 to
mediate cell attachment was previously demonstrated for fibroblasts
and endothelial cells (Kireeva et al., 1996), these studies show
cell attachment activity for both Fisp12 and Cyr61 in epithelial
cells in addition to endothelial cells and fibroblasts.
[0145] Like cell attachment to fibronectin and Cyr61 (Kireeva et
al. 1996). Fisp12-mediated cell attachment was inhibited when EDTA
was added to the culture medium. This inhibition was completely
abolished by the addition of excess MgCl.sub.2, indicating a
requirement for divalent cations in Fisp12-mediated cell
attachment. In addition to cell attachment, Fisp12 also promotes
cell spreading. Similar cell spreading was found when NIH 3T3 cells
were plated on dishes coated with either fibronectin, Cyr61, or
Fisp12, but not BSA. Endothelial and epithelial cells also spread
when plated on fibronectin, Cyr61, or Fisp12.
EXAMPLE 14
Migration of Fibroblasts
[0146] Cyr61 also affects chondrocytes, e.g., fibroblasts involved
in skeletal development. In particular, Cyr61 influences the
development, and perhaps maintenance, of cartilage, in contrast to
the variety of growth-related proteins that exclusively influence
development and maintenance of the bony skeleton. The chemotactic
response of NIH 3T3 cells to murine Cyr61 was examined using a
modified Boyden chamber (Neuroprobe Inc., catalog no. AP48).
Grotendorst, Meth. Enzymol. 147:144-152 (1987). Purified Cyr61
protein was serially diluted in DMEM containing bovine serum
albumin (BSA; 0.2 mg/ml) and added to the lower well of the
chamber. The lower well was then covered with a collagen-coated
polycarbonate filter (8 .mu.m pore diameter; Nucleopore Corp.,
Pleasanton, Calif.). Cells (6.times.10.sup.4) were then loaded into
the upper well. After 5 hours incubation (10% CO.sub.2, 37.degree.
C.), the filter was removed and the cells were fixed and stained
using Wright-Giemsa stain (Harleco formulation; EM Diagnostic
Systems. Gibbstown, NJ). Cells from the upper surface of the filter
were then removed by wiping with a tissue swab. The chemotactic
response was determined by counting the total number of migrating
cells detected in ten randomly selected high-power microscopic
fields (400-fold magnification) on the lower surface of the filter.
Duplicate trials were performed for each experiment and the
experiment was repeated three times to ensure reproducibility of
the data.
[0147] NIH 3T3 cells responded to Cyr61 as a chemotactic factor in
a dose-dependent manner in the Boyden chamber assay. Without Cyr61,
approximately 4.8 cells had migrated per high-power field. In the
presence of 0.5 .mu.g/ml murine Cyr61, about 5.2 cells were found
in each field. As the concentration of Cyr61 was raised to 1, 5 and
10 .mu.g/ml, the average number of migrating cells detected per
field rose to 7.5, 18.5, and 18.7. Thus, murine Cyr61 acts as a
chemoattractant for fibroblasts. The optimal concentration for the
chemotactic activity of Cyr61 is 1-5 .mu.g/ml in this assay; this
concentration range is consistent with the reported ranges at which
other ECM molecules provide effective chemotactic stimulation. For
example, Thrombospondin, at 5-50 .mu.g/ml has a chemotactic effect
on endothelial cells (Taraboloetti et al., J. Cell Biol.
111:765-772 (1990); fibronectin also functions as a chemotactic
agent at 1-30 .mu.g/ml (Carsons et al., Role of Fibronectin in
Rheumatic Diseases, in Fibronectin [Mosher, ed., Academic Press
1989]; Carsons et al., Arthritis. Rheum. 28:601-612 [1985]) as
determined using similar Boyden chamber assays. The human Cyr61
polypeptide may be used to chemoattract fibroblasts in a manner
analogous to murine Cyr61. Human CTGF has also been reported to
induce the migration of non-human mammalian cells such as NIH 3T3
cells (mouse fibroblasts) and BASM cells (bovine aortic smooth
muscle cells), as described in U.S. Pat. No. 5,408,040, column 7,
line 65 to column 11, line 7, incorporated herein by reference.
[0148] In an alternative embodiment, an assay for modulators of
cell migration, such as the migration of chondrocytes, involves a
combination of a suspected modulator of cell migration and Cyr61
being added to the lower well of a Boyden chamber. As a control,
Cyr61 is separately added to the lower well of another Boyden
chamber. Relative cell migrations are then measured. An increase in
cell migration in the presence of the suspected modulator relative
to cell migration in response to Cyr61 alone identifies a promoter
of chondrocyte cell migration, while a relative decrease in cell
migration in the presence of the suspected modulator identifies an
inhibitor.
EXAMPLE 15
Migration of Endothelial Cells--In Vitro Assays
[0149] The end product of in vitro angiogenesis is a well-defined
network of capillary-like tubes. When cultured on gel matrices.
e.g., collagen, fibrin, or Matrigel gels, endothelial cells must
first invade the matrix before forming mature vessels. (Matrigel is
a complex mixture of basement membrane proteins including laminin,
collagen type IV, nidogen/entactin, and proteoglycan heparin
sulfate, with additional growth factors. Kleinman et al., Biochem.
25:312-318 (1986). The invasive structures are cords which
eventually anastomose to form the vessel-like structures. The
angiogenic effect of human Cyr61 on confluent monolayers of human
umbilical vein endothelial cells is assessed by seeding the cells
onto three-dimensional collagen or fibrin gels, in the presence or
absence of Cyr61. HUVE cells do not spontaneously invade such gels
but do so when induced by agents such as tumor promoters.
[0150] Collagen gels were prepared by first solubilizing type I
collagen (Collaborative Research, Inc. Bedford, Mass.) in a sterile
1:1000 (v/v) dilution of glacial acetic acid (300 ml per gram of
collagen). The resulting solution was filtered through sterile
triple gauze and centrifuged at 16,000.times.g for 1 hour at
4.degree. C. The supernatant was dialyzed against 0.1.times.
Eagle's Minimal Essential Medium (MEM; GIBCO-BRL, Inc.) and stored
at 4.degree. C. Gels of reconstituted collagen fibers were prepared
by rapidly raising the pH and ionic strength of the collagen
solution. The pH and ionic strength adjustments were accomplished
by quickly mixing 7 volumes of cold collagen solution with one
volume of 10.times.MEM and 2 volumes of sodium bicarbonate (11.76
mg/ml) in a sterile flask. The solution was kept on ice to prevent
immediate gelation. The cold mixture was dispensed into 18 mm
tissue culture wells and allowed to gel for 10 minutes at
37.degree. C.
[0151] Fibrin gels were prepared by dissolving fibrinogen (Sigma
Chemical Co. St. Louis, Mo.) immediately before use in calcium-free
MEM to obtain a final concentration of 2.5 mg of protein/ml.
Clotting was initiated by rapidly mixing 1.35 ml of fibrinogen
solution with 15 .mu.l of 10.times.MEM containing 25 U/ml thrombin
(Sigma Chemical Co.) in a plastic tube. The mixture was transferred
immediately into 18 mm tissue culture wells and allowed to gel for
about 2 minutes at 37.degree. C.
[0152] In some wells, Cyr61 was mixed into the gel matrix before
gelation (final concentration 10 .mu.g/ml) while in other wells,
Cyr61 was not in the gel matrix but was added as part of the
nutrient medium (similar range of concentrations as in the matrix)
after the cells reached confluency. HUVE cells were seeded onto the
gel matrix surface at 5.times.10.sup.4 cells per well in Ham's F12K
medium [GIBCO-BRL. Inc.] containing 10% fetal bovine serum, 100
.mu.g/ml heparin, and 30 .mu.g/ml endothelial cell growth factor.
When the cells reached confluency, the medium was removed, the
cells were rinsed with PBS, and fresh medium without endothelial
cell growth factor was supplied. Some cultures received purified
recombinant Cyr61, while others received Cyr61 and polyclonal
anti-Cyr61 antibodies. Thus, the variety of cultures at confluency
included: a) cultures with no Cyr61; b) cultures with Cyr61 within
the matrix; c) cultures with Cyr61 supplementing the medium; and d)
cultures with Cyr61 supplementing the medium along with polyclonal
anti-Cyr61 antibodies.
[0153] Invasion of the gel matrix was quantified about 4-7 days
after treatment of the confluent cultures. Randomly selected fields
measuring 1.0 mm.times.1.4 mm were photographed in each well under
phase-contrast microscopy with a Zeiss Axiovert inverted
photomicroscope. Photographs were taken at a single level beneath
the surface monolayer. Invasion was quantified by measuring the
total length of all cell cords that penetrated beneath the surface
monolayer. Results were expressed as the mean length in microns per
field for at least 3 randomly selected fields from each of at least
3 separate experiments.
[0154] In order to examine the network of cords within the matrix
for capillary-like tube formation, cultures were fixed in situ
overnight with 2.5% glutaraldehyde and 1% tannic acid in 100 mM
sodium cacodylate buffer, pH 7.4. Cultures were then washed
extensively in 100 mM sodium cacodylate buffer, pH 7.4. The gels
were cut into 2 mm.times.2 mm fragments, post-fixed in 1% osmium
tetroxide in veronal acetate buffer (to minimize tissue swelling;
see Hayat, in Principles and Tec/niques of Electron Microscopy:
Biological Applications 1:38 [Litton Educational Publishing, Inc.
