U.S. patent application number 10/051044 was filed with the patent office on 2002-12-19 for use of fviia or a tissue factor antagonist for regulating gene expression and cell migration or chemotaxis.
Invention is credited to Ezban, Mirella, Petersen, Lars Christian, Siegbahn, Agneta.
Application Number | 20020193302 10/051044 |
Document ID | / |
Family ID | 27221063 |
Filed Date | 2002-12-19 |
United States Patent
Application |
20020193302 |
Kind Code |
A1 |
Ezban, Mirella ; et
al. |
December 19, 2002 |
Use of FVIIa or a tissue factor antagonist for regulating gene
expression and cell migration or chemotaxis
Abstract
The present invention relates to use of FVII and/or FVIIa and/or
another TF agonist and/or FVIIai and/or another TF antagonist in
therapeutic treatment of pathological conditions that can be
related to cell migration or treated by specific regulation of cell
migration or chemotaxis. The invention also relates to the use of
of FVII and/or FVIIa and/or another TF agonist and/or FVIIai and/or
another TF antagonist in therapeutic treatment of pathological
conditions that can be related to regulation of expression of at
least one gene in a cell, e.g., Cyr61 gene.
Inventors: |
Ezban, Mirella; (Copenhagen
O, DK) ; Petersen, Lars Christian; (Horsholm, DK)
; Siegbahn, Agneta; (Uppsala, DK) |
Correspondence
Address: |
Reza Green, Esq.
Novo Nordisk of North America, Inc.
Suite 6400
405 Lexington Avenue
New York
NY
10174-6401
US
|
Family ID: |
27221063 |
Appl. No.: |
10/051044 |
Filed: |
January 14, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10051044 |
Jan 14, 2002 |
|
|
|
PCT/DK00/00401 |
Jul 14, 2000 |
|
|
|
60148300 |
Aug 11, 1999 |
|
|
|
Current U.S.
Class: |
514/8.2 ;
514/13.3; 514/14.3; 514/19.8 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 35/00 20180101; A61P 17/02 20180101; C12Y 304/21021 20130101;
A61P 43/00 20180101; A61P 9/00 20180101; A61P 35/04 20180101; A61P
9/10 20180101; C12N 9/6437 20130101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 038/37; A61K
038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 12, 1999 |
DK |
PA 1999 01117 |
Jul 14, 1999 |
DK |
PA 1999 01023 |
Claims
1. A method for inducing or enhancing cell migration, comprising
the step of contacting said cell with a tissue factor agonist
2. The method of claim 1, wherein the tissue factor agonist is FVII
or FVIIa.
3. A method of reducing or inhibiting cell migration, comprising
the step of contacting the cell with a tissue factor
antagonist.
4. The method of claim 3, wherein the tissue factor antagonist is
modified FVII.
5. The method of claim 1 or claim 3, wherein said cell is a human
cell expressing tissue factor, including fibroblasts, smooth muscle
cells, tumour cells, haematopoietic cells, monocytes, macrophages
and epithelial cells.
6. The method of claim 5, wherein said cell further expresses PDGF
and PDGF receptors, especially PDGF beta-receptors.
7. The method according to claim 4, wherein the modified factor VII
is selected from factor VII modified with Dansyl-Phe-Phe-Arg
chloromethyl ketone, Phe-Phe-Arg chloromethylketone,
Dansyl-D-Phe-Phe-Arg chloromethyl ketone and D-Phe-Phe-Arg
chloromethylketone.
8. A method for inducing or enhancing wound healing in a patient,
comprising administering to said patient an effective amount of a
pharmaceutical composition comprising Factor VIIa or factor VII or
another tissue factor agonist.
9. A method for inhibiting or reducing cell migration, invasion,
migration-induced cell proliferation or angiogenesis in a patient
having a disease or condition associated with undesired cell
migration, invasion, migration-induced cell proliferation or
angiogenesis, comprising administering to said patient an effective
amount of a pharmaceutical composition comprising a tissue factor
antagonist.
10. A method according to claim 9, wherein the disease or condition
is primary tumour growth, tumour invasion or metastasis.
11. A method according to claim 9, wherein the tissue factor
antagonist is modified factor VII.
12. Use of a tissue factor agonist for the manufacture of a
medicament for inducing or enhancing cell migration.
13. Use according to claim 12, wherein the tissue factor agonist is
FVII or FVIIa or a combination thereof.
14. Use of a tissue factor antagonist for the manufacture of a
medicament for reducing or inhibiting cell migration.
15. The use of claim 14, wherein the tissue factor antagonist is
modified factor VII.
16. Use according to claim 15, wherein the modified factor VII is
selected from factor VII modified with Dansyl-Phe-Phe-Arg
chloromethyl ketone, Phe-Phe-Arg chloromethylketone,
Dansyl-D-Phe-Phe-Arg chloromethyl ketone and D-Phe-Phe-Arg
chloromethylketone.
17. A method of regulating the expression of at least one gene in a
cell, comprising the step of contacting said cell with a tissue
factor agonist or a tissue factor antagonist, under conditions that
result in a measurable change in said expression.
18. The method of claim 17, wherein the tissue factor agonist is
selected from the group consisting of FVII, FVIIa, and combinations
thereof.
19. The method of claim 17, wherein the tissue factor antagonist is
modified FVII.
20. The method of claim 19, wherein the modified factor VII is
selected from the group consisting of factor VII modified with
Dansyl-Phe-Phe-Arg chloromethyl ketone, Phe-Phe-Arg
chloromethylketone, Dansyl-D-Phe-Phe-Arg chloromethyl ketone and
D-Phe-Phe-Arg chloromethylketone.
21. The method of claim 17, wherein the gene is a gene belonging to
the CCN gene family.
22. The method of claim 17, wherein said gene is selected from the
group consisting of Cyr61, CTFG, dopamine D2 receptor, EST Incyte
PD 395116 and P2U nucleotide receptor.
23. The method of claim 21, wherein the gene is Cyr61 gene.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of PCT/DK00/00401 filed
on Jul. 14, 2000, and claims priority under 35 U.S.C. 119 of Danish
application no. PA 1999 01117 filed on Aug. 12, 1999, Danish
application no. PA 1999 01023 filed on Jul. 14, 1999, and U.S.
provisional application No. 60/148,300 filed on Aug. 11, 1999, the
contents of which are fully incorporated herein by reference.
FIELD OF INVENTION
[0002] A novel cell regulating activity of coagulation factor VII
(FVII) or a tissue factor antagonist such as, for example,
inactivated coagulation factor VIIa (FVIIai) of cells expressing
tissue factor (TF) has been described. The present invention
relates to a method for regulating cell migration or chemotaxis by
contacting the cell with FVIIa or another TF agonist, or FVIIai or
another TF antagonist and determining the migration of said cell.
The invention also relates to the use of FVIIa or another TF
agonist, or FVIIai or another TF antagonist for the preparation of
a medicament for regulation of cell migration in a patient.
Moreover the present invention relates to a method of treatment,
and a method of detecting the activity of compounds, in particular
drug candidates that interact with cell migration.
BACKGROUND OF THE INVENTION
[0003] The extrinsic pathway of blood coagulation is initiated when
FVIIa circulating in plasma binds to the integral-membrane protein,
tissue factor (TF). The role of TF in blood coagulation has been
extensively studied. The involvement of FVIIa as a proteolytic
enzyme in the blood coagulation cascade is believed to be confined
to the extracellular leaflet of TF expressing cells. An
intracellular activity of FVIIa was first implied when the sequence
of TF showed homology to the cytokine/interferon- or heamatopoietic
receptor superfamily. The subclass I of the heamotopoietic receptor
family includes receptors for growth hormone, prolactin,
interleukins 1 to 7, granulocyte-macrophage colony stimulating
factors, eiythropoitin and thrombopoitin. Subclass II includes TF
and receptors for interferon a and b.
[0004] The resemblance of TF to this class of receptors was further
substantiated with the appearance of the crystal structure.
Characteristic of this class of cytokine receptors that includes
receptors for interferon b and g and IL-10 is that their activation
lead to rapid tyrosine phosphorylation of the receptors themselves,
as well as a subset of intracellular proteins. Within minutes after
the initial tyrosine phosphorylation an array of mitogen-activated
(Ser/Thr) kinases (MAPK) is activated. These kinases are arranged
in several parallel signalling pathways. Thorough studies of the
putative intracellular signalling capacity of FVIIa have shown that
it induce mobilisation of intracellular free calcium (Ca.sup.2+) in
the human bladder carcinoma cell line, J82, which constitutively
express TF and in umbelical vein endothelial cells which were
pre-treated with interleukin-1 to express TF, but have failed to
show any cytokine-like activation of intracellular tyrosine
kinases. In conclusion FVIIa is believed, in a TF dependent manner,
to induce mobilisation of intracellular Ca.sup.2+ through
activation of phospholipase C. The mechanism by which FVIIa
activates phospholipase c is not known, but tyrosine kinase
activation has specifically been ruled out.
[0005] Recent reports from a number of laboratories indicate that
TF may influence an array of important biological functions other
than coagulation., such as angiogenesis, embryo vascularization and
tumor metastasis. At present, however, it is unclear how TF
contributes to these biological processes. The extracellular domain
of TF consists of two fibronectin-type III-like modules, as in the
typical class II cytokine receptor extracellular domain, raising
the possibility that TF may play a role in signal transduction, the
primary function of cytokine receptor. However, TF has a very short
cytoplasmic domain (only 21 amino acid residues in length) and
lacks membrane-proximal motifs that mediate binding of the
non-receptor Janus kinases (Jaks) that are essential for cytokine
receptor signaling. Nonetheless, several biochemical findings
suggest a signal transduction function for TF. Analysis ofthe human
TF protein sequence revealed a putative phosphorylation site in the
cytoplasmic domain, which is conserved in mouse, rat and rabbit TF.
Specific serine residues in the cytoplasmic tail of TF are
phosphorylated in cells following stimulation with protein kinase C
activator. The human TF cytoplasmic tail is phosphorylated in vitro
at multiple sites when incubated with lysates of U87-MG cells. A
potential role for the TF cytoplasmic domain in signal transduction
is also indicated in studies that showed prometastatic function of
TF is critically dependent on the TF cytoplasmic domain. Further,
TF cytoplasmic domain is shown to interact with actin-binding
protein 280 (ABP-280) and supports cell adhesion and migration
through recruitment of ABP-280 to TF-mediated adhesion
contacts.
