U.S. patent application number 11/462106 was filed with the patent office on 2007-03-08 for composition and method for treatment and prevention of restenosis.
This patent application is currently assigned to BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM. Invention is credited to Kenichi Fujise, Zakar H. Mnjoyan.
Application Number | 20070054853 11/462106 |
Document ID | / |
Family ID | 33451548 |
Filed Date | 2007-03-08 |
United States Patent
Application |
20070054853 |
Kind Code |
A1 |
Fujise; Kenichi ; et
al. |
March 8, 2007 |
Composition and Method for Treatment and Prevention of
Restenosis
Abstract
Compositions and methods are disclosed which employ PARIS
proteins that are useful for suppressing proliferation of smooth
muscle cells. Preferred PARISs are soluble proteins that are
secreted by vascular smooth muscle cells, and include PARIS-1
(neuronal pentraxin 1), PARIS-2 (SBP (MIC-1, GDF-15), PARIS-3
(BTG2) and PARIS-4 (soluble fractalkine). Methods of preventing or
treating restenosis by administering the new compositions are
disclosed. Also disclosed are methods for treating patients
undergoing angioplasty procedures, patients with atherosclerosis,
and patients with other proliferative disorders, in order to
suppress the growth of vascular smooth muscle cells or other cells
that play a role in the particular proliferative disorder or
condition. A method of screening mRNAs and identifying genes
encoding PARISs is also disclosed.
Inventors: |
Fujise; Kenichi; (Houston,
TX) ; Mnjoyan; Zakar H.; (Houston, TX) |
Correspondence
Address: |
CONLEY ROSE, P.C.
P. O. BOX 3267
HOUSTON
TX
77253-3267
US
|
Assignee: |
BOARD OF REGENTS OF THE UNIVERSITY
OF TEXAS SYSTEM
201 West 7th Street
Austin
TX
|
Family ID: |
33451548 |
Appl. No.: |
11/462106 |
Filed: |
August 3, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10448664 |
May 30, 2003 |
7101852 |
|
|
11462106 |
Aug 3, 2006 |
|
|
|
Current U.S.
Class: |
514/1.9 ;
514/18.9; 514/19.3 |
Current CPC
Class: |
C07K 14/47 20130101;
A61P 35/04 20180101; A61K 38/00 20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 38/17 20070101
A61K038/17 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made in whole or in part with funding
from the National Heart, Lung, and Blood Institute of the National
Institutes of Health (Grant No. HL068024). Accordingly, the United
States Government has certain rights in this invention.
Claims
1. An inhibitor of smooth muscle cell proliferation comprising at
least one protein or polypeptide chosen from the group consisting
of: PARIS-1, PARIS-2 and PARIS-3, a biologically active portion of
PARIS-1, PARIS-2, PARIS-3 or PARIS-4 having activity for directly
or indirectly inhibiting smooth muscle cell proliferation, and a
biologically active addition, deletion or substitution homolog
having at least 24% amino acid identity to SEQ. ID NO:1, SEQ. ID
NO:2, SEQ. ID NO:3 or SEQ ID NO:4 and having activity for directly
or indirectly inhibiting smooth muscle cell proliferation.
2. A composition for treating a cell proliferation disorder
comprising: an inhibitor of smooth muscle cell proliferation
comprising at least one protein or polypeptide chosen from the
group consisting of: PARIS-1, PARIS-2 and PARIS-3, biologically
active portions of PARIS-1, PARIS-2, PARIS-3 or PARIS-4, wherein
biological activity comprises inhibition of smooth muscle cell
proliferation, and polypeptides having at least 24% amino acid
identity to SEQ. ID NO:1, SEQ. ID NO:2, SEQ. ID NO:3 or SEQ ID
NO:4; and optionally, a carrier.
3. The composition of claim 2 further comprising PARIS-4.
4. The composition of claim 2 wherein said protein or polypeptide
has at least 40% amino acid identity with SEQ. ID NO:1, SEQ. ID
NO:2 and SEQ. ID NO:3.
5. A method of inhibiting smooth muscle cell proliferation
comprising contacting said cell with the composition of claim 2,
whereby proliferation of said smooth muscle cell is inhibited.
6. The method of claim 5 wherein said smooth muscle cell is a
vascular smooth muscle cell.
7. The method of claim 6, wherein said contacting comprises
administering to a site at risk of undesired smooth muscle cell
proliferation a cell growth inhibitory amount of said composition,
whereby a smooth muscle cell proliferative disorder is deterred or
prevented.
8. The method of claim 6, wherein said contacting comprises
administering to a patient at risk of restenosis an effective
amount of said inhibitor to inhibit vascular smooth muscle cell
proliferation resulting in deterrence or prevention of
restenosis.
9. The method of claim 8 wherein said patient is undergoing an
angioplasty procedure and said administering comprises
administering an effective amount of said inhibitor to said patient
before, during or after an angioplasty procedure to deter or
prevent restenosis.
10. The method of claim 9 wherein said administering includes
delivering said inhibitor to an angioplasty site in said
patient.
11. The method of claim 9 wherein said angioplasty procedure
includes placement of a stent at an angioplasty site in said
patient.
12. The method of claim 11 wherein said stent is a drug-eluting
stent capable of releasing said inhibitor in situ.
13. The method of claim 10 wherein said contacting comprises
administering said inhibitor to a patient at risk of
atherosclerosis progression, to suppress the proliferation of
vascular smooth muscle cells in said patient, whereby the risk of
atherosclerosis progression in the patient is reduced.
14. The method of claim 5 wherein said contacting comprises
administering said inhibitor to a patient at risk of keloid
formation, whereby keloid formation in said patient is
inhibited.
15. The method of claim 5 wherein said contacting comprises
administering said inhibitor to a patient suffering from cancer
originating from a smooth muscle cell, whereby proliferation of a
cancer cell is inhibited.