1970]) for 45 minutes, stained en bloc with 0.5% uranyl acetate in
veronal buffer for 45 minutes, dehydrated by exposure to a graded
ethanol series, and embedded in Epon in flat molds. Semi-thin
sections were cut perpendicular to the culture plane with an
ultramicrotome, stained with 1% toluidine blue, and photographed
under transmitted light using an Axiophot photomicroscope
(Zeiss).
[0155] In an alternative embodiment, a suspected modulator of
angiogenesis is combined with Cyr61 and the combination is added
before, or after, formation of a gel. In this embodiment, a control
is established by using Cyr61 alone. The migration of cells in
response to the suspected modulator and Cyr61 is then compared to
the migration of cells in response to Cyr61 alone. A promoter or
positive effector will increase cell migration while an inhibitor
or negative effector will decrease cell migration.
[0156] In an alternative in vitro assay for angiogenic activity, an
assay for endothelial cell migration was developed. This chemotaxis
assay has been shown to detect the effects of Cyr61 concentrations
on the order of nanograms per milliliter. Primary Human
Microvascular Endothelial Cells (HMVEC PO51; Clonetics. San Diego,
Calif.) were maintained in DME with 10% donor calf serum (Flow
Laboratories, McLean, Va.) and 100 .mu.g/ml endothelial cell
mitogen (Biomedical Technologies Inc., Stoughton, Mass.). The cells
were used between passages 10 and 15. To measure migration, cells
were starved for 24 hours in DME containing 0.1% BSA, harvested,
resuspended in DME with 0.1% BSA, and plated at 1.75.times.10.sup.4
cells/well on the lower surface of a gelatinized 0.5 .mu.m filter
(Nucleopore Corporation, Pleasanton, Calif.) in an inverted
modified Boyden chamber. After 1-2 hours at 37.degree. C., during
which time the cells were allowed to adhere to the filter, the
chamber was reverted to its normal position. To the top well of
separate chambers, basic Fibroblast Growth Factor (a positive
control), Cyr61, or a negative control solution (conditioned medium
known to lack chemoattractants or DME plus BSA, see below) was
added at concentrations ranging from 10 ng/ml to 10 .mu.g/ml.
Chambers were then incubated for 3-4 hours at 37.degree. C. to
allow migration. Chambers were disassembled, membranes fixed and
stained, and the number of cells that had migrated to the top of
the filter in 3 high-powered fields was determined. Tolsma er al.,
J. Cell. Biol. 122:497-511 (1993) (incorporated by reference), and
references cited therein. DME with 0.1% BSA was used as a negative
control and either bFGF (10 ng/ml) or conditioned media from
angiogenic hamster cell lines (20 .mu.g/ml total protein) were used
as positive controls. Rastinejad et al., Cell 56.345-355 (1989).
Each sample was tested in quadruplicate (test compound such as
Cyr61, positive control, conditioned medium as a negative control,
and DME plus BSA as a negative control) in a single experiment;
experiments were repeated at least twice.
[0157] To allow comparison of experiments performed on different
days, migration data is reported as the percent of maximum
migration towards the positive control, calculated after
subtraction of background migration observed in the presence of DME
plus BSA. Test compounds that depressed the random movement of
endothelial cells showed a negative value for the percent
migration. Very high concentrations of thrombospondin (TSP) caused
endothelial cells to detach from the membrane. Detachment was
detected by counting cells on the lower face of the membrane. When
cell loss exceeded 10%, the number of migrated cells was corrected
for this loss. The results indicate that 0.01-10 .mu.g/ml bFGF
induced the migration of a constant 92 cells per three high-powered
microscope fields Migration in the presence of Cyr61 revealed a
greater dependence on concentration. At 10 ng/ml, Cyr61 induced an
average of 64 cells to migrate per three high-powered fields
examined. At 100 ng/ml Cyr61, approximately 72 cells were found in
three fields; at 1 .mu.g/ml Cyr61, a peak of 87 cells had migrated;
at approximately 7 .mu.g/ml Cyr61, about 61 cells were observed;
and at 10 .mu.g/ml Cyr61, approximately 57 cells were found to have
migrated. The negative control revealed a constant basal level of
endothelial cell migration of 53 cells per three high-powered
microscope fields. In addition to these results, there is a perfect
correlation of the results from this in vitro assay and the results
from the in vivo cornea assay, described below.
[0158] To monitor toxicity, endothelial cells were treated with
each of the tested compounds at a range of concentrations, under
conditions identical to those used in the migration assay. Cells
were then stained with Trypan blue and cells excluding Trypan blue
were counted. The results showed that cells remained viable and
that the inhibition of migration could not be attributed to
toxicity. Where relevant, endothelial cells were pretreated for
36-48 hours with peptides at 20 .mu.M in DME with 0.1% BSA before
use in the migration assays. Toxicity was also tested over these
time frames and found to be negligible.
[0159] The ability of Cyr61 to induce matrix invasion and tube
formation by HUVE cells, as well as the ability of Cyr61 to induce
human microvascular endothelial cells to migrate, demonstrates the
angiogenic properties of this protein. It is anticipated that other
members of the ECM signalling molecule family of cysteine-rich
proteins, such as Fisp12 and CTGF, have similar properties that may
be used in methods of the invention for screening for, and
modulating, angiogenic conditions. In particular, one of ordinary
skill in the art understands that an in vitro assay for angiogenic
inhibitors involves the assay described above, including an
effective amount of Cyr61, with and without the candidate
inhibitor.
EXAMPLE 16
Migration of Endothelial Cells--An In Vitro Assay for Angiogenesis
Inhibitors
[0160] The inclusion of an effective amount of an ECM signalling
molecule, such as Cyr61, in the in vitro migration (i.e.,
chemotaxis) assay described in the preceding Example, provides an
assay designed to detect inhibitors of ECM signalling molecules and
angiogenesis. Because of the crucial role of neovascularization in
such processes as solid tumor growth and metastasis, the
development of assays to detect compounds that might antagonize
these processes would be useful.
[0161] The above-described in vitro migration assay was adapted to
include an ECM signalling molecule, Cyr61. Cyr61 was included at 1
.mu.g/ml, which was found to be the optimal dosage in titration
studies. As in the preceding Example, human microvascular
endothelial cells (Clonetics) were used. In one series of assays,
several carbohydrates and carbohydrate derivatives were analyzed.
These compounds included 10 mM mannose, 10 mM mannose-6-phosphate,
and 10 mM galactose. Results of these assays showed that Cyr61 plus
mannose yielded approximately 73 cells per set of three
high-powered microscope fields (see above). Cyr61 plus galactose
induced the migration of approximately 74 cells per set of three
high-powered fields. However, Cyr61 plus mannose-6-phosphate
yielded approximately 2 migrating cells for each set of three
high-powered fields examined. Control experiments demonstrate that
the inhibition of Cyr61 activity by mannose-6-phosphate is
specific.
[0162] The angiogenic activity of basic FGF (10 ng/ml) was also
tested, as described above, with and without mannose-6-phosphate.
In the presence of 10 mM mannose-6-phosphate, bFGF induced 51 cells
per set of three high-powered fields to migrate; in its absence,
bFGF induced the migration of approximately 52 cells. However, when
either Cyr61 or Insulin Growth Factor II (IGF-II) were tested,
mannose-6-phosphate reduced the number of migrating cells from
approximately 48 or 47 cells, respectively, to approximately 12 or
11 cells, respectively. The effect of mannose-6-phosphate on IGF II
activity was anticipated because mannose-6-phosphate is known to
compete with IGF II for their common receptor (the IGF II
receptor). Thus, mannose-6-phosphate specifically inhibits the
chemotactic activity of Cyr61 on human endothelial cells. Moreover,
because there is an essentially perfect correlation between the in
vitro migration assay and the in vivo angiogenesis assay, described
below, mannose-6-phosphate has been identified as an inhibitor of
angiogenesis based on the results of the assay disclosed herein.
Accordingly, the invention contemplates a method of inhibiting
angiogenesis comprising the step of administering an inhibitor the
angiogenic activity of Cyr 61 such as mannose-6-phosphate. Assays
such as that described above may also be used to screen for other
inhibitors of angiogenesis which may be useful in the treatment of
diseases associated with angiogenesis such as cancer, and diseases
of the eye which are accompanied by neovascularization.
[0163] In an embodiment of the invention, a method of screening for
modulators of angiogenesis involves a comparative assay. One set of
conditions involves exposure of cells to a combination of Cyr61 and
a suspected modulator of cell migration. As a control, a parallel
assay is performed that exposes cells to Cyr61 alone. A promoter of
cell migration elevates the rate of in vitro cell migration
relative to the rate of migration in the presence of Cyr61 alone;
the converse is true for an inhibitor of the chemoattracting
ability of Cyr61.
EXAMPLE 17
Migration of Endothelial Cells--An In Vivo Assay
[0164] An in vivo assay for endothelial cell migration has also
been developed. In general, the assay protocol is consistent with
the disclosure of Tolsma et al. 1993. To assess angiogenesis
associated with the formation of granulation tissue (i.e., the
newly formed, proliferative, fibroblastic dermal tissue around
wounds during healing), sponge implants were used as previously
described (Fajardo, et al., Lab. Invest. 58:718-724 [1988]).
Polyvinyl-alcohol foam discs (10-mm diam.times.1-mm thick) were
prepared by first removing a 2-mm diameter central core of sponge.