[0006] However, TF has also been shown to participate certain types
of cell signaling by serving as a cofactor for its physiological
ligand FVIIa in an extracellular signaling by a proteolytic
mechanism. For example, binding of FVIIa to cell surface TF is
shown to induce intracellular Ca.sup.2+ oscillations in a number of
TF expressing cells, transient phosphorylation of tyrosine in
monocytes, activation of MAP kinase, alteration in gene expression
in fibroblasts and enhanced expression of urokinase receptor in
tumor cells. Catalytically inactive FVIIa (FVIIai) fails to induce
many of the above signaling responses, from Ca.sup.2+ oscillations
to MAP kinase activation and gene reduction, and it appears that
the catalytic activity of FVIIa may be required for at least some
TF-FVIIa-mediated signal transduction. At present, not much is
known about signaling pathway(s) that are induced by
proteolytically active FVIIa and how the signals generated by FVIIa
could contribute to angiogenesis and tumor metastasis.
[0007] To study temporal program of transcription that underlies
the FVIIa-induced response, in the present study, we have examined
the response of human fibroblasts to FVIIa using a cDNA microarray.
The data revealed that the cellular expression of several genes was
detectably altered in fibroblasts upon exposure of to FVTIIa. One
such gene is Cyr61, a growth factor-inducible intermediate early
gene, whose product is shown to promote cell adhesion, augment
growth factor-induced DNA synthesis and stimulate cell migration in
fibroblasts and endothelial cells.
SUMMARY OF THE INVENTION
[0008] The present invention relates to usage of FVII and/or FVIIa
and/or another TF agonist and/or FVIIai and/or another TF
antagonist in therapeutic treatment of pathological conditions that
can be related to cell migration or treated by specific regulation
of cell migration or chemotaxis.
[0009] In another aspect the invention relates to the use of FVII
and/or FVII and/or another TF agonist and/or FVIIai and/or another
TF antagonist in therapeutic treatment of pathological conditions
that can be related to the regulation of expression of at least one
gene in a cell e.g. the Cyr61 gene.
[0010] In another aspect the invention relates to a method for
inducing or enhancing cell migration, comprising the step of
contacting said cell with a tissue factor agonist
[0011] In one embodiment, the tissue factor agonist is FVII or
FVIIa.
[0012] In another aspect the invention relates to a method of
reducing or inhibiting cell migration, comprising the step of
contacting the cell with a tissue factor antagonist.
[0013] In one embodiment the tissue factor antagonist is modified
FVII.
[0014] In one embodiment the cell is a human cell expressing tissue
factor, including fibroblasts, smooth muscle cells, tumour cells,
haematopoietic cells and epithelial cells.
[0015] In one embodiment the modified factor VII is selected from
factor VII modified with Dansyl-Phe-Pro-Arg chloromethyl ketone,
Dansyl-Glu-Gly-Arg chloromethyl ketone, Dansyl-Phe-Phe-Arg
chloromethyl ketone, Phe-Phe-Arg chloromethylketone,
Dansyl-D-Phe-Pro-Arg chloromethyl ketone, Dansyl-D-Glu-Gly-Arg
chloromethyl ketone, Dansyl-D-Phe-Phe-Arg chloromethyl ketone and
D-Phe-Phe-Arg chloromethylketone.
[0016] In another aspect the invention relates to a method for
inducing or enhancing wound healing in a patient, comprising
administering to said patient an effective amount of a
pharmaceutical composition comprising Factor VIIa or factor VII or
another tissue factor agonist or a combination thereof.
[0017] In another aspect the invention relates to a method for
inhibiting or reducing cell migration, invasion, migration-induced
cell proliferation or angiogenesis in a patient having a disease or
condition associated with undesired cell migration, invasion,
migration-induced cell proliferation or angiogenesis, comprising
administering to said patient an effective amount of a
pharmaceutical composition comprising a tissue factor
antagonist.
[0018] In one embodiment the disease or condition is primary tumour
growth, tumour invasion or metastasis.
[0019] In another aspect the invention relates to the use of a
tissue factor agonist for the manufacture of a medicament for
inducing or enhancing cell migration.
[0020] In another aspect the invention relates to the use of a
tissue factor antagonist for the manufacture of a medicament for
reducing or inhibiting cell migration.
[0021] In another aspect the invention relates to a method of
regulating the expression of at least one gene in a cell,
comprising the step of either contacting said cell with a tissue
factor agonist or contacting said cell with a tissue factor
antagonist.
[0022] In one embodiment the gene is a gene belonging to the CCN
gene family.
[0023] In another embodiment the gene is selected from the group
consisting of Cyr61, CTFG, dopamine D2 receptor, EST Incyte PD
395116 or P2U nucleotide receptor.
[0024] In one embodiment the gene is Cyr61 gene.
[0025] In one embodiment the regulation is inducing or enhancing
expression. In another embodiment the regulation is reducing or
inhibiting expression.
[0026] In one embodiment FVII or FVIIa or another tissue factor
agonist induces or enhances gene expression and modified FVII or
another tissue factor antagonist reduces or inhibits gene
expression, e.g. when the gene is a gene belonging to the CCN gene
family, or the gene is selected from the group consisting of Cyr61,
CTFG, dopamine D2 receptor, EST Incyte PD 395116 or P2U nucleotide
receptor.
[0027] In another embodiment FVII or FVIIa or another tissue factor
agonist reduces or inhibits gene expression, and modified FVII or
another tissue factor antagonist induces or enhances gene
expression, e.g., when the gene is EST PD674714.
[0028] Diseased states, which may be treated, are pathological
conditions such as, for example, atherosclerosis, tumour
deposition, tumour growth, tumour invasion, metastasis, or
angiogenesis. Other states that may be treated is, for example,
healing of wounds including regeneration of vessel walls and
treatment of burns, or inflammation, or the regulation of cell
migration in vitro such as, for example, growing of tissue.
LIST OF FIGURES (fra 6011)
[0029] FIGS. 1A and 1B: Flow cytometric analysis of TF expression
in fibroblasts (1A). The cells were stained with either a murine
monoclonal fluoresceinisothiocyanate (FITC)-conjugated mouse anti
IgG-antibody (unfilled area) that was used as negative control or a
monoclonal FITC-conjugated anti-tissue factor (TF) antibody (filled
area). FIG. 1B shows the procoagulant activity of fibroblasts.
Fibroblasts with TF expression generated a 10-fold increase in PCA
compared to monocytes without TF expression.
[0030] FIG. 2A: Effects of FVIIa and FFR-FVIIa on PDGF-BB induced
chemotaxis in human fibroblasts. .nu. show the chemotactic response
of fibroblasts to different concentrations of PDGF-BB. Fibroblasts
incubated with 100 nM FVIIa (.lambda.) or 100 nM FFR-FVIIa (.mu.)
migrated towards different concentrations of PDGF-BB. Results are
means and SEM of three separate experiments. P-values less than
0.05,* was considered statistically significant (Student's t
test).
[0031] FIGS. 3 A-D: The influence of different concentrations of
FVIIa or FFR-FVIIa on PDGF-BB induced chemotaxis in fibroblasts. v
show migration of fibroblasts to different concentrations of
PDGF-BB. Cells were incubated with 12.5 (A), 25 (B), 50(C) and 100
(D) nM FVIIa (.lambda.) or FFR-FVIIa (.mu.) and assayed in the
Boyden chamber towards different concentrations of PDGF-BB. Results
are mean and SEM of three different experiments. *=p<0.05,
**=p<0.01 and ***=p<0.001 Student's t test.
[0032] FIG. 4A: A mixture of three monoclonal antibodies to TF
blocks the effects of FVIIa and FFR-FVIIa on PDGF-BB induced
chemotaxis in fibroblasts. .nu. show migration towards PDGF-BB of
fibroblasts without TF antibodies, .lambda. fibroblasts
preincubated with TF antibodies and 100 nM FVIIa, and .mu.
fibroblasts preincubated with TF antibodies and 100 nM FFR-FVIIa.
Results are mean and SEM of three separate experiments.
[0033] FIGS. 5A and 5B: The influence of FXa on the chemotactic
response to PDGF-BB induced by FVIIa. Fibroblasts were preincubated
with 200 nM TAP (FIG. 5A) (.nu.) or with 0.2-2 .mu.M TAP (FIG. 5B)
(.nu.) and then with 100 nM FVIIa (.lambda.). TAP was present
during the entire experiments. Chemotaxis was induced by different
concentrations of PDGF-BB (5A) or by 0.1 ng/ml PDGF-BB (5B).
Results are mean and SD of two separate experiments.
[0034] FIG. 6A: The influence of thrombin on the chemotactic
response to PDGF-BB induced by FVIIa. Fibroblasts were preincubated
with 5 U/mL (final concentration) Hirudin and then with 100 nM
FVIIa. Hirudin was present during the entire experiments.
Chemotaxis was induced by different concentrations of PDGF-BB. .nu.
show cells incubated with Hirudin alone and .lambda. cells with
Hirudin and FVIIa. Results are mean and SD of two separate
experiments.
[0035] FIG. 7A: Effect of inhibition of PI3'-kinase on chemotaxis
in fibroblasts incubated with FVIIa. Cells were preincubated with
varying concentrations of LY294002 for 30 min at 37.degree. C., and
then with 100 nM FVIIa (.lambda.) or without FVIIa (.nu.). The
inhibitor was present throughout the chemotaxis assay. Chemotaxis
was induced by 0.1 ng/mL PDGF-BB. Results are mean and SD of two
separate experiments.
[0036] FIGS. 8A AND 8B: Effect of inhibition of PLC on chemotaxis
in fibroblasts incubated with FVIIa. Cells were incubated with
varying concentrations of U73122 (active PLC inhibitor) (8A) or
U73343 (inactive control) (8B) for 30 min at 37.degree. C. before
incubation with or without 100 nM FVIIa, and then assayed in the
Boyden chamber to a concentration gradient of 0.1 ng/mL PDGF-BB.
The agents were present during the entire experiments. .nu. show
cells with U73122 or U73343 alone, .lambda. cells with U73122 or
U73343 and FVIIa. Results are mean and SD of two separate
experiments.
[0037] FIG. 9: Release of inositol trisphosphate (IP.sub.3) from
fibroblasts stimulated with FVIIa, FFR-FVIIa alone or in
combination with PDGF-BB. Cells were labelled over night with myo
[.sup.3H] inositol, incubated with or without 100 nM FVIIa or
FFR-FVIIa in the absence or presence of 10 ng/mL or 100 ng/mL
PDGF-BB. Cells were then analysed for release in IP.sub.3. Open
bars show cells without FVIIa or FFR-FVIIa (control), hatched bars
show cells with FFR-FVIIa, and black bars show cells incubated with
FVIIa.