16. A method for identifying an inhibitor of smooth muscle cell
proliferation comprising: extracting RNAs from growing vascular
smooth muscle cells from a first animal model that is
restenosis-resistant with respect to balloon injury to a blood
vessel in said first animal model, to provide a first pool of
isolated RNAs; generating a first cDNA pool from said first pool of
RNAs; extracting RNAs from growing vascular smooth muscle cells
from a second animal model that is restenosis-prone with respect to
balloon injury of a blood vessel in said second animal model, to
provide a second pool of isolated RNAs; generating a second cDNA
pool from said second pool of isolated RNAs; identifying cDNAs that
are present in said first cDNA pool in a greater amount than in
said second cDNA pool, to provide a pool of upregulated cDNAs;
identifying a first set of genes that correspond to said
upregulated cDNAs; out of said first set of genes, identifying a
subset of genes that encode soluble proteins which are secreted to
a greater extent by vascular smooth muscle cells from said first
animal model than from said second animal model, to provide a group
of upregulated genes encoding soluble proteins; confirming that at
least one said encoded soluble protein is expressed to a greater
extent in vascular smooth muscle cells from said first animal model
than in vascular smooth muscle cells from said second animal model,
to identify at least one upregulated soluble protein inhibitor of
smooth muscle cell proliferation; optionally, expressing at least
one gene coding for said upregulated soluble protein; optionally,
purifying said at least one upregulated soluble protein, to provide
a purified upregulated soluble protein; and optionally, adding said
purified protein to vascular smooth muscle cells in cell culture
medium and confirming that said protein is active for retarding
proliferation of said vascular smooth muscle cells.
17. The method of claim 16 comprising determining that at least one
said upregulated gene is expressed at least 1.5-fold more in said
first animal model than in said second animal model.
18. The method of claim 16 further comprising identifying a
biologically active region of said upregulated soluble protein that
is capable of directly or indirectly inhibiting the proliferation
of smooth muscle cells.
19. The method of claim 19 comprising identifying at least one
soluble protein capable of being secreted by a proliferating human
vascular smooth muscle cell, having at least 24% amino acid
identity to at least one of said protein or proteins encoded by
said upregulated gene(s), and having activity for inhibiting smooth
muscle cell proliferation.
20. The method of claim 19 comprising identifying at least one
soluble protein capable of being secreted by a proliferating human
vascular smooth muscle cell, having at least 40% amino acid
identity to at least one of said protein or proteins encoded by
said upregulated gene(s), and having activity for inhibiting smooth
muscle cell proliferation.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/448,664 filed May 30, 2003, the disclosure of which is
hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Technical Field of the Invention
[0004] The present invention generally relates to compositions and
methods for prevention of proliferative disorders, including
restenosis, atherosclerosis and cancer. More particularly, the
invention relates to compositions containing molecules secreted by
cells and which are capable of inhibiting proliferation of those
and/or other cells. The invention also relates to therapeutic
methods employing such compositions.
[0005] 2. Description of Related Art
[0006] Percutaneous transluminal coronary interventions ("PCI")
such as angioplasty procedures are common practice today for
relieving atherosclerotic blockage caused by fatty acid deposits in
coronary arteries, whereby blood flow is restored in the affected
arteries. A relatively common complication of angioplasty is
restenosis, a renarrowing of the blood flow due to uncontrolled
proliferation of smooth muscle cells at the angioplasty site.
Post-angioplasty restenosis was first treated by balloon
redilatation and, when stents became available.sup.1, by stent
implantation.sup.2. However, close to 20% of patients developed
restenosis within the stent ("in-stent restenosis").sup.2, due to
neointimal VSMC growth.sup.3. In-stent restenosis was initially
treated by repeat angioplasty, rotational atherectomy, laser
angioplasty, "stent-in-stent", and other techniques, but all of
those procedures yielded suboptimal outcomes.sup.4. Brachytherapy
has been investigated for preventing restenosis.sup.12,13 after
primary angioplasty, however, at least 15% of patients treated with
brachytherapy still develop restenosis, suggesting that the
prevention of restenosis by brachytherapy is not entirely
efficacious.sup.13. It has been reported that brachytherapy only
moderately reduced the recurrence rate of in-stent restenosis (from
43.8% to 28.2%).sup.5, at the expense of adverse radiation exposure
both to patients and operators and of late-occurring, intralesional
thrombosis.
[0007] Among a number of pharmacological interventions attempted,
only a few preventive strategies, such as probucol.sup.6,
trapidil.sup.7, cilostazol.sup.8, n-3 fatty acid (eicosapentaenoic
acid).sup.9, and folic acid combined with vitamin B12 and
pyridoxine.sup.10, have been found acceptable. Even in the better
trials, restenosis still developed in 17.9-24.2% of patients. When
stent implantation was used to treat primary lesions in order to
prevent restenosis, a significant number (18%) of patients who
underwent stent implantation experienced restenosis
nevertheless.sup.1,2,11. It has been reported that stents coated
with sirolimus (also known as rapamycin) are more effective than
conventional stents in a randomized, double-blind clinical
trial.sup.55, and recently the FDA has approved a sirolimus-eluting
coronary stent for angioplasty procedures to open clogged coronary
arteries. Long-term effects and side-effect profiles of sirolimus
have not been determined in a large clinical trail, however.
[0008] Although brachytherapy and sirolimus-eluting stents may
effectively treat a selected group of patients with restenosis,
those treatment modalities are likely to remain very expensive and
exclusive. For example, the cost of sirolimus-eluting stents is
estimated to be four times higher than that of conventional stents,
while brachytherapy requires the involvement of radiation
oncologists and nuclear physicists. It has been estimated that up
to a million PCIs are being performed annually in North America
alone.sup.14. A therapeutically viable, lower-cost treatment that
can significantly reduce the risk of restenosis is greatly needed.
It has been calculated that a treatment that reduces risk of
restenosis by 25-33% risk reduction would save approximately
$1,400-$2,000 per patient in hospital, procedural and professional
fees, with a total savings in North America alone of $400-800
million a year.sup.15.
SUMMARY OF THE PREFERRED EMBODIMENTS
[0009] In the course of investigations leading up to the present
invention, it was discovered that certain soluble proteins
("PARISs") normally secreted by vascular smooth muscle cells
("VSMC") are also able to inhibit VSMC growth. Since it is well
known in the field of cardiovascular medicine that VSMC cells play
a critical role in restenosis and atherosclerosis, it is now
proposed that PARISs can be effectively employed to treat, deter or
even prevent restenosis and atherosclerosis progression. Some
PARISs also appear to be normally secreted by a variety of cells,
including non-vascular SMCs. In some cases the PARISs are secreted
to a lesser degree than in VSMCs. However, this unique group of
proteins hold promise as inhibitors of cell growth in a variety of
tissues, and may find use in treating or deterring cell
proliferation in a variety of proliferative disorders such as
keloid formation, venous grafts, coronary arteries of transplanted
hearts and cancers.