PBS or an RGDS peptide (other possible test compounds include
fragments of Cyr61, RGDS peptide, small molecules such as
mannose-6-phosphate) at 100 .mu.M were added to the sponge core
which was then coated with 5 .mu.l of sterile Hydron (Interferon
Sciences, New Brunswick, N.J.). After solidifying, the coated core
was returned to the center of the sponge which was then covered on
both sides with 5 .mu.m filters and secured in place with glue
(Millipore Corp., Bedford, Mass.). One control and one test disc
were then implanted subcutaneously in the lower abdomen of
anesthetized Balb/c female mice where granulation tissue could
invade the free perimeter of the disc. Wounds were closed with
autoclips and animals left undisturbed until sacrificed.
[0165] Quantitative estimates of thymidine incorporation in situ
into endothelial cells in the discs were obtained as previously
described (Polverini, et al., J. Immunol. 118:529-532 [1977]).
Sponge implants were evaluated at days 5, 7, 10, and 14 after
implantation. Thirty minutes before sacrifice, mice were injected
with a solution containing [.sup.3H]-thymidine in saline (specific
activity 6.7 Ci/mM; New England Nuclear/Du Pont, Wilmington, Del.)
to a level of 1 .mu.Ci per gram of body weight. Sponges were
removed and facially embedded to provide a uniform section of the
entire circumference. Tissues were fixed in 10% neutral buffered
formalin, dehydrated through a graded series of alcohols, and
embedded in glycol methacrylate (Polysciences, Miles, Ill.).
Autoradiograms were prepared by dipping sections mounted on
acid-cleaned glass slides into NTB type 2 emulsion (Eastman Kodak).
After exposure for 4 weeks at 4.degree. C., autoradiographs were
developed in half strength D-19 developer, fixed in Kodak Rapid
Fixer, and stained with hematoxylin and eosin. Quantitation of
endothelial cell labeling was performed by counting all endothelial
cells that lined capillaries and venules extending from the
periphery to the center of the sponge by rectilinear scanning under
oil immersion (.times.1.000). A total of 500-700 endothelial cells
were counted in each of two sponges containing either PBS, TSP, or
peptide fragments (i.e., thrombospondin fragments). Cells were
considered labeled if five or more grains were detected over the
nucleus. The percentage of labeled cells was calculated and a
chi-square analysis of data derived from control and experimental
sponges was performed.
[0166] The results of the foregoing assay established that
thrombospondin fragments could inhibit the process of angiogenesis.
More generally, one of ordinary skill in the art would appreciate
that the scope of the present invention extends to such in vivo
assays for suspected modulators of ECM signalling molecule
activities, such as the chemotactic ability of Cyr61 to induce cell
migration. As with other assays of the invention, a comparative
assay involves exposure of cells, in vivo, to a sponge laden with
Cyr61 in the presence or absence of a suspected modulator of Cyr61
activity. Following implantation, incubation, and removal, the
relative rates of cell migration are determined. A promoter of
Cyr61 activity will increase the rate of cell migration relative to
cell migration induced by Cyr61 alone; an inhibitor will decrease
the rate of cell migration relative to the level ascribable to
Cyr61 alone.
EXAMPLE 18
Mitogen Potentiation
[0167] In another aspect of the invention, murine Cyr61 enhanced
the mitogenic effect of growth factors on fibroblasts and
endothelial cells. When NIH 3T3 fibroblasts or HUE cells were
treated with a non-saturating dose of either basic Fibroblast
Growth Factor (bFGF) or Platelet-Derived Growth Factor (PDGF-BB),
the addition of murine Cyr61 significantly increased the
incorporation of radiolabeled thymidine compared to cells treated
with the growth factors alone. The thymidine incorporation assay is
a standard technique for determining whether cells are actively
growing by assessing the extent to which the cells have entered the
S phase and are synthesizing DNA. The Cyr61 enhancement of bFGF- or
PDGF-BB-induced thymidine incorporation was dose dependent,
requiring a minimum concentration of 0.5-1.0 .mu.g/ml of
recombinant protein for either cell type. The enhancement of DNA
synthesis by Cyr61 was inhibited by the addition of specific
anti-Cyr61 antiserum.
[0168] More specifically. NIH 3T3 fibroblast cells were plated on
24-well plates at 3.times.10.sup.4 cells/well and grown in DMEM
with 10% fetal bovine serum (Intergen Co. Purchase. NY) for 3-4
days and incubated with medium containing 0.2% FBS for the
following 48 hours. The following compounds, at the parenthetically
noted final concentrations, were then added to the plated cells in
fresh DMEM containing 0.2% fbs and [.sup.3H]-thymidine (1 .mu.Ci/ml
final concentration: ICN Biomedicals, Inc., Costa Mesa, Calif.):
bFGF (15 ng/ml), PDGF-BB (30 ng/ml), and murine Cyr61 (0.5-5
.mu.g/ml). These compounds were added to individual plates
according to the following pattern: 1) no supplementation: 2)
murine Cyr61; 3) bFGF; 4) murine Cyr61 and bFGF; 5) PDGF-BB; and 6)
murine Cyr61 and PDGF. After 18-20 hours of incubation, cells were
washed with PBS and fixed with 10% trichloroacetic acid. DNA was
dissolved in 0.1 N NaOH and thymidine incorporation was determined.
The results indicated that murine Cyr61, in the absence of a growth
factor, did not stimulate DNA synthesis as measured by tritiated
thymidine incorporation. Without any supplements. 3T3 cells
incorporated approximately 1.8.times.10.sup.4 cpm of
[.sup.3H]-thymidine, in the presence or absence of Cyr61. Cells
exposed to bFGF alone incorporated about 1.2.times.10.sup.5 cpm;
cells contacting bFGF and murine Cyr61 incorporated
2.times.10.sup.5 cpm. Cells receiving PDGF-BB incorporated about
1.2.times.10.sup.5 cpm; and cells exposed to PDGF-BB and murine
Cyr61 incorporated approximately 2.4.times.10.sup.5 cpm. Therefore,
murine Cyr61 did not function as a mitogen itself, but did
potentiate the mitogenic activity of bFGF and PDGF-BB, two known
growth factors.
[0169] The ability of murine Cyr61 to potentiate the mitogenic
effect of different levels of bFGF also revealed a threshold
requirement for the growth factor. Human umbilical vein endothelial
cells were plated essentially as described above for 3T3 cells and
exposed to a constant amount of murine Cyr61; controls received no
Cyr61. Different plates were then exposed to different levels of
bFGF, comprising a series of bFGF concentrations ranging from 0-10
ng/ml. Following culture growth in the presence of
[.sup.3H]-thymidine for 72 hours, cells exposed to 0-0.1 ng/ml of
bFGF exhibited a baseline level of thymidine incorporation
(approximately 4.times.10.sup.2 cpm), in the presence or absence of
Cyr61. At 1 ng/ml bFGF, however, HUVE cells increased their
thymidine incorporation in the presence of bFGF to 6.times.10.sup.2
cpm; in the presence of 1 ng/ml bFGF and murine Cyr61, HUVE cells
incorporated 1.3.times.10.sup.3 cpm. At 10 ng/ml bFGF, cells
exposed to bFGF incorporated about 1.8.times.10.sup.3 cpm
thymidine; cells receiving 10 ng/inl bFGF and Cyr61 incorporated
approximately 6.1.times.10.sup.3 cpm.
[0170] The capacity of murine Cyr61 to potentiate the mitogenic
activity of bFGF was verified by a thymidine incorporation assay
involving HUVE cells and various combinations of bFGF, Cyr61, and
anti-Cyr61 antibodies. Cells were plated and grown as described
above. The following combinations of supplements (final plate
concentrations noted parenthetically) were then pre-incubated for 1
hour before addition to individual plates: 1) pre-immune antiserum
(3%); 2) bFGF (15 ng/ml) and pre-immune antiserum (3%); 3)
pre-immune antiserum (3%) and Cyr61 (4 .mu.g/ml): 4) pre-immune
antiserum (3%), Cyr61 (4 .mu.g/ml), and bFGF (15 ng/ml); 5)
anti-Cyr61 antiserum (3%); 6) anti-Cyr61 antiserum and bFGF (15
ng/ml); 7) anti-Cyr61 antiserum (3%) and Cyr61 (4 .mu.g/ml); and 8)
anti-Cyr61 antiserum (3%). Cyr61 (4 .mu.g/ml), and bFGF (15
ng/ml).
[0171] Following incubation in the presence of [.sup.3H]-thymidine
as described above, cells exposed to pre-immune antiserum
incorporated about 2.times.10.sup.2 cpm thymidine; cells contacting
pre-immune antiserum and bFGF incorporated 1.3.times.10.sup.3 cpm;
cells receiving pre-immune antiserum and Cyr61 incorporated
1.times.10.sup.2 cpm; cells receiving pre-immune antiserum, Cyr61,
and bFGF incorporated 3.6.times.10.sup.3 cpm; cells exposed to
anti-Cyr61 antiserum incorporated 2.times.10.sup.2 cpm; cells
receiving anti-Cyr61 antiserum and bFGF incorporated about
1.3.times.10.sup.3 cpm; cells contacting anti-Cyr61 antiserum and
Cyr61 incorporated about 1.times.10.sup.2; and cells receiving
anti-Cyr61 antiserum. Cyr61, and bFGF incorporated 1.times.10.sup.3
cpm. These results indicate that pre-immune antiserum had no effect
on Cyr61-induced potentiation of bFGF mitogenic activity.
Anti-Cyr61 antiserum, however, completely abolished the
potentiation of bFGF by Cyr61. Moreover, the effect of anti-Cyr61
antiserum was specific to Cyr61-induced mitogenic potentiation
because anti-Cyr61 antiserum had no effect on the mitogenic
activity of bFGF per se. Therefore, Cyr61 can be used as a reagent
to screen for useful mitogens.