[0038] FIG. 10: Tyrosine phosphorylation of PLC-.gamma.1 in
response to PDGF-BB alone (control), FVIIa or FFR-FVIIa in
combination with PDGF-BB. Cells were incubated with 100 nM FVIIa or
FFR-FVIIa for one hour, and then with or without PDGF-BB at
indicated concentrations. Cells were lysed and tyrosine
phosphorylation of PLC-.gamma.1 detected as described in
methods.
[0039] FIG. 1. Northern blot analysis confirming the data obtained
with cDNA microarray assay. Ten .mu.g of total RNA (from the same
RNA samples that were used to isolate poly (A) RNA to generate
probes for hybridization of cDNA microarray) were patiented to
Northern blot analysis and probed with .sup.32P-labeled Cyr61 (a
partial length cDNA, obtained from Genomic Systems). Panel B. The
hybridization signals are quantified with PhosphorImager (Molecular
Dynamics).
[0040] FIGS. 2 and 2B. Time-dependentfactor VIIa-induced expression
of Cyr61. Quiescent monolayers of WI-38 cells were treated with
factor VIIa (5 82 g/ml) (2A) or PDGF-BB (10 ng/ml) (2B) for varying
time periods. Total RNA (10 .mu.g) was patiented to Northern blot
analysis and probed with radio labeled Cyr61. Ethidium bromide
staining of 28S ribosomal RNA of the corresponding blot is shown in
the bottom panel as RNA loading control.
[0041] FIG. 3. Dose-dependentfactor VIIa-induced expression of
Cyr61. Quiescent monolayers of WI-38 cells were treated with
varying doses of factor VIIa, 0, 0.1, 0.5, 2.0 and 5.0 .mu.g/ml for
45 min. Total RNA (10 .mu.g) was patiented to Northern blot
analysis and probed with radiolabeled Cyr61. Ethidium bromide
staining of 28S ribosomal RNA of the corresponding blot is shown in
the bottom panel as RNA loading control.
[0042] FIG. 4. Factor VIIa catalytic activity is required for the
induced expression of Cyr61. Quiescent monolayers of WI-38 cells
were treated with a control serum-free medium or serum-free medium
cotaining factor VIIa (5 .mu.g/ml) or active-site inactivated
factor VIIa (VIIai, 5 .mu.g/ml) for 45 min. Total RNA (10 .mu.g)
was patiented to Northern blot analysis and probed with
radiolabeled Cyr61 . Ethidium bromide staining of 28S ribosomal RNA
of the corresponding blot is shown in the bottom panel as RNA
loading control.
[0043] FIG. 5. Factor VIIa-induced expression of Cyr61 is not
abolished by specific inhibitors offactor Xa and thrombin.
Quiescent monolayers of WI-38 cells were treated with control
medium or the medium containing factor VIIa (5 .mu.g/ml; 100 nM for
45 min. Cells were preincubated with 200 nM recombinant TAP lane 3)
or hirudin (lane 4) for 30 min before exposure to factor VIIa for
45 min. Total RNA (10 .mu.g) was patiented to Nothern blot analysis
and probed with radiolabeled Cyr61. Ethidium bromide staining of
28S ribosomal RNA of the corresponding blot is shown in the bottom
panel as RNA loading control.
[0044] FIG. 6. Effect of actinomycin-D and cycloheximide on factor
VIIa-induced Cyr61 mRNA steady-state levels. Quiescent monolayers
of WI-38 cells were preincubated with a control vehicle,
actinomycin D (10 .mu.g/ml) or cycloheximide (10 .mu.g/ml) for 30
min before the cells were exposed to factor VIIa (5 .mu.g/ml) for
45 min. Total RNA (10 .mu.g) was patiented to Northern blot
analysis and probed with radiolabeled Cyr61. Ethidium bromide
staining of 28S ribosomal RNA of the corresponding blot is shown in
the bottom panel as RNA loading control.
[0045] FIG. 7. Factor VIIa induces the expression of CTGF.
Quiescent monolayers of WI-38 cells were treated with factor VIIa
(5 .mu.g/ml) for varying time periods. Total RNA (10 .mu.g) was
patiented to Northern blot analysis and probed with radio labeled
CTGF. Ethidium bromide staining of 28S ribosomal RNA of the
corresponding blot is shown As RNA loading control.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The present invention relates to the use of FVII or FVIIa or
another TF agonist for the manufacture of a pharmaceutical
composition for inducing or enhancing cell migration.
[0047] In a further aspect the present invention relates to the use
of FVII, FVIIa or another TF agonist for the manufacture of a
pharmaceutical composition for inducing or enhancing wound healing
or angiogenesis.
[0048] In a still further aspect the present invention relates to
the use of FVIIai or another TF antagonist for the manufacture of a
pharmaceutical composition for inhibiting or preventing cell
migration.
[0049] In one embodiment the cell migration is in a subject.
[0050] In a further aspect the present invention relates to the use
of FVIIai or another TF antagonist for the manufacture of a
pharmaceutical composition for inhibiting or preventing
angiogenesis, metastasis, tumour growth or tumour invasion.
[0051] In a further aspect the present invention concerns a method
for inducing or enhancing cell migration in a patient, which
comprises administering an effective amount of FVII or FVIIa or
another TF agonist to said patient.
[0052] In a still further aspect the present invention concerns a
method for inhibiting or preventing cell migration in a patient,
which comprises administering an effective amount of FVIIai or
another TF antagonist to said patient.
[0053] In a particular embodiment the effective amount is a daily
dosage from about 5 .mu.g/kg/day to about 500 .mu.g/kg/day.
[0054] In a further embodiment the TF antagonist comprises a
modified FVIIa, for example, FFR-FVIIa.
[0055] The present invention provides a mechanism for an activity
of FVII and/or FVIIa that relates to stimulation of cell migration.
Such a mechanism provides the basis for establishing the
involvement of FVII and/or FVIIa in pathological conditions in
which TF expressing cells like endothelial cells, epithelial cells,
fibroblasts, smooth muscle cells and monocytes/ macrophages
participate. The invention furthermore provides the basis for
identifying specific pharmacological targets that are useful for
therapeutic intervention.
[0056] Thus, the present invention relates to usage of FVII and/or
FVIIa and/or FVIIai in therapeutic treatment of pathological
conditions that can be related to cell migration or treated by
specific regulation of cell migration.
[0057] In another aspect, the present invention relates to a method
of detecting drug candidates that regulate cell migration, which
method comprise
[0058] a) culturing a TF expressing cell;
[0059] b) measuring the migration of the cell;
[0060] c) incubating the cell with a drug candidate, and
[0061] d) measuring the migration of the incubated cell and
determining any change in the level of migration compared to the
migration measured in step b, such change being indicative of
biologically active drug candidate in said cell.
[0062] Generally, the blood components, which participate in what
has been referred to as the coagulation "cascade" are proenzymes or
zymogens, enzymatically inactive proteins, which are converted to
proteolytic enzymes by the action of an activator, itself an
activated clotting factor. Coagulation factors that have undergone
such a conversion and generally referred to as "active factors",
and are designated by the addition of the letter "a" to the name of
the coagulation factor (e.g. factor VIIa).
[0063] The term "zinc-chelator" is intended to comprise a compound
that binds to factor VIIa and induces replacement of calcium ions
with zinc ions within factor VIIa, thereby inhibiting the activity
of factor VIIa or tissue factor-factor VIIa complex (TF-FVIIa).
[0064] A suitable TF antagonist according to the invention may be a
zinc-chelating compound, e.g. a dihydroxamate or a dihydrazide with
the hydroxamate or hydrazide groups located relative to each other
in such a position that they are able to chelate a zinc ion. The
zinc-chelating compound acts in combination with FVIIa.
Zn.sup.2+-ions exert their inhibitory action in competition with a
stimulatory effect of Ca.sup.2+-ions. It is predicted that
Zn.sup.2+-ions displace Ca.sup.2+-ions from one or more calcium
binding site(s) within FVIIa. Zinc-chelating compounds, e.g.
hydroxamates and hydrazides, are capable of acting as powerfull
supporters for binding of zinc ions in competition with calcium
ions. Specific compounds thereby potentiate zinc inhibition of the
activity of the factor VIIa/tissue factor complex. The activity of
factor VIIa in complex with tissue factor can be inhibited by a
mechanism in which a zinc chelator binds to factor VIIa and
facilitates replacement of Ca.sup.2+ with Zn.sup.2+. By this action
the chelator exerts a modulatory effect on TF at the normal
concentration of free Ca.sup.2+ and Zn.sup.2+ ions in the
blood.
[0065] The term "FVII" or "factor VII" means "single chain"
(zymogenic) coagulation factor VII. The term "Factor VIIa", or
"FVIIa" means "two chain" activated coagulation factor VII cleaved
by specific cleavage at the Arg152-Ile153 peptide bond. FVII and
FVIIa may be purified from blood or produced by recombinant means.
It is evident that the practice of the methods described herein is
independent of how the purified factor VIIa is derived and,
therefore, the present invention is contemplated to cover use of
any factor VII or FVIIa preparations suitable for use herein.
Preferred are human FVIIa. FVII or FVIIa is also intended to
include FVII variants wherein one or more amino acid residues has
(have) been replaced.
[0066] The term "modified factor VII", "inactivated FVII" or
"FVIIai" is intended to mean FVIIa having at least one modification
in its catalytic centre, which modification substantially inhibits
the ability of modified FVIIa to activate FX and FIX. The terms may
be used interchangeably. Such modification includes amino acid
substitution (or replacement) of one or more of the catalytic triad
residues Ser344, Asp142 and His193, and also includes modification
of catalytic triad residues with serine protease inhibitors such as
organo-phosphor compounds, sulfanylfluoride, peptide halomethyl
ketone or azapeptide. FFR-FVIIa is one example of a FVIIai
derivative obtained by blocking of the active centre of FVIIa with
the irreversible inhibitor,
D-phenylalanine-L-phenylalanine-L-argininine chloromethyl ketone
(FFR cmk). Other suitable FVIIai derivates are inactivated FVIIa
obtained or obtainable by blocking the active centre with
L-phenylalanine-L-phenylalanine-L-argininine chloromethyl ketone,
dansyl-L-phenylalanine-L-phenylalanine-L-argininine chloromethyl
ketone, or dansyl-D-phenylalanine-L-phenylalanine-L-argininine
chloromethyl ketone, Preferred is FFR-FVIIa (FVIIa inactivated by
FFR cmk).
[0067] The term "protein kinase" is intended to indicate an enzyme
that is capable of phosphoiylating serine and/or threonine and/or
tyrosine in peptides and/or proteins.
[0068] The term "drug candidate" is intended to indicate any
sample, which has a biological function or exerts a biological
effect in a cellular system. The sample may be a sample of a
biological material such as a microbial or plant extract, or it may
be a sample containing a compound or mixture of compounds prepared
by organic synthesis or genetic techniques.