[0010] Individually, the representative proteins disclosed herein
as PARIS-1, PARIS-2, PARIS-3 and PARIS-4, have little or no common
homology or mutual family associations. Each has previously been
assigned another name and a different implicated function has been
attributed to it. While some amino acid sequence information is
available for these proteins and some of their physical properties
have been described by others, these proteins are not well
characterized and their implicated biological functions are
different than the bioactivity disclosed herein for the first time
(i.e., their inhibitory effects on vascular smooth muscle cell
growth and, potentially, other cells.) In accordance with certain
embodiments of the present invention, compositions are provided
which contain one or more purified PARIS, and may include a
suitable carrier (e.g., sterile isotonic saline). For example, the
composition may be suitable for direct injection at the desired
site of action in a vessel. In certain embodiments the composition
is useful for preventing or treating restenosis. In certain
preferred embodiments the composition comprises at least one
soluble protein secreted from a VSMC and capable of inhibiting VSMC
growth, with or without a carrier. For the purposes of this
disclosure, the term "soluble protein" has its usual meaning and
includes secreted non-matrix proteins. The PARIS may be a natural
or synthetic protein, or a biologically active portion thereof.
[0011] Certain preferred PARIS proteins from rat have the amino
acid sequence identified as GenBank Accession No. P47971 (R.
norvegicus) (H. sapiens ortholog: Q15818) (PARIS-1), GenBank
Accession No. Q9Z0J6 (R. norvegicus) (H. sapiens ortholog:
NP.sub.--004855) (PARIS-2), GenBank Accession No. A40443 (R.
norvegicus) (H. sapiens ortholog: P78543) (PARIS-3), and GenBank
Accession No. O55145 (R. norvegicus) (H. sapiens ortholog:
NP.sub.--002987) (PARIS-4). Other PARIS proteins in accordance with
certain embodiments of the present invention share at least 40%
homology with the above-identified PARISs. 24% amino acid identity
with the above-identified rat proteins, preferably sharing at least
40% identity, and still more preferably more than 50% identity.
[0012] In another embodiment of the present invention a composition
is provided that contains at least two of the proteins: PARIS-1,
PARIS-2, PARIS-3 and PARIS-4.
[0013] In certain other embodiments of the present invention,
methods of using the above-described PARISs and compositions for
treatment of patients such as those undergoing angioplasty
procedures, patients with atherosclerosis, and patients with other
proliferative disorders, in order to suppress the growth of
vascular smooth muscle cells or other cells that play a role in the
particular proliferative disorder or condition are provided.
Advantageously, therapies employing PARISs are potentially less
expensive and more inclusive (i.e., they may be administered
without special instruments or personnel) than conventional
post-angioplasty restenosis treatments and preventatives. Another
advantage of employing a PARIS therapeutically is that since PARISs
are native proteins, or biologically active portions thereof, no
antigen-antibody immune reaction should occur. In some embodiments
the proliferative disorder is cancer. In some embodiments the
disorder is keloid formation.
[0014] In some embodiments a method of deterring or preventing a
smooth muscle cell ("SMC") proliferative disorder is provided which
includes administering to a site at risk of overgrowth by SMCs a
cell growth inhibitory amount of a composition described above. In
certain embodiments a method of inhibiting VSMC growth is provided
which comprises administering to a VSMC at least one PARIS protein,
preferably PARIS-1, PARIS-2, PARIS-3 or PARIS-4.
[0015] In some embodiments a method of preventing post-angioplasty
restenosis is provided which includes administering to an
above-described protein or composition to an angioplasty site.
[0016] In some embodiments a method of deterring or preventing
atherosclerosis progression is provided which includes
administering to a site at risk of overgrowth by vascular smooth
muscle cells a cell growth inhibitory amount of a protein or
composition as described above. The PARISs may be administered via
a PARIS-eluting stent or other local drug delivery system, or they
may be administered systemically, percutaneously, sublingually, or
rectally.
[0017] In still other embodiments of the present invention, a
screening method for detecting an inhibitor of SMC proliferation is
provided. The method comprises:
[0018] a) extracting RNAs from the growing vascular smooth muscle
cells from a first animal model that is restenosis-resistant with
respect to balloon injury to a blood vessel in the first animal
model (e.g., Harlan SD rat), followed by the generation of the pool
of which are labeled with a suitable fluorescent marker.
[0019] b) extracting RNAs from the growing vascular smooth muscle
cells from a second animal model (e.g., Sasco SD rat) that is
restenosis-prone with respect to balloon injury of a blood vessel
in the second animal model, followed by the generation of the pool
of cDNAs, which are labeled with a suitable fluorescent marker.
[0020] c) performing microarray analyses to identify genes that are
abundantly present in the first set of cDNA pool but scarcely
present in the second set of cDNA pool, followed by identification
of genes that encode soluble proteins that are secreted by the
vascular smooth muscle cells from the first animal model more
abundantly than from the second animal model. Alternatively,
another molecular biological technique could be substituted, such
as subtraction cloning, to identify genes that are differentially
present between the first and the second animal models.
[0021] d) assaying the protein levels to validate that proteins
encoded by these genes are in fact upregulated in the vascular
smooth muscle cells from the first, but not as much as in the
second, animal model.
[0022] e) expressing and purifying these proteins and confirming
that these proteins, in fact, suppress the growth of vascular
smooth muscle cells.
[0023] The method may also include identifying homologs or
biologically active portions of the/those protein(s). In certain
embodiments the expression level of the upregulated gene is greater
in growing cells from the first animal model than in those from the
second animal model, preferably at least 1.5-fold, and more
preferably at least 3-fold greater. These and other embodiments,
features and advantages of the present invention will become
apparent with reference to the following description and
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0025] FIG. 1 is a group of photographs showing the status of
neointimal formation in Harlan and Sasco SD rat carotid arteries 14
days after balloon injury. Panels A and D: Pre-angioplasty; Panels
B, C, E and F: Post-angioplasty. Panels A and B: Harlan X50; Panel
C: Harlan X200; Panels D and E: Sasco X50; Panel F: Sasco x200. The
asterisk (*) denotes the vessel lumen.