[0172] DNA synthesis for HUVE cells and NIH 3T3 fibroblasts was
measured by thymidine incorporation as described previously
(Kireeva et al., Mol. Cell. Biol. 16: 1326-1334 [1996]) with minor
modifications. HUVE cells were grown in 24-well plates to a
subconfluent state, serum-starved for 24 hours and treated with
F12K medium containing 10% fetal bovine serum (FBS), 1 .mu.Ci/ml
[.sup.3H]-thymidine and 10 ng/ml basic Fibroblast Growth Factor
(bFGF) (Gibco-BRL, Inc.) with various concentrations of Cyr61 and
Fisp12 as indicated. NIH 3T3 fibroblasts were grown to
subconfluence, serum-starved for 48 hours, and treated with Minimal
Essential Medium (MEM) containing 0.5% FBS, 1 .mu.Ci/ml
[.sup.3H]-thymidine, bFGF and various concentrations of Cyr61 or
Fisp12. Thymidine incorporation into the trichloroacetic
acid-insoluble fraction was determined after 24 hour incubation.
Logarithmically grown mink lung epithelial cells (Mv1lu, CCL64)
were treated with various concentrations of TGF-.beta.1 (Gibco-BRL)
and 2 .mu.g/ml of Cyr61 or Fisp12 for 18 hours; [.sup.3H]-thymidine
was then added to 1 .mu.Ci/ml for 2 hours. Thymidine incorporation
was determined as described above.
[0173] Purified recombinant Fisp12 protein did not exhibit any
mitogenic activity under any tested assay conditions. Rather,
Fisp12 was able to enhance DNA synthesis induced by fibroblast
growth factor in either NIH 3T3 fibroblasts or HUVE-cells. This
activity was nearly indistinguishable from that exhibited by
Cyr61.
[0174] Whereas in fibroblasts and endothelial cells, Cyr61 and
Fisp12 enhance growth factor-induced DNA synthesis, both proteins
can also enhance growth factor-mediated actions in another way. It
is known that TGF-.beta. acts to inhibit DNA synthesis in
epithelial cells (Satterwhite et al., 1994). It was observed that
both Cyr61 and Fisp12 enhanced the ability of TGF-.beta. to inhibit
DNA synthesis in mink lung epithelial cells. The data demonstrate
that both recombinant Cyr61 and Fisp12, purified from serum-free
sources, are not mitogenic by themselves, but have the ability to
synergize with the actions of polypeptide growth factors. Cyr61 and
Fisp12 enhance DNA synthesis induction by FGF, and enhance DNA
synthesis inhibition by TGF-.beta..
[0175] The present invention also comprehends the use of CTGF in
methods to potentiate the mitogenic effect of true growth factors,
or to screen for true growth factors. Those contemplated uses are
in contrast to the reported use of CTGF as a mitogen or growth
factor itself. U.S. Pat. No. 5,408,040, column 7, line 65, to
column 11, line 7, incorporated herein by reference
hereinabove.
[0176] Further, the invention comprehends methods of screening for
modulators of mitogen potentiation. A comparative assay exposes
subconfluent cells to an ECM signalling molecule such as Cyr61, a
growth factor, and a suspected modulator of an ECM signalling
molecule. As a control, similar cells are exposed to the ECM
signalling molecule and the growth factor. A further control
exposes similar cells to the growth factor and the suspected
modulator in the absence of the ECM signalling molecule. Based on
the relative cell proliferation rates, as measured by, e.g.,
[.sup.3H]-thymidine incorporation, an identification of a suspected
modulator as a promoter of mitogen potentiation (elevated cell
proliferation in the presence of all three molecules) or an
inhibitor of mitogen potentiation (decreased cell proliferation in
the presence of the three molecules) can be made.
EXAMPLE 19
Cornea Assay for Angiogenic Factors and Modulators
[0177] Another assay for modulators of angiogenesis is an in vivo
assay for assessing the effect of a suspected modulator in the
presence of an ECM signalling molecule-related biomaterial, such as
Cyr61, on angiogenesis is the Cornea Assay. The Cornea Assay takes
advantage of the absence of blood vessels in the cornea, which in
the presence of an angiogenic factor, results in the detectable
development of capillaries extending from the sclera into the
cornea. Friedlander et al., Science 270:1500-1502 (1995). This
ingrowth of new blood vessels from the sclera can be
microscopically monitored. Further, the visually determined rate of
migration can be used to assess changes in the rate of
angiogenesis. These cornea assays may be performed using a wide
variety of animal models. Preferably, the cornea assays are
performed using rats. By way of example, an assay for suspected
modulators of Cyr61 using this assay is disclosed. To perform this
assay, Cyr61 is initially titrated using primary capillary
endothelial cells to determine effective concentrations of Cyr61.
Subsequently, Cyr61, in the presence or absence of a suspected
modulator, is surgically implanted into the corneas of mammalian
laboratory animals, e.g., rabbits or rats. In a preferred
embodiment, Cyr61 (or Cyr61 and a suspected modulator) is embedded
in a biocompatible matrix, using matrix materials and techniques
that are standard in the art. Subsequently, eyes containing
implants are visually observed for growth of the readily visible
blood vessels within the eye. Control implantations may consist of
physiologically balanced buffers embedded in the same type of
matrix and implanted into eyes of the same type of laboratory
animal receiving the Cyr61-containing implants.
[0178] The development of an in vivo cornea assay for angiogenic
factors has advantages over existing in vitro assays for these
factors. The process of angiogenesis involves four distinct phases:
induction of vascular discontinuity, endothelial cell movement,
endothelial cell proliferation, and three-dimensional restructuring
and sprouting. In vitro assays can evaluate only two of these
steps: endothelial cell migration and mitogenesis. Thus, to provide
a comprehensive assay for angiogenic factors, an in vivo assay such
as the cornea assay is preferred.
[0179] The cornea assay has been used to confirm the effect of
angiogenic factors such as Cyr61, Fisp12, CTGF, and Nov, on the
process of angiogenesis. Moreover, modifying the cornea assay by
including any of these angiogenic factors and a suspected modulator
of their activity results in a cornea assay for modulators of
angiogenesis. For example, in one embodiment of the invention, dose
of an angiogenic factor such as Cyr61 could be used in cornea
assays for positive effectors of the angiogenic activity of Cyr61.
An appropriate dose of Cyr61 would initially be determined by
titration of the dose response relationship of Cyr61 with
angiogenic events. Inclusion of a control assay lacking Cyr61 would
eliminate compounds having a direct effect on angiogenesis. In an
alternative embodiment of the invention, an effective dose of an
angiogenic factor such as Cyr61 could be used to assay for negative
modulators of the activity of an angiogenic factor. In yet another
alternative embodiment, a corneal implant comprises Cyr61 and
another corneal implant comprises Cyr61 and a suspected modulator
of angiogenesis. Measurements of the development of blood vessels
in the implanted corneas provides a basis for identifying a
suspected modulator as a promoter of angiogenesis (elevated blood
vessel development in the cornea containing an implant comprising
the suspected modulator. A relative decrease in blood vessel
development identifies an inhibitor of angiogenesis.
[0180] The rat is preferred as the animal model for the cornea
assay. Disclosures in the art have established the rat model as a
well-characterized system for analyzing angiogenesis. Parameters
such as implant size, protein release dynamics, and suitable
surgical techniques, have been well characterized. Although any
strain of rat can be used in the cornea assay, preferred strains
will be well-characterized laboratory strains such as the
Sprague-Dawley strain.
[0181] Although rats of various sizes can be used in the cornea
assay, a preferred size for the rats is 150-200 g/animal.
Anesthesia is induced with Methoxyflurane and is maintained for
40-60 minutes with sodium pentobarbital (50 mg/kg, delivered
intraperitoneally). The eyes are gently opened and secured in place
by clamping the upper eyelid with a non-traumatic hemostat. Two
drops of sterile proparacaine hydrochloride (0.5%) are then placed
on each eye as to effect local anesthesia. Using a suitable
surgical blade such as a No. 11 Bard Parker blade, an approximately
1.5 mm incision is made approximately 1 mm from the center of the
cornea. The incision extends into the stroma but not through it. A
curved iris spatula approximately 1.5 mm in width and approximately
5 mm in length is then inserted under the lip of the incision and
gently blunt-dissected through the stroma toward the outer canthus
of the eye. Slight finger pressure against the globe of the eye
helps to steady the eye during dissection. The spatula penetrates
the stroma no more than approximately 2.5 mm. Once the cornea
pocket is made, the spatula is removed and the distance between the
limbus and base of the pocket is measured to make sure the
separation is at least about 1 mm.
[0182] To provide slow release of the protein after implantation in
the cornea, protein is mixed with poly-2-hydroxyethylmethacrylate
(Hydron), or an equivalent agent, to form a pellet of approximately
5 .mu.l. Implants made in this way are rehydrated with a drop of
sterile lactated Ringers solution and implanted as described above.
After implantation, the corneal pocket is sealed with erythromycin
ointment. After implantation, the protein-Hydron pellet should
remain near the limbus of the cornea (cornea-sclera border) and
vision should not be significantly impaired.
[0183] Following surgery, animals are examined daily for seven days
with the aid of a stereomicroscope to check for inflammation and
responses. To facilitate examination, the animal is anesthetized
with Methoxyflurane and the anesthetic is continuously administered
by nose cone during examination. During this seven day period,
animals are monitored for implant position and corneal exudate.