[0069] The term "TF agonist" comprises compounds inducing
[0070] a) signal transduction by direct binding to TF (e.g.
FVIIa),
[0071] b) stimulation of MAPK cascade,
[0072] c) abrogation of MAPK inhibition (e.g. PTPase inhibitors),
which agonists are drug candidates as defined above.
[0073] The term "TF antagonist" comprises
[0074] a) reagents which compete with FVIIa for binding to TF
without transmission, e.g. FVIIai,
[0075] b) reagents which bind to FVIIa and prevent binding to TF,
e.g. Zn hydroxamate,
[0076] c) reagents which inhibit signal transduction by interfering
with members of the MAPK cascade,
[0077] d) reagents which bind to FVIIa/TF and prevent
transmission,
[0078] e) reagents which bind to FVIIa/TF/FX and prevent
transmission,
[0079] f) reagents which block human factor X activation catalysed
by human tissue factor/factor VIIa complex,
[0080] which antagonists are drug candidates as defined above.
[0081] The term "pharmacological targets" is intended to indicate a
protein that can alter the migration of TF expressing cells.
[0082] The term "reporter gene" is intended to indicate a DNA
construct that, when transcribed, produces a protein that can be
detected.
[0083] The term "SRE promoter element" means a DNA sequence that
binds transcription factors induced by components present in
serum.
[0084] The term "TF expressing cell" means any mammalian cell that
expresses TF.
[0085] The term "protein phosphorylation" is intended to indicate
phosphorylation of serine and/or threonine and/or tyrosine in
peptides and/or proteins.
[0086] Modulation or regulation of cell migration is defined as the
capacity of FVIIa or another TF agonist, or FVIIai or another TF
antagonist to 1) either increase or decrease ongoing, normal or
abnormal, cell migration, 2) initiate normal cell migration, and 3)
initiate abnormal cell migration.
[0087] Modulation or regulation of gene expression encompasses the
capacity of FVIIa or another TF agonist, or FVIIai or another TF
antagonist to 1) either increase or decrease ongoing, normal or
abnormal, cell migration, 2) initiate normal cell migration, and 3)
initiate abnormal cell migration.
[0088] Modulation or regulation of gene expression encompasses an
increase or decrease in any parameter of gene expression of at
least about 1.5-fold, preferably at least about 2-fold, more
preferably at least about 3-fold, and most preferably at least
about 5-fold. Useful parameters of gene expression include, without
limitation, rate of transcription, level of mRNA accumulation, rate
of synthesis of the gene product, and level of protein
accumulation. Modulation of gene expression may also be reflected
in secondary indices known to those of ordinary skill in the art.
Any measurable change in any of these parameters indicates
regulation of expression.
[0089] In this context, the term "treatment" is meant to include
both prevention of an adverse condition and regulation of an
already occurring condition with the purpose of inhibiting or
minimising the condition. Prophylactic administration of FVIIa or
another TF agonist, or FVIIai or another TF antagonist is thus
included in the term "treatment".
[0090] In this context, the term "one unit" is defined as the
amount of factor VII present in 1 ml of normal plasma,
corresponding to about 0.5 .mu.g protein. After activation 50 units
correspond to about 1 .mu.g protein.
[0091] In this context, the term "patient" is defined as any
animal, in particular mammals, such as humans. The term "subject"
is used interchangeably with "patient"
1 Abbreviations TF tissue factor FVII factor VII in its
single-chain, unactivated form FVIIa factor VII in its activated
form RFVIIa recombinant factor VII in its activated form FVIIai
modified (inactivated) factor VII FFR-FVIIai factor VII inactivated
by reaction with D-Phe-L-Phe-L-Arg chloromethyl ketone
[0092] Tissue factor (TF) is the cellular receptor for factor FVIIa
(FVIIa) and the complex is principal initiator of blood
coagulation. We have studied the effects of FVIIa binding to TF on
cell migration and signal transduction of human fibroblasts that
express high amounts of TF. Fibroblasts incubated with FVIIa
migrated towards a concentration gradient of PDGF-BB at about one
hundred times lower concentration than do fibroblasts not ligated
with FVIIa. Anti-TF antibodies inhibited the increase in chemotaxis
induced by FVIIa/TF. Moreover, a pronounced suppression of
chemotaxis induced by PDGF-BB was observed with active
site-inhibited FVIIa (FFR-FVIIa). The possibility was excluded that
hyperchemotaxis was induced by a putative generation of FXa and
thrombin activity.
[0093] FVIIa induced the production of inositol-1,4,5-trisphosphate
to the same extent as PDGF-BB; the effects of FVIIa and PDGF-BB
were additive. FFR-FVIIa did not induce any release of
inositol-1,4,5,-trisphosphate. The cellular migration response to
PDGF-BB and FVIIa was totally blocked by a PLC-inhibitor,
suggesting that activation of PLC is important for the response.
Thus, binding of FVIIa to TF can independent of coagulation,
modulate cellular responses, such as chemotaxis, and the catalytic
activity of FVIIa is necessary.
[0094] TF is believed to exert a function in tumour cell
metastasis, but the mechanism is yet not known. However, Ott et al.
very recently identified actin-binding protein 280 (ABP-280) as a
ligand for the TF cytoplasmic domain, providing a molecular pathway
by which TF may support tumor cell metastasis. The molecular
signals and the biological functions transduced by FVIIa/TF are,
however, still poorly understood.
[0095] Human fibroblasts have a constitutive expression of TF.
These cells also express receptors for platelet-derived growth
factor (PDGF). PDGF induces in its target cells mitogenicity, actin
reorganization and directed cell migration (chemotaxis). We have
previously shown that PDGF-BB is an efficient chemotactic factor
for human fibroblasts and that the chemotactic response is mediated
by the .beta.-receptor class. Therefore, these cells were chosen to
study putative signal transduction and cell migration induced by
binding of FVIIa to TF.
[0096] Below we show for the first time a clear connection between
signalling induced by FVIIa binding to TF and the cellular response
to a growth factor. We present data that in human fibroblasts the
FVIIa/TF complex leads to a hyperchemotactic response to PDGF-BB.
Furthermore, active site-inhibited FVIIa (FFR-FVIIa) in a
dose-dependent way suppressed the directed migration towards
PDGF-BB. By the use of specific inhibitors to PLC and
phosphatidylinositol 3'-kinase (PI3'-kinase) we also demonstrate
that the hyperchemotactic response towards PDGF-BB induced by
FVIIa/TF signalling is dependent upon phospholipase C (PLC)
activity but independent of PI3'-kinase. FVIIa and PDGF-BB induced
the production of inositol-1,4,5-trisphosphate (IP.sub.3), one of
the second messengers released after activation of PLC, in an
additive manner.
[0097] TF is constitutively expressed on the plasma membrane of
many extravascular cells, such as stromal fibroblasts in vascular
adventitia and in fibrous capsules of liver, spleen and kidney.
Thus, expression of TF is found at sites physically separated from
the circulating blood and providing a haemostatic envelope. Upon
injury this barrier is thought to protect the organism against
bleeding. TF can, however, be induced in monocytes/macrophages,
vascular smooth muscle cells, endothelial cells and in a number of
tumour cells by a variety of agents, including cytokines and growth
factors. Induction at the transcriptional level occurs rapidly
after stimulation, identifying TF as a growth-related immediate
early gene.
[0098] In this study we have investigated the role of TF as a
signalling receptor. We show that human fibroblasts with a
constitutive expression of TF upon ligand binding of FVIIa migrate
towards extremely low concentrations of PDGF-BB. TF/FVIIa alone did
not induce enhanced spontaneous migration, i.e. random migration.
Thus, a combination of intracellular signal transduction by
FVIIa/TF and the growth factor PDGF-BB was necessary to achieve the
motility response. Not only binding to TF, but also the catalytic
activity of TF/FVIIa was mandatory, since active-site inhibited
FVIIa did not elicit enhanced migration response. Furthermore,
inhibitory monoclonal antibodies prevented enhancement of the
chemotactic response by FVIIa. We also excluded that indirect
signalling occured due to FXa or thrombin, since TAP and Hirudin
had no effect on FVIIa/TF induced chemotaxis. We instead found that
increasing concentrations of FFR-FVIIa actively inhibited PDGF-BB
induced chemotaxis. Fibroblasts incubated with FFR-FVIIa showed
completely normal random migration. The inhibitory effect of
FFR-FVIIa on PDGF-BB-induced chemotaxis was not observed in the
presence of the combination of anti-TF antibodies thereby ruling
out the possibility of FFR-FVIIa being toxic. The results suggest
rather, that in cells expressing PDGF .beta.-receptors and TF, the
FVIIa/TF complex is of importance for the chemotactic response to
PDGF-BB.
[0099] Our finding that FVIIa increases IP.sub.3 production, and
the previously reported data on FVIIa/TF induced Ca.sup.2+
oscillations especially in MDCK cells, strongly support the notion
that PLC is activated by FVIIa/TF signalling in a number of cells.
In addition, the hyperchemotactic response in human fibroblasts to
PDGF-BB induced by FVIIa/TF was blocked in a dose-dependent way by
a PLC-inhibitor. We have previously found a similar
hyperchemotactic response to PDGF-BB in PDGF .beta.-receptor Y934F
mutant cells, which showed increased phosphorylation and activation
of PLC-.gamma.1. In these cells, the enhanced phosphorylation of
PLC-.gamma.1 correlated with a threefold higher IP.sub.3 production
compared to wild-type PDGF .beta.-expressing cells. The combination
of FVIIa/TF and PDGF-BB induced about twofold increase in IP.sub.3
production in human fibroblasts. FVIIa/TF-induced IP.sub.3
production, however, did not correlate with phosphorylation of
PLC-.gamma.1. Tyrosine phosphorylation of PLC-.gamma.2 induced by
FVIIa/TF cannot be excluded, but seems unlikely since the
expression of PLC-.gamma.2 is very low in human fibroblasts.
Moreover, the intracellular part of TF is not endowed with
intrinsic protein tyrosine kinase activity. These results suggest
that FVIIa/TF induces activation of .beta. and/or .delta. PLC
isozymes. In the assay for IP.sub.3 release the cell culture medium
was supplemented with 0.1% FBS containing only about 0.1 nM FXa. We
found that a concentration of more than 20 nM FXa is necessary to
induce IP.sub.3 production. The mechanism by which .beta. or
.delta. PLC isozymes are activated remains to be elucidated. It is
believed that activation involves the cooperation between TF and a
membrane-associated protein.