[0026] FIG. 2 is a graph showing cell growth/neointima development
over a 14 day period following balloon injury in Sasco SD rats.
[0027] FIGS. 3A-C are graphs showing the results of morphometric
analysis of Harlan and Sasco SD rat carotid arteries 14 days after
balloon injury. FIG. 3A: Intimal area in control and balloon
injured Harlan and Sasco rats; FIG. 3B: Ratio of intimal to medial
area for control and injured Harlan and Sasco rats; FIG. 3C: Medial
area in control and balloon injured Harlan and Sasco rats.
[0028] FIGS. 4A-B are photomicrographs showing that Harlan and
Sasco SD rat vascular smooth muscle cells do not differ
morphologically. FIG. 4A: Harlan and Sasco cells in subconfluent
and confluent cultures. FIG. 4B: .alpha.-actin and DAPI staining
showing differentiation of Harlan and Sasco and VSMCs, and U2OS
cells as negative control.
[0029] FIG. 5 is a graph comparing cell growth of Sasco and Harlan
SD rat VSMCs in tissue culture over a 6 day period under the same
cell culture conditions.
[0030] FIG. 6 is a bar graph showing thymidine incorporation by
Sasco SD VSMCs and Harlan DS VSMCs under the same cell culture
conditions.
[0031] FIG. 7 is a graph showing trypan blue uptake in TNF-.alpha.
stimulated Harlan and Sasco SD VSMCs in culture.
[0032] FIG. 8 is a pair of graphs showing growth of Sasco VSMCs or
rat A7r5 VSMCs over a 144 hr period in either Harlan or Sasco
conditioned media. (Left panel) Sasco VSMCs; (Right panel) Rat A7r5
VSMCs.
[0033] FIG. 9 is a schematic illustration showing the steps of a
gene microarray experiment to identify the highly expressed genes
in growing Harlan and Sasco SD rat VSMCs.
[0034] FIG. 10 is a bar graph showing the relative incidence of
various groups of genes identified in accordance with the
experiment of FIG. 9. Closed bars: Harlan SD VSMCs. Open bars:
Sasco SD VSMCs.
[0035] FIG. 11 is a bar graph comparing the real time RT-PCR,
showing the up-regulation of PARIS-4 mRNA (ng) in growing Harlan
VSMCs compared to Sasco VSMCs.
[0036] FIG. 12 is a photograph of a Northern blot analysis of rat
PARIS-4 mRNA showing its relative concentration in various rat
tissues.
[0037] FIG. 13 is a photograph of a Western blot analysis of rat
PARIS-4 protein showing its relative expression in various rat
tissues.
[0038] FIG. 14 is a bar graph showing the results of an ELISA
analysis of rat PARIS-4 protein in Harlan VSMCs, Sasco VSMCs, rat
proximal tubule epithelial cells (RPTECs), hepatoma, human VSMCs
and plain media with fetal calf serum.
[0039] FIGS. 15A-C are bar graphs comparing the amount of PARIS-4
protein found in the conditioned media of Harlan VSMCS and Sasco
VSMCs. FIG. 15A: media after 24 hrs incubation. FIG. 15B: media
after 48 hrs incubation. FIG. 15C: PARIS-4 secretion rate
(hr.sup.-1).
[0040] FIG. 16 is a graph showing PARIS-4 production by growing
Harlan and Sasco VSMCs over a 200 hr. period in cell culture.
[0041] FIG. 17 is a pair of graphs showing cell growth in the
presence or absence of recombinant rat PARIS-4. (Left panel) Sasco
VSMCs. (Right panel) Rat A7r5 VSMCs.
[0042] FIGS. 18A-D illustrate the immunohistochemical method
employed and the results of immunohistochemical analysis of the
neointima of balloon-injured rat carotid arteries to detect
overexpression of PARIS-4. FIG. 18A illustrates the TSA-enhanced
immunohistochemistry method. FIG. 18B Photograph (X100) TSA-stained
restenotic tissue (Sasco SD rat) (Left panel) no anti-PARIS-4
antibody. (Right panel) Anti-PARIS-4 antibody present. M denotes
media. FIG. 18C is a comparison of PARIS-4 expression in Harlan and
Sasco restenotic tissues (X40). (Left panel) Sasco balloon-injured
carotid arteries. (Right panel) Harlan balloon-injured carotid
arteries. * denotes clot formation within the lumen of the artery.
Open arrows indicate the absence of PARIS-4 signal in Sasco
neointima (left panel); closed arrows indicate the strong PARIS-4
signals seen in Harlan neointima (right panel). FIG. 18D shows DAB
signal intensities expressed in an arbitrary unit. (Right panel)
DAB signal of Harlan neointima. (Left panel) DAB signal of Sasco
neointima.
[0043] FIGS. 19A-C illustrate the feasibility of large scale
production PARIS-4 protein production using baculovirus-Sf9 cell
system. FIG. 19A depicts schematically a procedure for producing a
recombinant DNA construct AcMNPV-PARIS-4 and -LUC, containing
PARIS-4 and Luciferase, respectively. FIG. 19B the results of a
Western blot analysis of the proteins produced by the recombinant
DNA of FIG. 19A. FIG. 19C is a photograph of a Coomassie Blue
stained gel electrophoresis of the Ni-NTA purified proteins from
the constructs of FIG. 19B.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0044] Research efforts on post-angioplasty restenosis and
atherosclerosis have conventionally focused primarily on the
molecules that cause smooth muscle cells to grow. Little attention
has been paid previously to molecules that prevent cells from
growing (i.e., inhibitors or negative regulators of cell
proliferation). It is believed that no molecules have been
previously reported that are secreted from vascular smooth muscle
cells and which inhibit vascular smooth muscle cell growth. The
four proteins (PARIS 1-4) described herein are believed to be the
first such inhibitors that are capable of inhibiting smooth muscle
cell growth in vitro and in vivo.