Animals exhibiting corneal exudate are sacrificed. A preferred
method of euthanasia is exsanguination. Animals are initially
anesthetized with sodium pentobarbital (50 mg/kg) and then
perfused, as described below.
[0184] After seven days, animals are perfused with colloidal carbon
(e.g., India Ink). Anesthesia is induced with Methoxyflurane, and
is maintained with sodium pentobarbital (50 mg/kg,
intraperitoneally). Each animal is perfused with 100-200 ml warm
(37.degree. C.) lactated Ringers solution per 150 g of body mass
via the abdominal aorta. Once the snout of the animal is completely
blanched. 20-25 ml of colloidal carbon is injected in the same way
as the Ringers solution, until the head and thoracic organs are
completely black. Eyes are then enucleated and fixed. Corneas are
excised, flattened, and photographed.
[0185] Each protein is typically tested in three doses, in
accordance with the practice in the art. Those of ordinary skill in
the art realize that six positive corneal responses per dose are
required to support an identification of an angiogenic response. An
exemplary cornea assay includes three doses of the protein under
study, with six rats being tested at each dose. Additionally, six
animals are exposed to a buffer-Hydron implant and serve as
negative controls. Exposure of at least three animals to a known
angiogenic factor-Hydron implant serve as positive controls.
Finally, to demonstrate the specificity of any observed response,
six animals are exposed to implants containing a single dose of the
protein under study, an excess of neutralizing antibody, and
Hydron.
[0186] A cornea assay as described above was performed to assess
the ability of Cyr61 to induce angiogenesis. Four animals were
given negative control implants containing a buffer-Hydron pellet
(both eyes). Each of these animals failed to show any blood vessel
development in either eye after seven days. Six animals received
implants containing a biologically effective amount of Fibroblast
Growth Factor (0.15 .mu.M) in one eye and a control pellet in the
other eye; all six showed angiogenic development in the eye
receiving FGF, none showed neovascularization in the eye receiving
the negative control. Seven animals received 1 .mu.g/ml Cyr61, in
one eye and all seven of these eyes showed blood vessel growth; one
of the seven eyes receiving a negative control showed angiogenic
development. Finally, four animals received implants locally
releasing 1 .mu.g/ml Cyr61 (Hydron prepared with a 10 .mu.g/ml
Cyr61 solution) and a specific anti-Cyr61 antibody in three-fold
excess over Cyr61; none of the eyes of this group showed any
angiogenic development. Thus, the in vivo assay for angiogenesis
identifies angiogenic factors such as FGF and Cyr61. The assay also
is able to reveal inhibition of angiogenic development induced ECM
signalling molecules such as Cyr61.
EXAMPLE 20
Blood Clotting
[0187] ECM signalling molecules are also useful in correcting
hemostasis, or abnormal blood clotting. A defect in blood clotting
caused by, e.g., low level expression of cyr61 which thereby allows
Tissue Factor Pathway Inhibitor (TFPI) to act unchecked can be
corrected by expression or use of recombinant Cyr61 protein.
[0188] Cyr61 can interact with TFPI, a protein that inhibits
extrinsic blood coagulation. TFPI inhibits blood clotting in a two
step process. First. TFPI binds to factor Xa and the TFPI:Xa
complex then interacts with the Tissue Factor (TF): Factor VIIa
complex, thereby inhibiting the latter complex. The TF:Factor VIIa
complex is the complex that activates factors IX and X. By
inhibiting TF:VIIa. TFPI regulates coagulation by preventing the
activation of Factors IX and X, required for blood clotting. The
interaction of Cyr61 with TFPI inhibits the activity of TFPI, thus
promoting blood coagulation. Cyr61 is, thus, a tissue factor
agonist.
[0189] Because of the interaction of Cyr61 and TFPI, Cyr61 can
control the ability of TFPI to inhibit coagulation, thereby
regulating hemostasis. A defect in Cyr61 may lead to the inability
to inhibit TFPI at the appropriate time, resulting in excessive
inhibition of tissue factor, thereby preventing clot formation.
Deregulated expression of Cyr61 will conversely inhibit the
activity of TFPI constitutively, and thus tissue factor is
constantly active, resulting in excessive clotting. When the
expression of cyr61 in endothelial cells is deregulated, one
possible outcome is thrombosis.
[0190] In addition to Cyr61, other ECM signalling molecules, such
as Fisp12 and CTGF, have been shown to exert effects on cells
participating in angiogenesis. Consequently, it is anticipated that
a variety of ECM signalling molecule-related biomaterials, alone or
in combination, may be used in the methods of the invention
directed towards modulating hemostasis.
EXAMPLE 21
Ex Vivo Hematopoietic Stem Cell Cultures
[0191] To investigate the effect of Cyr61 on the growth of
primitive multipotent stem cells, several assays that distinguish
these cells from more mature progenitor cells in a hematopoietic
culture are employed. These assays make use of physicochemical
(fibronectin-binding) or growth and development-related (generation
of progenitor blast colonies) differences between immature and
mature subsets of cells.
[0192] Two cell lines which require conditioned media for growth
are used as a source of hematopoietic stem cells (HSC). These
cloned, factor-dependent murine lines are B6Sut (cloned from long
term bone marrow culture and capable of growing in liquid medium
without differentiation, but multipotent in agar, as described in
Greenberger et al., Proc. Natl. Acad. Sci. [USA] 80:2931 [1983]),
and FDCP-mix (cloned from long term bone marrow culture cells
infected with the recombinant virus src-MoMuLV, and are multipotent
in agar cultures, as described in Spooncer et al., Nature 310:2288
[1984]). B6Sut cells are propagated in Kincaid's medium with 10%
fetal calf serum (FCS) and 10% 6.times.-concentrated
WEHI-conditioned medium. Greenberger et al. FDCP-mix cells are
propagated in Fischer's medium with 20% horse serum and 10%
6.times.-concentrated WEHI-conditioned medium. The cell lines are
propagated at 37.degree. C. 5% CO.sub.2.
[0193] Various ex vivo or in vitro cultures are assayed for
population growth in the presence or absence of exogenously
supplied murine Cyr61 or polyclonal anti-Cyr61 antibodies. Under
limiting dilution conditions, the cobblestone area forming cell
(CAFC) assay is used to identify cells with long term repopulating
ability. Ploemacher et al., Blood 74:2755 (1989); Ploemacher et
al., Blood 78:2527 (1991). Cells identified as having long term
repopulating ability by the CAFC assay are then analyzed by
measuring three parameters: Rate of population doubling, mitotic
index, and rate of DNA synthesis.
[0194] Long term cultures with or without supplementation with
Cyr61, are assayed for their levels of primitive HSC in the CAFC
assay. van der Sluijs et al., Exp. Hematol. 22:1236 (1994). For
example, M2-10B4 stromal cells, B6Sut, and FDCP-mix are each
subjected to the CAFC assay in the following manner, described for
the M2-10B4 cell line. Stromal cell layers are prepared by
inoculating 5.times.10.sup.5 M2-10B4 stromal cells (a cell line
cloned from bone marrow stroma, Sutherland et al., Blood 78:666
[1991]) into each well of a 96-well culture plate in DMEM with 10%
FCS. When the cells approach confluency, they are rinsed with PBS
and irradiated (20 Gy of gamma-irradiation, 1.02-1.04 Gy/minute) to
prevent replication of an hematopoietic cells within the stroma,
without affecting the stroma's ability to support
hematopoiesis.
[0195] Hematopoietic stem cells are added to the irradiated stromal
cells in DMEM with 10% FCS, in the presence or absence of Cyr61 (10
.mu.g/ml final concentration). Population doubling rates are
determined. e.g., by microscopic examination of cell morphology to
determine the numbers of long term repopulating cells (and more
mature short term progenitor cells) present in the various
experimental long term cultures. Subsequent investigation of the
expansion and differentiation capacities of the potential long term
HSC cultures is used for confirmation of suitable candidate cell
lines.
[0196] The mitotic index is determined according to procedures
standard in the art. Keram er al., Cancer Genet. Cytogenet. 55:235
(1991). Harvested cells are fixed in methanol:acetic acid (3:1,
v:v), counted, and resuspended at 10.sup.6 cells/ml in fixative.
Ten microliters of this suspension is placed on a slide, dried, and
treated with Giemsa stain. The cells in metaphase are counted under
a light microscope, and the mitotic index is calculated by dividing
the number of metaphase cells by the total number of cells on the
slide. Statistical analysis of comparisons of mitotic indices is
performed using the 2-sided paired t-test.
[0197] The rate of DNA synthesis is measured using a thymidine
incorporation assay. Various cultures are propagated in 1 .mu.Ci/ml
[.sup.3H]-thymidine (ICN Biomedicals, Inc., Costa Mesa, Calif.) for
24-72 hours. Harvested cells are then rinsed with PBS and fixed
with 10% trichloroacetic acid. DNA is dissolved in 0.1 N NaOH, and
thymidine incorporation is determined, for example by liquid
scintillation spectrophotometry.
[0198] The use of an ECM signalling molecule-related biomaterial,
such as Cyr61, can be used in the ex vivo expansion of
hematopoietic stem cell cultures. In addition, more than one ECM
signalling molecule-related biomaterial may be used to expand these
cultures. For example, Cyr61, with its expression targeted locally,
may be combined with Fisp12, which exhibits a more expansive
targeting as evidenced by the presence of Fisp12 in culture media.
As an alternative, CTGF may be substituted for Fisp12, its mouse
ortholog. One of skill in the art would be able to devise other
combinations of ECM signalling molecule-related biomolecules that
are within the spirit of the invention.