[0100] Lately, the connection of TF with the cytoskeleton was
identified. A molecular interaction between the cytoplasmatic
domain of TF and the actin filament-binding protein ABP 280 was
shown. Furthermore, TF was found to be in close contact with actin
and actin filament-binding proteins, such as .alpha.-actinin and
ABP280 in lamellipodia and ruffled membrane areas in spreading
epithelial cells. ABP 280, a member of the filamin subfamily, is
required for normal function of lamellipodia and thus highly
important for cell motility. PI3'-kinase and PLC isozymes are
implicated in chemotactic responses, such as mobilisation of
actin-binding proteins. In previous studies we observed that the
PI3'-kinase pathway in PDGF-P receptor induced chemotaxis seems
less important in cells with over-expression and enhanced activity
of PLC-.gamma.1. This was also the case for cells with FVIIa bonded
to TF. This indicates that the magnitude of activation of
PI3'-kinase and PLC isozymes will determine which of these pathways
will dominate. Taken together, our data show that cell migration is
an important morphogenic function induced by FVIIa/TF
signalling.
[0101] Chemotaxis plays a pivotal role in wound healing,
angiogenesis and metastasis. Chemotaxis is also an important
component in the development of atherosclerotic plaques. In these
processes a variety of cells express TF as well as PDGF and PDGF
receptors. Restenosis is a major complication following
interventional procedure of obstructed arteries. PDGF has been
implicated in the vessel wall's response (neointima formation) to
mechanical injury by mediating the migration and proliferation of
smooth muscle cells and fibroblasts. We have shown now for the
first time that FVIIa binding to TF-expressing cells have an
increased chemotactic response to PDGF, which is independent of the
coagulation.
[0102] At present, not much is known about signaling pathway(s)
that are induced by proteolytically active VIIa and how the signals
generated by VIIa could contribute to cellular processes. One
possibility is that FVIIa could induce the expression of growth
regulators that act downstream to induce cellular processes. To
investigate this possibility, in the present study, we have
examined changes in the transcriptional program in human
fibroblasts in response to exposure to VIIa using a cDNA microarray
that contain more than 8,000 individual human genes. We chose
fibroblasts since these cells normally encounter serum, which
contain growth factors and activated clotting factors in the
context of vascular injury due to physical (e.g., surgery) and
pathophysiological conditions. The temporal program of gene
expression observed in response to serum suggests that fibroblasts
are programmed to interpret the abrupt exposure to serum nor as a
general mitogenic stimulus but as a specific physiological signal.
Characterization of transcriptional activation in response to serum
and growth factors also suggest that fibroblasts are an active
participant in a conversation among the diverse cells which
collectively control inflammation, angiogenesis and wound
healing.
[0103] cDNA microarray analysis with mRNA isolated from fibroblasts
exposed to VIIa for 90 min shows upregulation of Cyr61. Northern
blot analysis confirmed the VIIa-induced expression of Cyr61 in
fibroblasts. Although not as robust as in fibroblasts, VIIa also
increases the expression of Cyr61 in vascular smooth muscle cells.
Induction of Cyr61 expression is dependent on the FVIIa's catalytic
activity since FVIIai fail to induce the expression of Cyr61 .
Although factor Xa and thrombin could also induce the expression of
Cyr61(data not shown), these compounds are not involved in
FVIIa-induced expression of Cyr61 . We found no evidence for the
generation of traces factor Xa and thrombin in our experimental
system. Further, specific inhibitor of factor Xa and thrombin had
no significant effect on the FVIIa-induced expression of Cyr61.
[0104] Cyr61 is an immediate-early gene that is transcriptionally
activated by serum growth factors in fibroblasts. It encodes a
secreted 40 kDa, cysteine-rich and heparin-binding protein that
associates with extracellular matrix and cell surfaces. Cyr61 is a
member of an emerging gene family of conserved and modular proteins
characterized by the presence of an N-terminal secretory signal,
followed by four modular structural domains and 38 cysteine
residues that are largely conserved among members of the family.
The protein family now consists of six distinct members, including
Cyr61, connective tissue growth factor (CTGF) and an avian
proto-oncoprotein, Nov (thus named as CCN family) (The CCN family
is further described in Lau et al., Exp. Cell Res 248: 44-57,
1999). Cyr61 protein is shown to (i) promote the attachment and
spreading of endothelial cells in a manner similar to that of
fibronectin, (ii) enhance the effects of bFGF and PDGF on the rate
of DNA synthesis of fibroblasts and vascular endothelial cells
(iii) promotes cell migration in both fibroblasts and endothelial
cells. Recent studies show that Cyr61 acts as a ligand to integrin
.alpha..sub..gamma..beta..sub.3, an adhesion receptor known to be
involved in signaling that regulates a number of cellular processes
including angiogenesis and tumor metastasis. Purified Cyr61 protein
was shown to stimulate directed migration of human microvascular
endothelial cell in culture through a
.alpha..sub..gamma..beta..sub.3-dependent pathway and induce
neovascularization in rat corneas. Furthermore, expression of Cyr61
in tumor cells promotes tumor growth and vascularization.
[0105] Based on the present data that show FVIIa induces Cyr61
expression in fibroblasts, it is believed that FVIIa-induced Cyr61
is responsible, acting through integrin
.alpha..sub..gamma..beta..sub.3, for FVIIa-mediated cell migration
and tumor metastasis. Thus, Cyr61 links FVIIa-TF proteolytical
signal to the integrin-signaling pathway. The observations that
VIIa catalytic activity is required for migration of smooth muscle
cells and tumor cells, and tumor metastasis are consistent with the
other observation that FVIIa catalytic activity is required for the
induction of Cyr61.
[0106] In addition to Cyr61, VIIa could also induce other
regulators that could mediate FVIIa-induced biological responses.
FVIIa binding to cell surface TF in pancreatic cancer cells was
shown to selectively over-express uPAR gene. Earlier we have shown,
using differential display technique, up-regulation of
transcription of poly(A)polymerase gene in fibroblasts exposed to
FVIIa. Although it would have been interesting to find out whether
the cDNA microarray also show differential expression of PAP, the
filter did not contain the PAP cDNA. In addition to Cyr61, our cDNA
microarray also show differential expression of four other genes
(see results), but the differential expression ratio was very close
to the borderline significance. Since in preliminary experiments we
could not confirm their differential expression by Northern blot
analysis and also the absence of any suggestive relevant data on
the ability of these gene products to mediate FVIIa-induced
biological responses, we did not analyze their expression further.
However, since CTGF is a structurally related molecule to Cyr61 and
elicit same biological responses as Cyr61, we have examined the
expression of CTGF even though the relative ratio of CTGF
expression in FVIIa-treated sample vs the control sample in the
cDNA microarray is 1.8(2 is a conservative estimate to be a real
magnitude in the assay). The data revealed that FVIIa also induced
the expression of CTGF and the kinetics of VIIa-induced expression
of CTGF was similar to that of Cyr61.
[0107] Although CTGF behaves very similar to Cyr61, subtle
differences exist between them. For example, (a) CTGF has shown to
be mitogenic in itself whereas Cyr61 has no intrinsic mitogenic
activity but augments growth factor-induced DNA synthesis (b) Cyr61
stimulates chemotaxis whereas CTGF stimulates both chemotaxis and
chemokinesis (c) although both Cyr61 and CTGF are ECM-associated
signalling molecules, CTGF is shown to secrete into culture medium.
Thus, it is possible that FVIIa regulates cellular functions
locally via Cyr61 whereas acts at a distance from its site through
the secretion of CTGF.
[0108] The regimen for any patient to be treated with FVIIa or
another TF agonist or FVIIai or another TF antagonist as mentioned
herein should be determined by those skilled in the art. The daily
dose to be administered in therapy can be determined by a physician
and will depend on the particular compound employed, on the route
of administration and on the weight and the condition of the
patient. An effective amount is suitably a daily dosage from about
5 .mu.g/kg/day to about 500 .mu.g/kg/day, preferably from about 10
.mu.g/kg/day to 300 .mu.g/kg/day, more preferred from about 15
.mu.g/kg/day to 200 .mu.g/kg/day, most preferred from about 20
.mu.g/kg/day to 100 .mu.g/kg/day.
[0109] The FVIIa or another TF agonist or FVIIai or another TF
antagonist should be administered in one single dose, but it can
also be given in multiple doses preferably with intervals of 4-6-12
hours depending on the dose given and the condition of the
patient.
[0110] The FVIIa or another TF agonist or FVIIai or another TF
antagonist may be administered intravenously or it may be
administered by continuous or pulsatile infusion or it may be
administered directly to the relevant site such as, for example,
injected directly into a tumour. FVIIa or another TF agonist or
FVIIai or another TF antagonist is preferably administered by
intraveneous injections and in an amount of about 100-100,000 units
per kg body weight, and preferably in an amount of about 250-25,000
units per kg body weight corresponding to about 5-500 .mu.g/kg, a
dose that may have to be repeated 2-4 times per 24 hours.
[0111] Conventional techniques for preparing pharmaceutical
compositions, which can be used according to the present invention
are, for example, described in Remington's Pharmaceutical Sciences,
1985.
[0112] The compositions used according to this invention are
prepared by methods known per se by the skilled artisan.
[0113] In short, pharmaceutical preparations suitable for use
according to the present invention is made by mixing FVII, FVIIa or
another TF agonist or FVIIai or another TF antagonist, preferably
in purified form, with suitable adjuvants and a suitable carrier or
diluent. Suitable physiological acceptable carriers or diluents
include sterile water and saline. Suitable adjuvants, in this
regard, include calcium, proteins (e.g. albumins), or other inert
peptides (e.g. glycylglycine) or amino acids (e.g. glycine, or
histidine) to stabilise the purified factor VIIa. Other
physiological acceptable adjuvants are non-reducing sugars,
polyalcohols (e.g. sorbitol, mannitol or glycerol), polysaccharides
such as low molecular weight dextrins, detergents (e.g.
polysorbate) and antioxidants (e.g. bisulfite and ascorbate). The
adjuvants are generally present in a concentration of from 0.001 to
4% w/v. The pharmaceutical preparation may also contain protease
inhibitors, e.g. apronitin, and preserving agents.
[0114] The preparations may be sterilised by, for example,
filtration through a bacteria-retaining filter, by incorporating
sterilising agents into the compositions, by irradiating the
compositions, or by heating the compositions. They can also be
manufactured in the form of sterile solid compositions, which can
be dissolved in sterile water, or some other sterile medium
suitable for injection prior to or immediately before use.
[0115] In different aspects the present invention concerns:
[0116] A method of regulating the expression of at least one gene
in a cell, comprising the steps of:
[0117] a) contacting said cell with factor VII (a) or a tissue
factor antagonist
[0118] b) determining the expression of said gene in said cell.