Restenosis-Resistant and Restenosis-Prone Animal Models
[0045] Rat substrains with restenosis-resistant and
restenosis-prone phenotypes were employed an animal models in the
present investigations. It had been observed in a number of animal
surgeries at the University of Texas Health Science Center at
Houston that more robust neointima sometimes appeared to form in
the carotid arteries of one substrain of rat than in those of
another substrain after the same balloon angioplasty procedures
were performed. In order to investigate that possibility,
substrains of Sprague-Dawley rat were obtained from two different
vendors (i.e., Harlan and Sasco). The degree of neointimal
proliferation in 12 rats from each group was determined using a
standard carotid artery balloon injury-restenosis model, as
described by Clowes et al..sup.16 Three rats from each group (a
total of 6 rats) were sacrificed on days 1, 3, 7 and 14 after the
surgical intervention. Right and left carotid arteries were
harvested, fixed in 4% neutral buffered formalin, and then embedded
in paraffin. Sectioned tissues were stained with hematoxylin-eosin
(HE) and subjected to morphometric analysis and cell counting. In
FIG. 1, groups of photomicrographs showing the status of neointimal
formation in Harlan and Sasco rat carotid arteries 14 days after
balloon injury are as follows: Panels A and B: Harlan X50; Panel C:
Harlan X200; Panels D and E: Sasco X50; Panel F: Sasco X200. Panels
A and D: Pre-angioplasty; Panels B, C, E and F: Post-angioplasty.
In Panels C and F, the asterisk (*) denotes the vessel lumen.
[0046] The experimental protocol was as follows: Male
Sprague-Dawley (SD) rats (400-450 gram) were purchased from the
Sasco branch of Charles River Laboratories ("Sasco", Kingston,
N.Y.) and from Harlan Inc. ("Harlan", Indianapolis, Ind.). All the
animals were housed individually and cared for in accordance with
institutional animal welfare guidelines. Rats were allowed standard
rat chow and water ad labium and were on 12 hour-light-dark cycles.
In brief, individual rats (total number: 24) were weighed, then
anesthetized with halothane (Halocarbon Laboratories, River Edge,
N.J.), using a vaporizer (Vapomatic Sterline, Mass.). Surgical
areas on the inside left hind limb and the ventral neck region were
shaved, cleaned with providine, and a sterile drape placed over the
animal with opening at the surgical sites. A midline neck incision
was made to expose the left common carotid artery; the iliac artery
was exposed through another incision above its junction with the
femoral artery, then ligated at the distal end. The right common
carotid artery served as a control. A 2F Fogarty arterial
embolectomy catheter (Baxter Healthcare Corporation, Santa Ana,
Calif.) was inserted into the iliac artery and passed through the
aorta to the distal portion of the left common carotid artery.
Placement of the catheter was checked via midline incision in the
neck. The balloon catheter was inflated with a manually driven
inflator device (Encore, Scimed, Maple, Minn.) to 2.5 atmospheres,
then retracted in the inflated position to the origin of the left
common carotid artery at the aorta. The catheter was deflated,
returned to its original position, inflated, and retracted through
the carotid artery twice again. The Forgarty catheter was removed
via the iliac artery, which was loigated proximal to the incision
site; skin was closed in both the neck and hind limb, and the
incision sites were treated with topical antibiotics. Six rats (3
each from Harlan and Sasco) were killed on the 1, 3, 7 and 14 days
after the surgical intervention. Rats were euthanized with carbon
dioxide gas inhalation, the abdomen opened, and a catheter inserted
through the aorta to the arch. The 20 mL of heparinized PBS was
infused at 5 mL/min, at which time, the right and left carotid
arteries were dissected out. The middle one third of each carotid
artery was harvested and placed in 4% neutral buffered formalin
solution (Fisher Scientific, Pittsburgh, Pa.). Paraffin embedded
tissues were sectioned at 4 .mu.m thickness and Hematoxylin-Eosin
(HE) staining was performed on each tissue. As is shown in FIG. 1,
Harlan and Sasco SD rats had obvious, strikingly different patterns
of response to the balloon injury. The neointima of Harlan SD rats
was uniformly thinner than that of Sasco SD rats.
[0047] Referring now to FIG. 2, Sasco SD rats developed neointima
in response to balloon injury far more aggressively than did Harlan
SD rats. Hematoxin-eosin stained sections of rat carotid arteries
(balloon-injured) were subjected to the determination of the number
of the nuclei in the neointima. 0: Uninjured rat carotid arteries.
The double asterisks (**) denotes P<0.01 by Student's two sample
T-test. Although there were no differences in neointimal cell
numbers between Sasco and Harlan SD rats in 0, 1, and 3 days after
the injury, a striking difference in the number of the nuclei in
the neointima was obvious on 7.sup.th and 14.sup.th days after the
injury. At 14.sup.th day, Sasco had 1064.8.+-.195 cells in the
neointima while Harlan had only 469.+-.46.4 cells, P<0.01)
[0048] The counting of HE-positive nuclei in the intima revealed
that the intima of Sasco SD rats contained significantly more cells
on 7.sup.th and 14.sup.th days than did that of Harlan SD rats
(FIG. 2).
[0049] FIGS. 3A-C show the results of morphometric analysis of
Harlan and Sasco carotid arteries 14 days after balloon injury
showed the restenosis-resistant phenotype in Harlan SD rats. The HE
stained section of rat carotid arteries were analyzed by the
BIOMAX-BX40 triocular microscope (Olympus, Tokyo, Japan) connected
to CCD-IRIS video camera (Sony, Tokyo, Japan) which allowed the
projection of the entire observed field on the screen of a TV
monitor. The areas [mm2] of the intima and media were measured with
the aid of the JAVA computerized image analysis system (Jandel
Scientific, Corte Madera, Calif.). FIG. 3A: Intimal areas were
significantly larger in Sasco than in Harlan (N=6) FIG. 3B: Intima
to media ratio was calculated as (Intimal area)/(Medial area). FIG.
3C: The media of Harlan was slightly (not significantly) larger
both before and after the balloon injury. The double asterisk (**)
denotes P<0.01 by Student's two sample T-test. A morphometric
analysis showed both the intima-to-media ratio (IMR) and intimal
area to be significantly larger in Sasco SD rats than in Harlan SD
rats (FIG. 3). These data suggested that the carotid arteries of
Harlan and Sasco SD rats responded quite differently to the same
vascular injury and that Sasco SD rats had significantly more
neointimal formation than did Harlan SD rats (FIGS. 1-3).
[0050] The Harlan and Sasco SD animal lines were investigated as to
their lineage. Harlan and Sasco SD rats have the same ancestors,
rats from Sprague-Dawley Co. established by Dr. Dawley in the mid
1970's. Harlan Co. and Sasco Co. (later purchased by Charles River
Laboratories) initiated their own breeding programs in 1981 and
1979, respectively. The breeding protocols of their companies have
not changed since colonies were first established (Communication
with scientists of Harlan Co. and Charles River Laboratories).