[0199] Those of ordinary skill in the art will recognize that the
successful expansion of hematopoietic stem cell cultures in the
presence of ECM signalling molecules such as Cyr61 provides a basis
for a method of screening for suspected modulators of that
expansion process. As in the other methods of the invention, a
suspected modulator is combined with an ECM signalling molecule
such as Cyr61 and exposed to primitive cells. In parallel, the ECM
signalling molecule is exposed to similar cells. The relative rates
of expansion may be used to identify a promoter, or inhibitor, of
the ability of the ECM signalling molecule to expand pluripotent
hematopoietic stem cell cultures.
[0200] Cyr61 alone or in combination with other hematopoietic
growth factors, may also be used to expand stem cell populations
taken from a patient and which may, after expansion, be returned to
the patient or other suitable recipient patient after for example,
chemotherapy or other treatment modalities that result in the
depletion of blood cells in a patient. Stem cell populations
expanded according to the present invention may also be used in
bone marrow transplants in a patient in need thereof.
EXAMPLE 22
Organ Regeneration
[0201] The role of Cyr61 in the various cellular processes invoked
by changes in the cellular growth state indicate that this protein
would be effective in promoting organ regeneration. Towards that
end, studies were conducted to determine the expression profile of
murine cyr61 in remaining liver tissue following a partial
hepatectomy. (The response of remaining liver tissue following
partial hepatectomy is a model for the liver's response to a
variety of injuries, including chemical injuries, e.g., exposure to
toxic levels of carbon tetrachloride.)
[0202] BALB/c 3T3 (Charles River) mice were subjected to partial
hepatectomies removing approximately 67% of their liver tissue.
Higgins et al., Archs. Path. 12:186-202 (1931). Twenty microgram
aliquots of RNA were removed from the remaining liver tissue at
varying times following the operation and liver RNA was isolated by
tissue homogenization followed by guanidinium isothiocyanate,
cesium chloride precipitation. Sambrook et al. RNAs were then
immobilized on nitrocellulose filters and probed with radiolabeled
clones containing various regions of murine cyr61 cDNA. Results
were visualized by autoradiography and indicated that removal of
liver tissue induced cyr61 mRNA expression, particularly in cells
found near the injury site. Consequently, induction of cyr61
expression, e.g., by recombinant techniques, might promote the
regeneration of organs such as liver. For example, cyr61 expression
can be controlled, e.g., by introducing recombinant cyr61
constructs that have been engineered to provide the capacity to
control expression of the gene, e.g., by the use of tissue-specific
promoters, e.g., the K14 promoter for expression in skin. The
recombinant cyr61 may be introduced to cells of the relevant organ
by gene therapy techniques using vectors that facilitate homologous
recombination (e.g., vectors derived from Herpesviruses,
Adenovirus, Adeno-associated Virus, Cytomegalovirus, Baculovirus,
retroviruses, Vaccinia Virus, and others). Techniques for
introducing heterologous genes into eukaryotic cells, and
techniques for integrating heterologous genes into host chromosomes
by homologous recombination, are well known in the art.
[0203] The development of skin, another organ, is also affected by
Cyr61. The expression of cyr61 is induced in cells in the vicinity
of skin injuries. Also, as described above, Cyr61 has a chemotactic
effect (i.e., Cyr61 induces cell migration) on endothelial cells
and fibroblasts. Further. Cyr61 induces the proliferation of
endothelial cells and fibroblasts. Both processes are involved in
the healing of skin wounds. Accordingly, Cyr61 administration,
e.g., by localized or topical delivery, should promote skin
regeneration.
[0204] Cyr61 is also highly expressed in lung epithelium. These
cells are frequently injured by exposure to environmental
contaminants. In particular, lung epithelium is frequently damaged
by air-borne oxidants. The administration of Cyr61, e.g., in
atomizers or inhalers, may contribute to the healing of lung
epithelium damaged, e.g., by environmental contaminants.
EXAMPLE 23
Chondrogenesis--ECM Signalling Molecules are Expressed in
Mesenchyme
[0205] Some ECM signalling molecules are also expressed in cells,
such as mesenchyme cells, that ultimately become a part of the
skeletal system. In this Example. Cyr61 is identified as one of the
ECM signalling molecules expressed in mesenchyme cells. Limb
mesenchymal cells were grown in micromass culture as described
above on glass coverslips (Fisher) for 3 days. Cultures were fixed
in 4% paraformaldehyde in PBS, incubated for 30 minutes at room
temperature with 1 mg/ml bovine testicular hyaluronidase (type IV,
Sigma) in 0.1N sodium acetate (pH 5.5) with protease inhibitors
phenymethylsulfonyl fluoride (PMSF, 1 mM), pepstatin (1 .mu.g/ml),
leupeptin (1 .mu.g/ml), aprotinin (1 .mu.g/ml), aminocaproic acid
(50 mM), benzamidine (5 mM), and EDTA (1 mM), blocked with 10% goat
serum in PBS and incubated overnight at 4 C with primary antibodies
against Cyr61 (Yang et al., 1991), fibronectin (Gibco) and tenascin
(Gibco). Controls were incubated with anti-Cyr61 antibodies
neutralized with 1 .mu.g/ml purified Cyr61. Cultures were
subsequently incubated with FITC-conjugated goat-anti-rabbit
secondary antibody (Zymed), for 1 hour at room temperature.
[0206] For whole mount immunohistochemical staining, mouse embryos
from gestational days 10.5 to 12.5 were fixed in 4%
paraformaldehyde in PBS, dehydrated in methanol/PBS and stored at
-20 C in absolute methanol. After rehydration, embryos were
incubated with anti-Cyr61 antibodies as described in Hogan et al.,
Development 120:53-60 (1994), incorporated herein by reference.
Controls were incubated with anti-Cyr61 antibodies neutralized with
1 .mu.g/ml purified Cyr61. Immunostained embryos were fixed,
cleared and photographed.
[0207] Consistent with the transient expression of the cyr61 mRNA
in somitic mesenchymal cells that are differentiating into
chondrocytes (O'Brien et al., 1992), the Cyr61 protein was found in
the developing embryonic skeletal system. Cyr61 was localized by
whole mount immunohistochemical staining to the proximal limb bud
mesenchyme in gestational day 10.5 to 12.5 embryos. The Cyr61
protein was localized to the developing vertebrae, the calvarial
frontal bone and the first brachial arch, as well as in the heart
and umbilical vessels, forming an expression pattern that was
consistent with the cyr61 mRNA expression pattern (O'Brien et al.,
1992).
[0208] Cyr61 protein could be detected by immunoblot analysis in
whole limb buds and in micromass cultures of limb bud mesenchymal
cells. The level of Cyr61 protein remained at relatively constant
levels throughout the 4 day culture period during which
chondrogenesis occurred. Using quantitative immunoblot analysis.
Cyr61 was estimated to represent approximately 0.03% of total
cellular and extracellular proteins in the mesenchymal cell
cultures. Cyr61, tenascin (Gibco), and fibronectin were localized
to the cartilage nodules by immunofluorescent staining in the
mesenchymal cell cultures. Cyr61 and tenascin were primarily
localized among the intranodular cells, while a fibrillar staining
pattern was also observed around and between the cartilage nodules
with anti-fibronectin antibodies. A similar immunofluorescent
staining pattern was observed in transverse sections of the
micromass cultures for all three antibodies. Together, these
results show that endogenous Cyr61 is localized in the developing
limb bud mesenchyme, both in vivo and in vivo.
EXAMPLE 24
Chondrogenesis--ECM Signalling Molecules Promote Cell Adhesion
[0209] Cyr61 is a secreted protein that mediates the adhesion of
fibroblasts and endothelial cells to non-tissue culture-treated
plastic surfaces (Kireeva et al., Mol. Cell. Biol. 16:1326-1334
[1996]). The attachment of limb bud mesenchymal cells on non-tissue
culture dishes coated with BSA. Cyr61, tenascin, and fibronectin,
were compared.
[0210] Cyr61, fibronectin (Gibco), or tenascin (Gibco) were diluted
in 0.1% protease-free bovine serum albumin (BSA) in PBS with 0.5 mM
PMSF, to a final concentrations of 10 or 50 .mu.g/ml. A 10 .mu.l
drop/well was placed in a non-tissue culture treated 24-well plate
(Corning), and incubated at room temperature for 2 hours. The wells
were blocked with 1% BSA in PBS for 1 hour at room temperature, and
rinsed with serum-free MEM (Modified Eagle's Medium). Limb
mesenchymal cells, suspended at 5.times.10.sup.5 cell/ml in
serum-free MEM, were added at a volume of 400 .mu.l/well, and
incubated at 37 C, 5% CO.sub.2 for 1 or 3 hours. At each time
point, the cell suspension was removed, the wells were rinsed with
MEM and the remaining adherent cells were photographed.
[0211] Cells attached poorly to BSA-coated dishes, but adhered as
clusters of rounded cells to Cyr61--and tenascin-coated dishes
within 1 hour of plating. In contrast, cells plated on
fibronectin-coated dishes attached uniformly and started to spread.
When cells were allowed to attach for 3 hours, many more adherent
cells were observed. Furthermore, intercellular clustering and
rounded cell morphology were maintained in cells plated on Cyr61
and tenascin, while cells plated on fibronectin spread to form a
monolayer. These observations show that Cyr61 mediates the adhesion
and maintenance of a rounded cellular morphology which is conducive
for mesenchymal cell chondrogenesis (Zanetti et al., Dev. Biol.