[0119] The above method, wherein said cell is a human vascular cell
expressing tissue factor, including fibroblasts and smooth muscle
cells.
[0120] The method, wherein said gene is selected from the group
consisting of Cyr61 , CTFG, dopamine D2 receptor, EST incyte PD
395116 or P2U nucleotide receptor.
[0121] The method, wherein said tissue factor antagonist is
modified factor VII (a) known as factor VIIai.
[0122] A method wherein the expression of said gene is
enhanced.
[0123] A method wherein the expression of said gene is inhibited or
minimized.
[0124] A method of enhancing the expression of said gene comprising
contacting the cell with factor VIIa.
[0125] A method of inhibiting the expression of said gene
comprising contacting the cell with modified factor VII known as
FVIIai.
[0126] The method wherein said gene is EST PD674714.
[0127] A method for regulating cell migration, comprising the steps
of:
[0128] a) contacting said cell with factor VIIa or a tissue factor
antagonist;
[0129] b) determining the migration of said cell.
[0130] The method, wherein said cell is a human cell expressing
tissue factor, including fibroblasts, smooth muscle cells, tumour
cells, haematopoietic cells and epithelial cells.
[0131] The method, wherein the tissue factor antagonist is modified
factor VIIa known as factor VIIai.
[0132] The method, wherein the modified factor VII is selected from
Dansyl-Phe-Pro-Arg chloromethyl ketone, Dansyl-Glu-Gly-Arg
chloromethyl ketone, Dansyl-Phe-Phe-Arg chloromethyl ketone and
Phe-Phe-Arg chloromethylketone.
[0133] A method of enhancing cell migration, comprising contacting
the cell with FVIIa or a tissue factor agonist.
[0134] A method of reducing or inhibiting cell migration,
comprising contacting the cell with a tissue factor antagonist.
[0135] A method for inducing or enhancing wound healing in a
patient, comprising administering to said patient an effective
amount of a pharmaceutical composition comprising Factor VIIa or a
tissue factor agonist.
[0136] A method for inhibiting the invasiveness of tumour cells
comprising contacting said cells with an effective amount of a
tissue factor antagonist.
[0137] A method for inhibiting cell migration, invasion,
migration-induced cell proliferation or angiogenesis in a patient
having a disease or condition associated with undesired cell
migration, invasion, migration-induced cell proliferation or
angiogenesis, comprising administering to said patient an effective
amount of a pharmaceutical composition comprising a tissue factor
antagonist.
[0138] The method, wherein the disease or condition is primary
tumour growth, tumour invasion or metastasis.
[0139] The method, wherein the tissue factor antagonist is modified
factor VII known as FVIIai.
[0140] Use of factor VIIa or a tissue factor antagonist for the
manufacture of a medicament for regulating cell migration.
[0141] Use, wherein factor VIIa is used for the manufacture of a
medicament for enhancing cell migration.
[0142] Use, wherein a tissue factor antagonist is used for the
manufacture of a medicament for reducing or inhibiting cell
migration.
[0143] The method, wherein the tissue factor antagonist is modified
factor VIIa known as factor VIIai.
[0144] Use, wherein the modified factor VII is selected from
Dansyl-Phe-Pro-Arg chloromethyl ketone, Dansyl-Glu-Gly-Arg
chloromethyl ketone, Dansyl-Phe-Phe-Arg chloromethyl ketone and
Phe-Phe-Arg chloromethylketone.
[0145] The present invention is further illustrated by the
following examples that, however, are not to be construed as
limiting the scope of protection. The features disclosed in the
foregoing description and in the following examples may, both
separately and in any combination thereof, be material for
realising the invention in diverse forms thereof.
EXAMPLES
Example 1
Preparation of FVII
[0146] Human purified factor VIIa suitable for use in the present
invention is preferably made by DNA recombinant technology, e.g. as
described by Hagen et al., Proc.Natl.Acad.Sci. USA 83: 2412-2416,
1986 or as described in European Patent No. 200.421 (ZymoGenetics).
Factor VIIa produced by recombinant technology may be authentic
factor VIIa or a more or less modified factor VIIa provided that
such factor VIIa has substantially the same biological activity for
blood coagulation as authentic factor VIIa. Such modified factor
VIIa may be produced by modifying the nucleic acid sequence
encoding factor VII either by altering the amino acid codons or by
removal of some of the amino acid codons in the nucleic acid
encoding the natural FVII by known means, e.g. by site-specific
mutagenesis.
[0147] Factor VII may also be produced by the methods described by
Broze and Majerus, J.Biol.Chem. 255 (4): 1242-1247, 1980 and Hedner
and Kisiel, J.Clin.Invest. 71: 1836-1841, 1983. These methods yield
factor VII without detectable amounts of other blood coagulation
factors. An even further purified factor VII preparation may be
obtained by including an additional gel filtration as the final
purification step. Factor VII is then converted into activated
FVIIa by known means, e.g. by several different plasma proteins,
such as factor XIIa, IX a or Xa. Alternatively, as described by
Bjoern et al. (Research Disclosure, 269 September 1986, pp.
564-565), factor VII may be activated by passing it through an
ion-exchange chromatography column, such as Mono Q.RTM. (Pharmacia
fine Chemicals) or the like.
Example 2
Preparation of FVIIai
[0148] Modified factor VII suitable for use in the present
invention is made, e.g. as described in International Publications
Nos. 92/15686, 94/27631, 96/12800 and 97/47651 ZymoGenetics/Novo
Nordisk).
Example 3
Effects of FVIIa and FFR-FVIIa on the Chemotactic Response of
Fibroblasts to PDGF-BB
[0149] Fibroblasts expressing active TF (FIG. 1A and FIG. 1B) were
incubated with 100 nM of FVIIa and seeded in the upper part of the
modified Boyden chamber; while media containing 10% FBS and PDGF-BB
at different concentrations were added below the 150 .mu.m
micropore filter. The migration of the cells under conditions where
medium containing 10% FBS without PDGF-BB was added below the
filter was used as a measure of random migration, and calculated as
100% migration. A significant migration response was recorded at a
concentration of 0.01 ng/ml PDGF-BB in cells stimulated by FVIIa
compared to 1 ng/ml PDGF-BB for cells not ligated with FVIIa, i.e.
a 100-fold difference in concentration (FIG. 2A). At 0.01-0.1 ng/ml
PDGF-BB the migration response to FVIIa increased dose dependently,
starting at 25 nM and with a maximal effect at 50-100 nM FVIIa
(FIGS. 3A-D). No enhancement of random migration was observed after
activation with FVIIa. To test whether the proteolytically active
FVIIa was mandatory for the hyperchemotactic response to PDGF-BB,
fibroblasts were also incubated with 100 nM FFR-FVIIa and assayed
in the Boyden chamber in the same way (FIG. 2A). No increased
chemotaxis was observed with FFR-FVIIa at low concentrations of
PDGF-BB, 0.0-1 ng/ml. In contrast, a pronounced suppression of
chemotaxis induced by 10-50 ng/ml PDGF-BB was achieved by 100 nM
FFR-FVIIa (FIG. 2A and 3A-D). When fibroblasts were preincubated
with a mixture of three different TF antibodies and then with FVIIa
or FFR-FVIIa, the migration response to PDGF-BB was identical to
the response of fibroblasts without the presence of ligand bonded
to TF (FIG. 4A). An irrelevant monoclonal IgG antibody did neither
prevent hyperchemotaxis induced by FVIIa nor the inhibition of the
migration response induced by FFR-FVIIa (data not shown). The
presence of the IgG antibodies or the three TF antibodies did not
change random migration of the fibroblasts (data not shown).
Example 4
The Hyperchemotactic Response is not Mediated by FXa or by
Thrombin
[0150] Since FVIIa-induced signal transduction leading to the
hyperchemotactic response to PDGF-BB was dependent on the catalytic
activity of FVIIa it was important to determine whether signalling
occurred directly or via FXa or thrombin generated by the FVIIa/TF
complex. The enhanced migration response transduced by FVIIa/TF was
not blocked by 0.2-10 .mu.M Tick anticoagulant peptide (TAP), which
specifically blocks the active site of FXa and prevents a further
activation of the coagulation cascade leading to thrombin formation
(FIGS. 5A,5B). Neither addition of 5 U/ml Hirudin, a specific
thrombin inhibitor, had any effect on FVIIa/TF induced
hyperchemotaxis (FIG. 6A). TAP and Hirudin did not influence the
migration of fibroblast in response to PDGF without the presence of
the ligand FVIIa (FIGS. 5A, 5B, 6A). Thus, it is unlikely that the
effect of FVIIa on chemotaxis is mediated via the activation of FX
or thrombin.
Example 5
The Hyperchemotactic Response to PDGF-BB is Influenced by
PLC-Dependent Pathways, but Independent of PI3'-Kinase.
[0151] Activation of PI3'-kinase has recently been shown to be
important for PDGF .beta.-receptor induced chemotaxis. Therefore,
we investigated whether LY294002, a specific PI3'-kinase inhibitor,
was able to block the chemotactic response induced by FVIIa/TF
signalling. Fibroblasts were pretreated with LY294002 at indicated
concentrations for 30 minutes at 37.degree. C. before the addition
of 100 nM FVIIa and assayed in the Boyden chamber as described. The
concentration of PDGF-BB was kept constant at 0.1 ng/ml throughout
the assay, i.e. a very low concentration at which FVIIa/TF induced
a significant chemotactic response. LY294002 was present during the
entire experiments. FIG. 7A shows that the migration response to
PDGF-BB mediated by FVIIa/TF-signalling was unaffected by the
inhibition of PI3'-kinase.
[0152] To investigate whether the FVIIa/TF-induced chemotactic
response involved the activation of phosphatidylinositol specific
phospholipase C (PLC), we preincubated the fibroblasts with
different concentrations of U73122, a specific PLC-inhibitor, for
30 minutes at 37.degree. C. before adding 100 nM FVIIa; the cells
were then patiented to the chemotaxis assay in the presence of the
inhibitor. A close analogue, U73343, without effects on PLC was
used as negative control. The concentration of PDGF-BB was kept
constant at 0.1 ng/ml also in these experiments. Pretreatment of
the cells with the active PLC-inhibitor U73122 inhibited the
hyperchemotactic response to 0.1 ng/ml PDGF-BB in a dose-dependent
way, with a total inhibition at 1 .mu.M (FIGS. 8A and 8B). No
effect on chemotaxis was observed when the inactive analogue U73343
was used.