Despite sharing the same ancestors, Harlan and Sasco SD rats
exhibit clearly different phenotypes, as summarized in Table 1.
Notably, Sasco SD rats are significantly heavier than Harlan SD
rats later in life, despite the comparable food
consumption.sup.17,18. Sasco SD rats also exhibit different
behavioral.sup.19, neuroanatomical.sup.20,21,
endocrinological.sup.21, immunological.sup.22, and
cardiovascular.sup.22 phenotypes (Table 1). These observations
suggest that Harlan and Sasco SD rats represent two genetically
divergent substrains derived from the same ancestors.
TABLE-US-00001 TABLE 1 HARLAN AND SASCO SPRAGUE-DAWLEY RATS.
Substrains Harlan SD Sasco SD Vendor Harlan Co. Charles River
Laboratories Breeding Facilities Houston, TX and other locations
Kingston, NY History Colony originally established in Colony
originally established in 1981 when Harlan purchased Madison WI in
1979 by Sasco Co., Sprague-Dawley company which apparently
purchased the established by Dr. Dawley (His breeding pairs from
Sprague-Dawley wife's maiden name was Sprague). company. Sasco Co.
was subsequently Five breeding facilities established purchased by
Charles River in 1992; under the same breeding protocol Moved to
Omaha, NE in 1994, Moved since 1981. to Kingston, NY in 1996. The
same breeding protocol has been used since 1979. General Clinical
In-life palpable Male (41%), Female (89%).sup.56 Male (42%), Female
(84%) comparison mass Chronic renal Male (78%), Female (10%) Male
(38%), Female (11%) disease Pituitary mass Male (8%), Female (48%)
Male (48%), Female (86%) 105 week survival Male (15%), Female (45%)
Male (20%), Female (25%) Weight at Male (528 g), Female (329
g).sup.56,57 Male (788 g), Female (562 g) termination Food
consumption Male (0.13 kcal/day/weight), Male (0.115), Female
(0.109) Female (0.217) Neurological Long-term Less
pronounced.sup.58 More pronounced function facilitation (LTF)
Spinal projection Locus coeruleus projected more to Locus coeruleus
projected more to of neurons dorsal horn; Nucleus subcoeruleus
ventral horn; Nucleus subcoeruleus projected more to dorsal
horn.sup.59 projected more to ventral horn Locus coeruleus
.alpha.-2 adrenoceptor mediated.sup.60 Not .alpha.-2 adrenoceptor
mediated derived antinociception Inflammatory ACTH production
Smaller.sup.61 Larger reaction in response to LPS IL-6 production
in Larger.sup.61 Smaller response to IL-1.beta. Cardiovascular
Hypertensive Higher BP.sup.61 Lower BP response with NOS inhibition
Legend: SD: Sprague-Dawley; LTF: a prolonged, serotonin-dependent
augmentation of respiratory motor output following episodic
hypoxia; ACTH: adrenocorticotropin; LPS: lipopolysaccharide; NOS,
nitric oxide synthase.
[0051] As described above and shown in FIGS. 1-3, Harlan and Sasco
rat carotid arteries respond to vascular injury in very different
ways. Since these substrains exhibit various phenotypic differences
(Table 1), the phenotypic difference in the response of the artery
to the injury may be due to the genetic difference in endothelial
cells, VSMCs, platelets, leukocytes, or any other cells that play a
role in restenosis. Given the fact that a number of published
studies strongly suggest the critical role of VSMCs in restenosis,
it was hypothesized that the phenotypic difference in the response
of rat carotid arteries to injury is, at least partially caused by
the genetic differences in Harlan and Sasco VSMCs. In order to test
that hypothesis, the following series of experiments were carried
out on VSMCs isolated from the normal carotid arteries of Harlan
and Sasco SD rats.
EXAMPLE 1
VSMCs from Harlan SD Rats Grow More Slowly
[0052] Equal numbers of Harlan and Sasco SD rat VSMCs were seeded
in 6-well plates, synchronized, and subjected to the same growth
media. As shown in FIGS. 4A-B, Harlan and Sasco VSMCs do not differ
morphologically. In order to test whether a drastic phenotypic
difference observed in Harlan and Sasco SD rats in response to
balloon injury was due to, at least partially, the genetic
difference of two rat VSMCs to growth stimuli, Harlan and Sasco
VSMCs were first isolated from carotid arteries of these animals.
FIG. 4A: Under microscopy, both VSMCs looked identical under
subconfluent and confluent conditions. FIG. 4B: Differentiation
assay showed that both Harlan and Sasco differentiated in
Differentiation Media (0.5% serum; 50 .mu.g/mL heparin) equally
showing .alpha.-actin in >90% of cells in 7 days. For
comparative purposes, the U2OS cancer cell line from human
osteosarcoma was cultured under similar conditions and did not
differentiate.
[0053] Although the VSMCs from the two substrains did not differ
morphologically (FIGS. 4A-B) and were placed in the exact same
microenvironment, VSMCs from Harlan SD rats grew much more slowly
than did those from Sasco SD rats (FIG. 5). In order to determine
the growth rate of Harlan and Sasco VSMCs under the same
environment, 1.times.10.sup.5 cells (the passage 5) were seeded in
duplicate in 6-well dishes, synchronized by serum starvation, and
stimulated with serum on Day 0. The number of cells in each well
was determined every 24 hours for 6 days. *: P<0.05, ***:
P<0.005, ****: P<0.001 by Student's two sample T-test. A
graph showing the results of the growth assay reveals the slower
growth of Harlan VSMCs in comparison with Sasco VSMCs.
EXAMPLE 2
VSMCs from Harlan SD Rats Take Up Much Less Thymidine
[0054] In order to test the hypothesis that the observed difference
in the growth rates of Harlan and Sasco VSMCs was due to a
difference in the rate of DNA synthesis, a standard thymidine
incorporation assay was performed. Referring to FIG. 6, the
thymidine incorporation assay shows the larger uptake of thymidine
by Sasco SD VSMCs than by Harlan SD VSMCs upon serum stimulation. A
standard thymidine incorporation assay was performed using VSMCs
from two Sasco SD and three Harlan SD rats of the same (6) passage.