139.383-395 [1990]: Solursh et al., Dev. BIol. 94:259-264 [1982]),
similar to that previously reported for tenascin (Mackie et al., J.
Cell Biol. 105:2569-2579 [1987]).
[0212] As mentioned previously, ECM signalling molecules such as
Cyr61 may be used in methods of screening for modulators of cell
adhesion, including, but not limited to, the adhesion of
chondrocytes. The comparative assay, described above, measures the
relative adhesion levels of cells exposed to a combination of an
ECM signalling molecule and a suspected modulator of cell adhesion
and cells exposed to the ECM signalling molecule alone, whereby the
relative levels provide a basis for identifying either a promoter
or an inhibitor of cell adhesion.
EXAMPLE 25
Chondrogenesis--ECM Signalling Molecules Promote Cell
Aggregation
[0213] Since aggregation is an essential step for chondrogenic
differentiation (Solursh, M., In The role of extracellular matrix
in development, pp. 277-303 (Trelstad, R., ed.) (Alan R. Liss, New
York 1984)), the ability of Cyr61 to mediate intercellular
aggregation in suspension cultures of mesenchymal cells was
assessed. The number of cells remaining at various times after
isolation were counted. Untreated mesenchymal cells in suspension
began to aggregate soon after isolation, as the number of single
cells was decreased to 30% of the initial number within a 2 hour
incubation period. Cell aggregation was significantly inhibited in
cultures treated with affinity-purified anti-Cyr61 antibodies,
indicating that endogenous Cyr61 is important for mesenchymal cell
aggregation. To rule out the possibility that the affinity-purified
anti-Cyr61 antibodies might contain undefined components that
interfere with aggregation, anti-Cyr61 antibodies, described above,
were pre-incubated with purified Cyr61 protein prior to addition to
cells. These pre-incubated antibodies affected cell aggregation no
more than the IgG and Cyr61 buffer controls, indicating that the
anti-Cyr61 antibodies achieved their inhibition of cell aggregation
by neutralizing the endogenous Cyr61 protein of mesenchymal
cells.
[0214] In addition to the cell aggregation in suspension cultures
described above, the effect of Cyr61 on mesenchymal cell
aggregation in micromass cultures was also examined. When purified
Cyr61 protein (0.3 .mu.g/ml) was added to limb mesenchymal cells,
precocious cellular aggregation was observed within 24 hours,
unlike control cells which had not received Cyr61. Neither
Cyr61-treated nor control cultures had differentiated into
cartilage nodules at this time. By culture day 3, the development
of intermodular cellular condensations between the distinct
cartilage nodules was more extensive in Cyr61-treated cultures.
These intermodular condensations subsequently undergo
chondrogenesis, observed as Alcian blue-staining cartilaginous
matrix on culture day 4. Taken together, these results indicate
that Cyr61 is able to promote cell-cell aggregation, a necessary
step in chondrogenesis of mesenchymal cells in micromass
culture.
EXAMPLE 26
Chondrogenesis--ECM Signalling Molecules Promote Cell
Proliferation
[0215] Some ECM signalling molecules, such as Cyr61, affect
chondrogenesis, as revealed by effects on limb bud mesenchyme cells
in micromass culture, as described above. Ahrens et al., Dev. Biol.
60:69-82 (1977), has reported that these cells, in micromass
culture, undergo chondrogenesis in a manner similar to the in vivo
process. Mesenchyme cells were obtained from mouse embryonic limb
buds by trypsin digestion (1 mg/ml, 1:250 dilution of porcine
pancreatic trypsin, Sigma Chemical Co.). Cells were explanted in
plastic tissue culture wells and allowed to attach for 2 hours at
37.degree. C., 5% CO.sub.2. Cells were then incubated for 24 hours
at 37.degree. C. 5% CO.sub.2 in MEM with 10% FBS, penicillin (50
U/ml), and streptomycin (50 .mu.g/ml). At this point, the
composition of the medium was changed by substituting 4% NuSerum
(Collaborative Biomedical Products, Inc.) for 10% FBS. Individual
cultures then received Cyr61, fibronectin, heparin, (each at
approximately 1 .mu.g/ml) or buffer as a negative control. An
additional control was provided by adding a 1:100 dilution of
affinity-purified anti-Cyr61 antibody (approximately 13 .mu.g/ml
stock solution), elicited and purified by standard techniques.
Harlow et al.
[0216] Cell proliferation was assessed by the thymidine assay,
described above, and by microscopic cell counts. Chondrogenesis was
assessed by quantifying the incorporation of [.sup.35S]-sulfate
(ICN Biomedicals, Inc.) into sulfated glycosaminoglycans, and by
qualitatively determining the extent of chondrogenesis by cell
staining with Alcian Blue. Cultures, described above, were labeled
with 2.5 .mu.Ci/ml [.sup.35S]-sulfate for 18 hours, washed twice in
PBS, fixed with Kahle's fixative (Pepper et al., J. Cell Sci.
109:73-83 [1995]) and stained for 18 hours in 0.5% Alcian Blue, pH
1.0. The extent of chondrogenesis is correlated with the intensity
of Alcian Blue staining. San Antonio et al., Dev. Biol. 115:313-324
(1986). The results show that Cyr61 specifically increased limb bud
mesenchyme cell proliferation and aggregation, leading to enhanced
chondrogenesis.
[0217] In addition to demonstrating that purified Cyr61 enhanced
growth factor-induced DNA synthesis in fibroblasts and endothelial
cells, the effects of Cyr61 on cell proliferation were directly
examined. Cell proliferation during the 4 day culture period was
determined by counting cell number and by incorporation of
[.sup.3H]-thymidine. To determine cell number, cells were harvested
by trypsin/EDTA (Sigma) and counted with a Coulter counter. In
parallel cultures, [.sup.3H]-thymidine (1 .mu.Ci/ml; ICN) was added
to the media for 18 hours and incorporation in the TCA-insoluble
layer was determined by liquid scintillation counting. Purified
Cyr61 protein added to limb mesenchymal cells both increased cell
number and enhanced DNA synthesis after 2 and 3 days in culture,
although the total cell number in Cyr61-treated and Cyr61-untreated
cultures leveled off at the same level after 4 days.
[0218] The role of Cyr61 in chondrogenesis may also improve the
integration of prosthetic devices. For example, skeletal injuries
and conditions frequently are treated by the introduction of a
prosthesis e.g., hip prosthesis, knee prosthesis. Beyond questions
of histocompatibility, the successful implantation of a prosthetic
device requires that the foreign element become integrated into the
organism's skeletal structure. The capacity of Cyr61 polypeptides
to affect cell adhesion, migration, and proliferation, and the
ability of Cyr61 polypeptides to induce the differentiation of
mesenchyme cells into chondrocytes, should prove valuable in the
treatment of skeletal disorders by prosthesis implantation. For
example, integration of a prosthetic device by chondrocyte
colonization would be promoted by therapeutic treatments involving
the administration of Cyr61 in a pharmaceutically acceptable
adjuvant, carrier or diluent, using any of the administration
routes known in the art or by coating the prosthesis device with
Cyr61 polypeptides in a suitable carrier. The carrier may also be a
slow-release type vehicle to allow sustained release of the
polypeptides.
[0219] As noted in previously, the methods of the invention include
a method of screening for modulators of cell proliferation,
including chondrocytes. A comparison of the relative rates of cell
proliferation in the presence of a control comprising an ECM
signalling molecule alone (e.g., Cyr61) and in the presence of a
combination of an ECM signalling molecule and a suspected modulator
of cell proliferation provides a basis for identifying a suspected
modulator as a promoter, or inhibitor, of chondrocyte
proliferation.
EXAMPLE 27
Chondrogenesis--ECM Signalling Molecules Promote Chondrogenesis
[0220] Chondrogenic differentiation was quantitated by
incorporation of [.sup.35S]-sulfate (ICN) into sulfated
glycosaminoglycans and assessed qualitatively by Alcian Blue
staining. Cultures were radiolabeled with 2.5 .mu.Ci/ml
[.sup.35S]-sulfate for 18 hr, fixed with Kahle's fixative and
stained with 0.5% Alcian Blue, pH 1.0 (Lev et al., 1964). The
extent of chondrogenesis is correlated with the intensity of Alcian
Blue staining (San Antonio et al., 1986). [.sup.35S]-Sulfate
incorporation in the fixed cell layer was quantitated by liquid
scintillation counting.
[0221] Exogenous purified Cyr61 protein promoted limb mesenchymal
cell aggregation and resulted in greater Alcian blue-staining
cartilaginous regions in micromass cultures, suggestive of a
chondrogenesis-promoting effect. This effect was quantified by the
incorporation of [.sup.35S]-sulfate into sulfated
glycosaminoglycans (San Antonio et al., 1986) in Cyr61-treated
micromass cultures. Exogenous Cyr61 enhanced [.sup.35S]-sulfate
incorporation in a dose-dependent manner, resulting in a 1.5-fold
and 3.5-fold increase with 0.3 and 5 .mu.g/ml Cyr61, respectively,
and was correlated qualitatively by increased Alcian Blue staining.
The increase observed at the 5 .mu.g/ml Cyr61 dose (120 nM) is an
under-estimation of the actual extent of chondrogenesis, since some
of the large cartilage nodules which were formed at this dose
detached from the dish. Cultures treated with 10 .mu.g/ml Cyr61
formed a more massive mound of cartilage.