Example 6
FVIIa/TF Induce Activation of PLC
[0153] To further explore the importance of PLC activity for the
hyperchemotactic response, we also analysed the direct effects of
FVIIa/TF on PLC activity in fibroblasts. Activation of PLC leads to
production of two second messengers, inositol-1,4,5-trisphosphate
(IP.sub.3) and diacylglycerol. Fibroblasts were incubated with myo
[.sup.3H] inositol overnight, and then with 100 nM FVIIa or
FFR-FVIIa for 60 minutes, followed by incubation with or without
PDGF-BB at indicated concentrations. Treatment with 100 nM FVIIa
alone for 60 minutes induced IP.sub.3 release in fibroblasts at the
same level as 10 ng/ml and 100 ng/ml PDGF-BB alone did (FIG. 9).
Moreover, the combination of 100 nM FVIIa and 10 ng/ml or 100 ng/ml
PDGF-BB doubled the IP.sub.3 release. The active site-inhibited
FVIIa did not induce release of IP.sub.3. These results clearly
show that PLC is activated upon binding of FVIIa to TF.
Example 7
Phosphorylation of PLC-.gamma.1 is not Enhanced by TF/FVIIa
Signalling in Fibroblasts
[0154] In order to determine whether the PLC-.gamma.1 isoform,
which is activated by certain tyrosine kinase receptors, was
responsible for the increased PLC activity induced by FVIIa/TF,
tyrosine phosphorylation of PLC-.gamma.1 was studied. Fibroblasts
were incubated in the absence or presence of 100 nM FVIIa or
FFR-FVIIa for one hour, followed by the stimulation with 0, 2, 10
or 100 ng/ml PDGF-BB. After 5 minutes of incubation, the cells were
lysed and PLC-.gamma.1 was immunoprecipitated, separated by
SDS-PAGE and immunoblotted with antiphosphotyrosine antibodies.
Whereas a significant increase in tyrosine phosphorylation of
PLC-.gamma.1 was recorded with increasing concentrations of
PDGF-BB, addition of FVIIa alone to the fibroblasts did not induce
any tyrosine phosphorylation of PLC-.gamma.1 (FIG. 10). Moreover,
the combination of FVIIa and PDGF-BB at different concentrations
did not induce any further phosphorylation compared to stimulation
with PDGF-BB alone (FIG. 10). FFR-FVIIa had no effect on
PLC-.gamma.1 tyrosine phosphorylation (FIG. 10). Thus, other PLC
isoforms than PLC-.gamma.1 are responsible for the increased PLC
activity after FVIIa stimulation.
Example 8
Methods
[0155] Cell cultures. Human foreskin fibroblasts, AG1518 and AG1523
were grown to confluence in Eagle's MEM supplemented with 10% fetal
bovine serum (FBS). Before use, the cells were detached by
trypsinization (2.5 mg/ml for 10 min at 37.degree. C.), washed in
Hank's balanced salt solution, and resuspended in Eagle's MEM with
10% FBS or in Ham's medium supplemented with 0.1% FBS.
[0156] Proteins. Human FVIIa (Novo Nordisk A/S, Gentofte, Denmark),
was expressed and purified as described.sup.29. FFR-FVIIa (Novo
Nordisk) was obtained by blocking of FVIIa in the active site with
D-Phe-L-Phe-L-Arg chloromethyl ketone. Recombinant Tick
anticoagulant peptide (TAP) was kindly provided by Dr. P. Vlasuk,
Corvas (San Diego, Calif.). Hirudin was purchased from Sigma.
LY294002, U73122 and U73343 were obtained from Biomol (Plymouth
Meeting, Pa.). Anti-TF monoclonal antibodies, TF8-5G9, TF9-5B7 and
MTFH-1 (Morrissey, J. H., Fair, D. S., Edgington, T. S. Monoclonal
antibody analysis of purified and cell-associated tissue factor.
Thromb. Res. 52,247-261 (1988)) was a kind gift of Dr. James H.
Morrissey, Oklahoma Medical Research Foundation. The
phosphotyrosine antibody, PY99 was from Santa Cruz, Calif.
[0157] Flow cytometry. The surface expression of TF was analysed by
immunofluorescence with a flow cytometer (Coulter Epics XL-MCL,
Beckman Coulter, Fullerton, Calif., Coulter Electronics, USA). The
instrument was calibrated daily with Immuno-Check.TM. or Flow
Check.TM. calibration beads (Coulter). For indirect
immunofluorescence experiments AG1518 or AG1523 fibroblasts were
washed twice with PBS containing 0.1% bovine serum albumin (BSA),
incubated for 30 minut.about.ps on ice with a
fluorescein-isothiocyanate (FITC)-labelled anti-human TF monoclonal
antibody (4508CJ, American Diagnostica, Greenwich, Conn. USA). The
anti-Aspergillus niger glucose oxidase monoclonal IgG1 (Dakopatts)
was used as a negative control. Mean channel fluorescence intensity
(MFI) and percentage of positive cells were determined for each
sample.
[0158] Determination of TF activity. The procoagulant activity of
TF was determined as described by Lindmark et al. (Lindmark, E.,
Tenno, T., Chen, J., Siegbahn, A. IL-10 inhibits LPS-induced human
monocyte tissue factor expression in whole blood. Br. J. Haematol.
102, 597-604 (1998)). Briefly, aliquots containing
0.2.times.10.sup.5 AG1518 or AG1523 fibroblasts were washed twice
with PBS, placed in the wells of a 96-well microtitreplate (Nunc,
Roskilde, Denmark). The procoagulant activity was measured in a
two-stage amidolytic assay where a chromogenic substrate, S-2222
(Chromogenix, Molndal, Sweden), is cleaved by FXa, which in turn is
activated from FX by the TF/FVIIa complex. A reaction mixture
containing final concentrations of 0.6 mM S-2222, 2 mM CaCl.sub.2
and coagulation factors from the factor concentrate
Prothromplex-T.TM. TIM4 (Baxter, Vienna, Austria) at a final
concentration of 1 U/ml FVII and 1.2 U/ml FX, was added to the
wells, and change in absorbance at 405 nm following a 30 minutes
incubation at 37.degree. C. was determined. The measurements were
done in triplicate.
[0159] Chemotaxis assay. The migration response of fibroblasts was
assayed by means of the leading front technique in a modified
Boyden chamber, as previously described (Siegbahn, A., Hanimacher,
A., Westermark, B., Heldin, C-H. Differential effects of the
various isoforms of platelet-derived growth factor on chemotaxis of
fibroblasts, monocytes, and granulocytes. J. Clin. Invest. 85,
916-920 (1990) and Nistr, M., Hammacher, A., Mellstrom, K.,
Siegbahn, A., Ronnstrand, L., Westermark, B., Heldin, C-H. A
glioma-derived PDGF A chain homodimer has different functional
activities from a PDGF AB heterodimer purified from human
platelets. Cell 52, 791-799 (1988)). Micropore filters (pore size 8
.mu.m) were coated with a solution of type-1 collagen at room
temperature over night. The filters were air dried for 30 minutes
immediately before use. Human foreskin fibroblasts AG1523, were
grown to confluence in Eagle's MEM supplemented with 10% FBS. The
cells were detached by trypsinization (2.5 mg/ml for 10 minutes at
37.degree. C.) and suspended in Eagle's MEM with 10% FBS. The
fibroblasts were incubated for 10 minutes with or without FVIIa or
FFR-FVIIa before assay. One hundred microliters of the cell
suspension (2.times.10.sup.5 cells/ml) was added above the filter
of the Boyden chamber. PDGF-BB was diluted in assay media (Eagle's
MEM with 10% FBS) and added below the filter in the chamber. The
cells were incubated for 6 hours at 37.degree. C. in a humidified
chamber containing 95% air/5% CO.sub.2. FVIIa or FFR-FVIIa were
present during the entire experiment. The filters were then
removed, fixed in ethanol, stained with Mayer's Hemalun, and
mounted. Migration was measured as the distance of the two furthest
migrating fibroblast nuclei of one high-power field (12.5.times.24)
in focus. The migration distance in each filter was calculated as
the mean of the readings of at least three different parts of the
filter. Experiments were performed with two to four separate
filters for each concentration of chemoattractant. For each set of
experiments, the migration of fibroblasts toward the assay media
served as control.
[0160] In cases when anti-TF monoclonal antibodies or inhibitors to
coagulation factors, TAP and Hirudin, were used, cells were
preincubated for 10 minutes with these agents, then with or without
FVIIa or FFR-FVIIa before the chemotaxis assay was performed.
Antibodies, TAP or Hirudin were also present during the entire
chemotaxis experiment. In experiments where the effects on the
migration response of different inhibitors, LY294002, U73122 or
U73343, were tested, cells were preincubated for 30 minutes with
the inhibitors at indicated concentrations, and the inhibitors were
also present throughout the experiments.
[0161] Assay for release of inositol trisphosphate (IP.sub.3).
Six-well plates with semi-confluent cultures of AG1518 human
fibroblasts, were incubated over night ( approx. 20 hours) with 2
.mu.Ci of myo(.sup.3H) inositol (Amersham) in 2 ml Ham's F12 with
0.1% FBS. Medium was changed to Ham's F12 with 0.1% FBS (containing
2 mM CaCl.sub.2) and 20 mM LiCl and the cells were incubated for 15
minutes at 37.degree. C. Cells were then incubated in the absence
or presence of 100 nM FVIIa or 100 nM FFR-FVIIa for one hour.
PDGF-BB (0, 10 or 100 ng/ml) was added and the incubation was
continued for 10 minutes at 37.degree. C. The IP.sub.3 assay was
performed as previously described by Eriksson et al. (Eriksson, A.,
N.ang.nberg, E., Ronnstrand, L., Engstrom, U., Hellman, U., Rupp,
E., Carpenter, G., Heldin, C-H., Claesson-Welsh, L. Demonstration
of functionally different interactions between phospholipase
C-.gamma. and the two types of platelet-derived growth factor
receptors. J. Biol. Chem. 270, 7773-7781 (1995)).
[0162] Assay for agonist-induced PLC-.gamma.1 phosphorylation.
Semi-confluent cultures of AG1518 were serum starved overnight
(approx. 20 hours) in medium containing 0.1% FBS, and then
incubated in the absence or presence of 100 nM FVIIa or FFR-FVIIa
for one hour followed by incubation with 0, 2, 10 or 100 ng/ml
PDGF-BB for 5 minutes at 37.degree. C. Cells were lysed and
PLC-.gamma.1 was precipitated, essentially as previously described
(Hansen, K., Johnell, M., Siegbahn, A., Rorsman, C., Engstrom, U.,
Wernstedt, C., Heldin, C-H., Ronnstrand, L. Mutation of a Src
phosphorylation site in the PDGF .beta.-receptor leads to increased
PDGF-stimulated chemotaxis but decreased mitogenesis. EMBO J. 15,
5299-5313 (1996)) with anti-PLC-.gamma.1 antiserum generated by
immunizing rabbits with a peptide corresponding to the
carboxyterminus of bovine PLC-.gamma.1 (Artega, C. L., Johnson, M.