After synchronization by serum-starvation, cells were stimulated
with 5% serum with 1 .mu.Ci/mL of methyl-H.sup.3-thymidine for 24
hours. Counts were normalized to protein concentrations. *:
P<0.05; **: P<0.01. These data suggest that Harlan VSMCs
synthesize DNA more slowly than do Sasco VSMCs.
EXAMPLE 3
VSMCs from Harlan SD Rats are More Susceptible to Noxious
Stimuli
[0055] In order to test the hypothesis that the difference in
growth between Harlan and Sasco VSMCs was not only due to a
difference in DNA synthesis rate, but also due to a difference in
susceptibility to cytotoxicity, we performed a cell death assay in
which Harlan and Sasco SD rat VSMCs were challenged with tumor
necrosis factor-.alpha. (TNF-.alpha.) and the number of dead cells
were assessed by a trypan-blue exclusion assay. As is shown in FIG.
7, Harlan VSMCs were significantly more susceptible to TNF-.alpha.
challenge than were Sasco VSMCs. Harlan and Sasco VSMCs were seeded
in duplicate at 2.times.10.sup.5/well in 6-well dishes. Cells were
stimulated by 10 nM TNF-.alpha. in M231 media with 0.5% serum
supplements for indicated periods of time. Cells were harvested by
trypsinization, washed, and pelleted into trypan blue solution and
counted under hemocytometer. At least 200 cells were counted.
Trypan blue positive cells could not transport out trypan blue dye
and were dead. The asterisk (*) denotes a statistical difference of
P<0.05 by Student's two sample T-test. It was concluded that
Harlan SD cells are more susceptible to TNF-.alpha.-induced cell
death than Sasco SD cells.
EXAMPLE 4
VSMCs from Harlan Rats Secrete a Soluble Substance that Retards
Growth of VSMCs
[0056] In order to test the hypothesis that soluble factors from
VSMCs influence the growth patterns of Harlan and Sasco VSMCs, the
same growth assay was performed with conditioned media from either
Harlan or Sasco VSMC cultures. Harlan conditioned media suppressed
the growth of Sasco VSMCs and A7r5 VSMCs in contrast to Sasco
conditioned media. Double asterisks denote P<0.001 by ANOVA
(General linear model) between cells treated with Sasco conditioned
media and cells treated with Harlan conditioned media.
[0057] The experimental protocol was as follows: 1.times.10.sup.4
cells (either Sasco VSMCs or A7r5 VSMCs, which is rat vascular
smooth muscle cell line [ATCC, Manassas, Va.]) were seeded on
6-well plates in duplicate. Next day, media were exchanged for
Harlan or Sasco conditioned media, which were obtained by exposing
2.0 million Harlan/Sasco VSMCs to fresh Media 231 for 24 hours. The
number of cells in each well was determined every 24 hours for 6
days. Strikingly, when VSMCs were incubated with the Harlan
conditioned media, their growth rate was slower than that of VSMCs
incubated with the Sasco conditioned media (FIG. 8A). This
experiment was repeated thrice with the same results. These data
suggest that there are soluble factors secreted from Harlan and
Sasco VSMCs negatively and positively regulating VSMC growth,
respectively, and that these secreted factors explain, at least
partially, the sluggish growth of Harlan VSMCs in comparison with
that of Sasco VSMCs. The same experiment was performed using the
rat smooth muscle cell line A7r5, and the same results were
obtained.
[0058] As described above and in FIGS. 1-3, it was shown that Sasco
SD rat carotid arteries produced much more neointima in response to
balloon-injury than did Harlan SD rat carotid arteries. It is of
interest to note that in related studies it was also determined
that media and adventitia layers do not differ between the Harlan
and Sasco animal models after balloon injury (data not shown). It
was suggested that the genetic differences between Harlan and Sasco
SD rat VSMCs may explain the restenosis-resistant phenotype of
Harlan SD rats. When VSMCs were isolated from Sasco and Harlan SD
rat carotid arteries and studied, it was shown that the behavior of
these two groups of VSMCs in vitro was entirely concordant with
that of carotid arteries in vivo (FIGS. 4-7). These data suggested
that the difference in neointimal proliferation in Harlan and Sasco
SD rats could be at least partly explained by genetic differences
of Harlan and Sasco VSMC response to growth stimuli. Furthermore,
it was possible that Harlan VSMCs grew more slowly than did Sasco
VSMCs because Harlan and Sasco VSMCs secreted negative and positive
growth regulatory molecule(s) into the extracellular environment,
respectively. At that time, the transcripts of Harlan VSMCs were
compared with those of Sasco VSMCs, which were exposed to growth
stimuli, using microarray assay system. This study sought to
identify the secreted negative growth regulatory molecule(s), if
any, by evaluating genes upregulated in Harlan VSMCs.
EXAMPLE 5
Microarray Analysis of Transcripts from Harlan and Sasco SD
Rats
[0059] In order to evaluate the hypothesis that the significant
difference in growth pattern of VSMCs observed in vitro and in vivo
between Harlan and Sasco SDs (FIGS. 1-8) was due to the difference
in the degree and/or presence of the expression of
growth-controlling genes, the status of transcripts in growing
VSMCs from Harlan SD were compared to that of Sasco SD rats using
Affymetrix Rat Arrays (RGU344), as illustrated in FIG. 9. Extreme
care was taken to keep the growth condition of both groups
identical. The purity of the smooth muscle cell preparation was
always confirmed by .alpha.-actin staining and was shown to be
always higher than 90%.
[0060] The experimental protocol of the Affymetrix rat gene array
analysis to identify the restenosis related genes was as
follows:
[0061] Harlan and Sasco SD rats were purchased from Harlan Co. and
Charles River Laboratories, respectively, and housed individually
and cared for in an identical fashion, according to the
institutional guidelines on standard rat chow (Ralston Purina,
Richmond, Ind.) and water ad labium with 12 hour light-dark
cycles.
[0062] (2) Right and left carotid arteries were then harvested.
Adventitial layers were carefully removed by brunt dissection under
dissecting microscope. The endothelial layers were removed by
rubbing a cotton-tipped swab against the endothelial surface of
opened arteries several times.
[0063] (3) VSMCs were allowed to grow in a 10-cm dish on M231 Media
with serum supplements (Cascade Laboratories, Portland, Oreg.).