[0222] A review of the literature indicated that chondrogenesis in
limb mesenchymal cell micromass cultures was increased 2-fold with
the addition of 10 .mu.g/ml heparin (San Antonio et al. 1987; Resh
et al., 1985) and 3-fold with 50 .mu.g/ml tenascin (200 nM) (Mackie
et al., 1987). The results demonstrated that purified Cyr61 was
effective at concentrations (10-100 nM) similar to or less than
those of other molecules known to promote chondrogenesis in this
cell system.
[0223] Since a certain threshold cell density must be reached for
initial aggregation to occur (Umansky, 1966; Ahrens et al., 1977),
embryonic mesenchymal cells plated at low densities are normally
unable to differentiate into chondrocytes, although the addition of
exogenous factors such as heparin or poly-L-lysine (San Antonio et
al., 1986; San Antonio et al., 1987) have been shown to support
chondrogenesis in cells plated under these conditions. Therefore,
the ability of Cyr61 to promote differentiation of mesenchymal
cells plated at densities above and below the threshold for
chondrogenesis was assessed. Cells plated at 2.5.times.10.sup.6
cell/ml incorporated little [.sup.35S]-sulfate. However, when Cyr61
was added, these sub-threshold density cultures formed nodules and
incorporated sulfate to a level similar to that in cultures plated
at 3.times.10.sup.6 cells/ml, which supports chondrogenesis.
Therefore, Cyr61 can promote chondrogenesis in mesenchymal cells
plated at non-chondrogenic, sub-threshold densities.
[0224] It is conceivable that when mesenchymal cells are plated in
a high density micromass, the extent of chondrogenesis may be
maximal and cannot be enhanced further by exogenous factors, which
also may not be accessible to all cells. However, addition of
exogenous Cyr61 resulted in a 2-fold enhancement in
[.sup.35S]-sulfate incorporation in cultures plated at densities
ranging from 3 to 10.times.10.sup.6 cell/ml. Therefore, Cyr61 can
further enhance chondrogenesis in high density micromass cultures,
which have apparently not reached a maximal degree of
differentiation.
[0225] It is possible that the increased [.sup.35S]-sulfate
incorporation in Cyr61-treated cultures is at least partly due to
an increase in cell number, since Cyr61 also promotes cell
proliferation. If this were true, then normalization of sulfate
incorporation with respect to cell number would eliminate any
differences between control and Cyr61-treated cultures. This was
not found to be the case. Cyr61-treated cultures still showed an
approximately 2-fold increase in normalized sulfate incorporation
over control, indicating that Cyr61 promotes a net increase in
chondrogenesis. On culture day 2, the sulfate/cell number ratio was
lower in Cyr61-treated cultures compared to controls and is
reflective of a low level of [.sup.35S]-sulfate incorporation
relative to cell number, since mesenchymal cells are mostly
proliferating rather than differentiating in these early stage
cultures (Ede. 1983).
[0226] The presence of endogenous Cyr61 in these cells, both in
vivo and in vitro, indicates that Cyr61 may indeed function
biologically to regulate chondrogenic differentiation. The ability
of exogenously added purified Cyr61 to promote intercellular
aggregation and to increase [.sup.35S]-sulfate incorporation and
Alcian-blue staining in limb mesenchymal cells demonstrates that
Cyr61 can act as a chondrogenesis enhancing factor. As shown above
in Example 11, anti-Cyr61 antibodies can neutralize both the cell
adhesion and DNA-synthesis enhancement activities of Cyr61.
Anti-Cyr61 antibodies were added to the mesenchymal cell culture
media or mixed the cell suspension prior to plating. Chondrogenesis
was inhibited in the cultures treated with anti-Cyr61 antibodies,
as demonstrated by decreases of [.sup.35S]-sulfate incorporation to
50% and 30% of controls, when antibodies were added to the media,
and mixed with the cells, respectively. These observations were
correlated with decreased Alcian Blue staining. However, mixing of
the anti-Cyr61 antibodies with mesenchymal cells prior to plating
resulted in complete detachment in some of the treated cultures
within 24 hours.
[0227] To eliminate the possibility of an unidentified component in
the antibody preparation as a cause of cell detachment, anti-Cyr61
antibody was preincubated with 1 .mu.g/ml purified Cyr61 protein
prior to mixing with cells. The inhibition of chondrogenesis in
mesenchymal cells mixed with neutralized anti-Cyr61 antibodies was
abolished.
[0228] Generally, the invention contemplates a method of screening
for modulators of chondrogenesis. A comparative assay involves the
exposure of chondrocytes to either (a) a combination of a suspected
modulator of chondrogenesis and an ECM signalling molecule such as
Cyr61, or (b) the ECM signalling molecule alone. Measurements of
the relative rates of chondrogenesis then provide a basis for
identifying the suspected modulator of chondrogenesis as a promoter
or inhibitor of that process.
[0229] The results described in this Example demonstrate that
endogenous Cyr61 is present in mesenchymal cells and is important
for their chondrogenesis. Accordingly, the use of an ECM signalling
molecule, such as Cyr61, to induce bone healing is contemplated by
the invention. For example, a biologically effective amount of
Cyr61 is introduced into a matrix such as a sponge, as described
above, and this material is then applied to set bone fractures or
used to gather together the fragments of a comminuted bone
fracture. A biodegradable matrix may be employed, or the matrix may
be removed at an appropriate later time. Alternatively, Cyr61 may
be applied directly to bone. In addition, Cyr61 may be applied to
inanimate objects such as biocompatible prosthesis, as described in
Example 26.
EXAMPLE 28
Genetics
[0230] Another way to control the effects of an ECM signalling
molecule-related biomaterial is to inactivate it by creating
dominant negative mutations in the relevant gene in actively
growing and dividing cells. One approach involves the use of
recombinant techniques, e.g., to create homozygous mutant genotypes
in ex vivo cultures such as HSC cultures. Introduction of these
cells into an organism, e.g., a human patient, would then provide
an opportunity for the introduced mutant cells to expand and alter
the expression of the ECM signalling molecule in vivo. Mutants
homozygous for such a mutation could affect the expression of an
endogenous wild type ECM signalling molecule in other cells.
Heterozygous mutants might produce altered ECM signalling molecules
capable of interacting with the wild type ECM signalling molecule,
also being expressed, in such a way that the ECM signalling
molecule's activities are modulated or abolished.
[0231] Furthermore, because of the role played by ECM signalling
molecules such as Cyr61 in regulating chondrogenesis (i.e.,
skeletal development), genetic manipulations that alter the
expression of human Cyr61 may prove medically important for
prenatal screening methods and gene therapy treatments related to
skeletal conditions, in addition to angiogenic conditions. For
example, the cyr61 gene is expressed when mesenchymal cells of both
ectodermal and mesodermal origins differentiate to form
chondrocytes. Thus, one of the roles that Cyr61 might play is to
regulate the commitment of mesenchyme cells to chondrocyte cell
lineages involved in skeletal development. Consistent with this
view, transgenic mice overexpressing cyr61 ectopically are born
with skeletal abnormalities. In all cases examined, the presence of
the skeletal deformities correlates with expression of the
transgene. These results suggest that the human form of Cyr61 may
also regulate chondrogenesis and skeletal development. It is also
possible that the human cyr61 gene may correspond to a genetic
locus already known to affect skeletal development or birth defects
relating to bone morphogenesis. Knowledge of the human Cyr61
protein sequence, presented in SEQ ID NO:4 herein, and the coding
sequence of the cDNA, presented in SEQ ID NO:3 herein, provide the
basis for the design of a variety of gene therapy approaches.
[0232] This information also provides a basis for the design of
probes useful in genotypic analyses. e.g., Restriction Fragment
Length Polymorphism analyses. Such analyses are useful in the
fields of genetic counselling, e.g., in diagnosing diseases and
conditions and the likelihood of their occurrence, as well as in
forensic analyses.
[0233] By way of example, the materials of the present invention
are useful in the prenatal screening for a variety of conditions or
disorders, including blood disorders, skeletal abnormalities, and
cancerous conditions. Well known techniques for obtaining fetal
cells, e.g., amniocentesis, provide the materials needed for
diagnosis. In one embodiment of the invention, the fetal cells are
expanded and DNA is isolated. In another embodiment, fetal cells
are lysed and polymerase chain reactions are performed using
oligonucleotide primers according to the invention. Using either
approach, the DNA is then subjected to analysis. One analytical
approach involves nucleotide sequence determination of particular
regions of cyr61 or of the entire gene. The available human cyr61
coding sequence, presented in SEQ ID NO:3 herein, facilitates the
design of sequencing primers that brings nucleotide sequence
analysis into the realm of practical reality. An alternative to
nucleotide sequence analysis is an investigation of the expression
characteristics of the fetal nucleic acid. The capacity of the
fetal nucleic acid to be expressed might be dispositive in the
diagnosis of Cyr61-related angiogenic, chondrogenic, or oncogenic
disorders.
[0234] The invention also comprehends a kit comprising Cyr61. The
kits according to the invention provide Cyr61 in a form that is
useful for performing the aforementioned methods of the invention.
Kits according to the invention contain isolated and purified
recombinant human Cyr61 in a suitable buffer, optionally stabilized
by the addition of glycerol for storage at -20.degree. C. In
addition to the Cyr61 provided in the kit, the invention also
contemplates the inclusion of any one of a variety of buffering
agents, salts of various types and concentrations, and additional
protein stabilizing agents such as DTT, all of which are well known
in the art. Other kits according to the invention incorporate
isolated and purified murine Cyr61. Kits incorporating a Cyr61
polypeptide and an inhibitory peptide or an anti-Cyr61 antibody, as
described above, are also contemplated.
Sequence CWU 1
1
* * * * *