D., Todderud, G., Coffey, R. J., Carpenter, G., Page, D. L.
Elevated content of the tyrosine kinase substrate phospholipase
C-.gamma.1 in primary human breast carcinomas. Proc. Natl. Acad.
Sci. USA 88, 10435-10439 (1991). Samples were separated by SDS-PAGE
and immunoblotted with the phophotyrosine antibody PY99.
[0163] Statistical analysis. Data were analysed using the
Statistica TM for Windows package (StatSoft, Tulsa, Okla. USA). A
Student's t-test for dependent samples was used to determine
statistical significance between different data sets. P values of
<0.05 were considered statistically significant.
[0164] Proteins. Recombinant human VIIa, a gift from Novo Nordisk
(Gentofte, Denmark), was reconstituted in sterile water at a
concentration of 1 to 1.3 mg/ml. The stock VIIa solutions were
checked for contaminating trace levels of endotoxin using limulus
amebocyte lysate (Bio Whittaker) and none was detected (detection
level 30 pg). Recombinant tick anticoagulant protein (TAP) was
kindly provided by George Vlasuk (Corvas, San Diego, Calif.) and
recombinant hirudin was obtained from either Sigma (St.Louis, Mo.)
or Calbiochem (San Diego, Calif.). Purified human factor Xa and
thrombin were, obtained from Enzyme Research Laboratories
(Southbend, Ind.).
[0165] cDNA microarray. WI-38 cells were cultured to 80% confluency
and serum deprived for 24 hours to enter quiescent state as
described above. The culture medium was replaced with fresh
serum-free DMEM (supplemented with 5 mM CaCl.sub.2) and allowed to
stabilize for 2 h in culture incubator. Then, the cells were
treated with purified recombinant VIIa (5 .mu.g/ml) for 90 min. At
the end of 90 min treatment, total RNA was isolated from untreated
(control) and VIIa-treated cells using Trizol (GIBCO BRL). Poly (A)
RNA was purified by a double pass over Oligo Tex mRNA isolation
columns as described in manufacturer's technical bulletin (Qiagen).
Eight hundred ng (800 ng) of highly purified poly (A) RNA from the
control and VIIa-treated cells were sent for cDNA microarray
analysis service (Human UniGEM V microarray, Genome Systems Inc,
St. Louis, Mo.).
[0166] Northern Blot Analysis. Total RNA was prepared using TRIZOL
reagent from quiescent monolayer of WI-38 cells that were exposed
to VIIa and other materials as described in Results. Northern blot
analysis was carried out using standard procedure. Briefly, 10
.mu.g of total RNA was size fractionated by gel electrophoresis in
1% agarose/6% formaldehyde gels and transferred onto the
nitrocellulose membrane by a capillary blot method. Northern blots
were prehybridized at 42.degree. C. with a solution containing 50%
formamide, 5.times.SSC, 50 mM Tris.HCl, pH 7.5, 0.1% sodium
pyrophosphate, 1% SDS, 1% polyvinylpyrrolidone, 1% Ficoll, 25 mM
EDTA, 100 .mu.g/ml denatured salmon sperm DNA and 1% BSA and
hybridized with .sup.32P-labeled Cyr61 cDNA probe (106 cpm/ml). The
hybridized membranes were exposed to either Dupont NEF or Fuji RX
X-ray film. For quantification purposes, the membranes were exposed
to phosphor screen for 1 to 4 h, and the exposed screens were
analyzed in a Phosphorlmger (Molecular Dynamics) using
"Image-quant" software. To obtain mean values, the units (counts)
obtained from different experiments were normalized to an internal
control (counts present in control-treated sample).
[0167] Chromogenic Assay. WI-38 cells were cultured in 96-well
culture plate and made them quiescent as described above. After
washing the cells, FVIIa (5 .mu.g/ml) in 1,00 .mu.g of calcium
containing buffer was added to the culture wells containing cells
or wells coated with buffer (no cells). After 30 min incubation, 25
.mu.g of chromogenic substrates for factor Xa and thrombin, i.e.,
Chromozym X and Chromozym TH were added to the wells. After 3 h of
color development, the plate was read in a microplate reader. As
controls, cells were incubated with trace concentrations of factor
Xa (50 to 0.1 ng/ml) or thrombin (0.1 to 0.002 U/ml). No
differences were found in absorbance at 450 nm between VIIa added
to cells, or VIIa added to wells not containing cells. The reading
was lower than the readings obtained with lowest concentration of
factor Xa or thrombin and represents VIIa chromogenic activity.
Example 9
[0168] cDNA microarray. Quiescent fibroblasts were exposed to a
control serum-free medium or the serum-free medium supplemented
with VIIa (5 .mu.g/ml) for 90 min (three T-75 flasks for each
treatment). After the treatment, total RNA was harvested and poly
(A) RNA was isolated. Six hundred ng of mRNA was labeled with
either Cy3 or Cy5 fluorescence and then hybridized to the UniGem
Human V chip containing 8,000 sequence verified ESTs, representing
up to 5,000 known human genes (service performed by Genome System
Inc for a fee). The control plate, in which known concentrations of
reference cDNA was spiked into the probe generation reaction to
measure sensitivity and monitor the reverse transcription reaction,
purification determine hybridization efficiency and overall view of
the quality and performance of the assay indicated the success of
hybridization process. Global analysis of experimental data
revealed minimal differences in hybridization signals between the
control and VII-treated samples-Only a small number of genes showed
moderate differential expression. We found upregulation of 5 genes
(3.5 to 2-fold higher in VIIa treatment) whereas one gene was
down-regulated upon VIIa treatment (2.4-fold lower) (+/-2 is a
conservative estimate for determining the minimum magnitude of real
ratios). The identity of the 3.5-fold upregulated gene was not
revealed due to the proprietary nature. Other VIIa-upregulated
genes are Cyr61 (2.5-fold), dopamine D2 receptor (2.2-fold), EST
Incyte PD 395116 (2-fold) and P2U nucleotide receptor (2-fold). It
is interesting to note that CTGF, a gene belonging to the Cyr61
family, was 1.8-fold higher in VIIa-treated cells compared to
control cells. The downregulated transcript in VIIa-treated cells
was EST PD674714. We selected Cyr61 for further analysis.
Example 10
[0169] Confirmation of differential expression of Cyr61. To
validate the data obtained in microarray, we have patiented the RNA
samples from the control and VIIa-treated cells (the same RNA
samples that have been used to prepare poly (A) RNA for probe
generation in the microarray) to Northern blot analysis and probed
with radiolabeled Cyr61 cDNA. The data show that Cyr61 cDNA probe
hybridized to a single transcript (approximately 2.0 kb) of RNA
isolated from the control and VIIa-treated cells. However, the
intensity of hybridization signal was much higher in RNA isolated
from VIIa-treated cells (FIG. 1). Quantitation of hybridization
signal revealed that expression of Cyr61 was 2.8-fold higher in
cells exposed to VIIa over the control treated cells.
Example 11
[0170] Kinetics of VIIa-induced expression of Cyr61. To determine
the kinetics of Cyr61 expression, quiescent fibroblasts were
treated for varying time periods with 5 .mu.g/ml VIIa. Total RNA
was extracted and patiented to Northern blot analysis. As shown in
FIG. 2, Cyr61 expression was increased in time-dependent manner in
VIIa-treated cells. The expression was peaked at about 45 min and
thereafter declined to the base level in 2 to 3 h. Since it had
been reported that expression of Cyr61 in mouse fibroblasts after
stimulation with serum and growth factor was sustained for several
hours (up to 8 to 10 h) before repression occurs, we have examined
the effect of serum and PDGF on kinetics of Cyr61 expression in
quiescent human fibroblasts, WI-38. As shown in FIG. 2B, Cyr61 is
expressed only transiently upon stimulation with PDGF and become
fully repressed 2 h after the addition of stimuli. Similar results
obtained with serum-induced expression of Cyr61 (data not
shown).
Example 12
[0171] Factor VIIa-dose dependent induced expression of Cyr61. To
determine dose-dependency of VIIa, quiescent fibroblasts were
treated with varying doses FVIIa (0.1 to 5 .mu.g/ml) for 45 min and
then total RNA samples from the cells were patiented to Northern
blot analysis. As shown in FIG. 3, treatment of fibroblasts with as
low as 0.1 .mu.g/ml FVIIa was sufficient to induce the expression
of Cyr61 and a plasma concentration of FVII(a) (0.5 .mu.g/ml, 10 nM
resulted in a prominent response, close to the maximal.
Example 13
[0172] Factor VIIa-catalytic activity is required for Cyr61
induction. To test whether VIIa catalytic activity is required for
the induction of Cyr61, WI-38 cells were treated with VIIa and
active-site inactivated FVIIa (FVIIai) for 45 min and the
expression of Cyr61 was evaluated by Northern blot analysis. As
shown in FIG. 4, FVIIai failed to induce the expression of Cyr61
suggesting the requirement of FVIIa proteolytic activity. In this
context, it may be important to point out that FVIIai was shown to
bind cell surface TF with the same or higher affinity than FVIIa.
It is unlikely that VIIa-induced expression of Cyr61 in our
experiments was the result of generation of down-stream coagulation
factors, FXa and thrombin. By using sensitive chromogenic assays,
we found no evidence for the generation of factor Xa and thrombin
in our experimental system (detection sensitivity 10 pg). Further,
the specific inhibitors of factor Xa and thrombin, i.e., tick
anticoagulant protein and hirudin, respectively, failed to abolish
VIIa-induced expression of Cyr61 (FIG. 5).
Example 14
[0173] Involvement of transcriptional mechanism for the induction
of Cyr61 mRNA steady-state levels by VIIa. To investigate whether
transcription is involved in VIIa-mediated increase in Cyr61 mRNA
steady-state levels, quiescent WI-38 cells were incubated with
actinomycin-D (10 .mu.g/ml) for 30 min before the addition of VIIa
for 45 min. As shown in FIG. 6, actinomycin-D inhibited the
stimulator effect of VIIa. This finding indicates a transcriptional
mechanism for induction of Cyr61.
[0174] To investigate whether de novo protein synthesis is required
for the induction of Cyr61 nRNA by VIIa, WI-38 cells were
pretreated with the protein synthesis inhibitor cycloheximide
before the cells were exposed to VIIa for 45 min. As shown in FIG.
6, the stimulatory effect of VIIa was not blocked by cycloheximide.
In fact, cycloheximide markedly increased the VIIa-induced Cyr61
mRNA steady-state levels.
* * * * *