[0064] (4) When cells were confluent, they were propagated into one
chamber slide and three 10-cm dishes. Cells in the chamber slide
were allowed to differentiate on the Differentiation media (0.5%
serum, 50 .mu.g/mL Heparin) for 7 days and stained with
anti-.alpha.-actin antibody. The purity of VSMCs over 90% was
confirmed.
[0065] (5) When VSMCs on 10-cm dishes were 80% confluent, cells
were harvested.
[0066] (6) The total RNA were extracted using a RNeasy.TM. Midi kit
(Qiagen).
[0067] (7) The double-stranded complementary DNA (cDNA) was then
synthesized, using SuperScript Choice.TM. system (Gibco BRL),
followed by phenol-chloroform extraction and ethanol precipitation.
Synthesis of biotin-labeled cRNA was performed using Enzo BioArray
High Yield RNA Transcript Labeling Kit.TM. (Affymetrix), followed
by the fragmentation of the cRNA.
[0068] (8)-(9) The cRNAs were then subjected to target
hybridization. One array (GeneChip.RTM.; Rat Genome U34 Set,
Affymetrix) per rat was used normally, except for one Harlan rat
for which two arrays were used to test reproducibility of the
hybridization
[0069] Hybridization was performed at 45.degree. C. in an
Affymetrix Hybridization Oven 640 for 16 hours.
[0070] Post-hybridization wash, stain, and post-stain wash were
performed in Fluidics Station 400 in a standard fashion.
[0071] Finally, arrays were scanned by Affymetrix Scanner System.
Then data analysis was performed using d-chip software, as
described previously.sup.23. Replicate data for the same sample was
weighted gene-wise using inverse squared standard error as
weight.sup.23. An unpaired two-group comparison for each probe set
was performed. This analysis considered both measurement error (as
measured by the replicate data) and variation among samples. Genes
were determined to have altered gene expression levels if they had
a 2-fold or greater change in the means of the 2 groups (the
"first" approach, described below) and if a gene was determined to
be present in either both groups or in one of the groups (the
"second" approach described below).
[0072] A profound difference in gene expression between Harlan and
Sasco SD VSMCs was observed. In FIG. 10, the closed bars denote
Harlan; open bars denote Sasco. The data acquired by the experiment
described in FIG. 9 was analyzed as follows. A Rat Genome U34
GeneChip.RTM. contains approximately 7000 full-length sequences and
1000 EST clusters, allowing validation of the array data with the
previous works on restenosis and to identify novel genes that may
play an important role in restenosis. At least 6 independent array
experiments were performed in this study, the results of which are
essentially identical.
[0073] First, signal intensities of certain genes were compared
between Harlan and Sasco and identified genes/ESTs that had more
than 3-fold increase (either in Harlan or in Sasco). Data with high
standard deviations were excluded from analysis.
[0074] Second, the signals that were only present in Harlan and the
signals that were only present in Sasco were examined. Genes that
were identified by two different algorithms, genes that had
multiple "hits" within a single algorithm, and genes that were
upregulated (either in Harlan or Sasco)>10 folds were considered
to be important genes in the process of restenosis.
[0075] Third, these genes were categorized by the intracellular
locations and functions and tabulated (Tables 2 and 3). For ESTs
without homology to rat genes, Blast search using human and mouse
database was performed to identify a human/mouse homolog of the
particular EST.
[0076] Fourth, for each gene identified to be important, a MedLine
search was performed to determine (a) whether these genes have been
implicated in restenosis or negative growth regulation, (b) how
much characterization was made, and (c) if a particular gene would
be a candidate gene in our hypothesis, i.e., secreted soluble
non-matrix proteins that may play a role in the negative regulation
of VSMC growth. There were striking differences in genes between
Harlan and Sasco SD VSMCs. Secreted proteins in Sasco were
predominantly growth-promoting while those in Harlan, although
poorly characterized, were predominantly negative regulators of
cell growth and proliferation. Sasco VSMCs had a number of
extracellular matrix (ECM) genes upregulated, along with genes for
adhesion molecules, receptors, housekeeping genes, kinases and
survival factors. Harlan VSMCs had a number of genes upregulated
for transcriptional factors (almost always negative regulator of
growths), cytoskeleton and channels. Many genes identified as being
associated with restenosis have already been implicated in
restenosis by other investigators.
[0077] Genes that exhibited a more than 3-fold increase in signal
intensity in Harlan and Sasco SD rat VSMCs are presented in
simplified fashion in Tables 2 and 3, respectively. Categories of
genes overexpressed in Harlan and Sasco SD VSMCs are displayed in
bar graph form in FIG. 10.
[0078] There was a marked difference in the levels of several
transcripts in these animals. The microarray data show a striking
difference in gene expression between Harlan and Sasco SD VSMCs.
There were 34 and 32 genes whose expression levels were upregulated
more than 3-fold in Harlan and Sasco SD rats, respectively. The
genes upregulated in the Harlan SD rats were drastically different
both categorically and functionally from those in the Sasco SD
rats. While Sasco VSMCs upregulated a number of genes for ECM
proteins, cell surface receptors (most of them associated with
growth and proliferation), house-keeping genes, kinases, and
survival factors, Harlan VSMCs upregulated only a small number of
these genes. On the contrary, Harlan VSMCs upregulated many genes
for transcriptional factors (most of them were associated with
growth arrest and apoptosis) and cytoskeletal proteins, which were
barely present in Sasco VSMCs. These data suggest that Harlan and
Sasco VSMCs, when placed in the exact same growth environment,
express drastically different sets of genes. The discoveries that
Sasco VSMCs upregulated receptors for growth-promoting molecules,
housekeeping genes and survival-related genes and that Harlan VSMCs
upregulated transcriptional factors for negative growth regulation
and cytoskeleton proteins are intriguingly consistent with the in
vivo finding that neointimal formation was far more aggressive in
Sasco than in Harlan SD rats (FIG. 1-3). Taken together, the array
data appear to reflect what really happens in vivo in Sasco and
Harlan SD rat carotid arteries in response to balloon vascular
injury.
[0079] The data obtained in the present studies is consistent with
previous investigations of restenosis. Two striking features of the
data set derived from the current microarray experiments are,
first, that genes upregulated in Harlan VSMCs have been implicated
by other investigators in the prevention of restenosis and, second,
that genes upregulated in Sasco VSMCs have been shown to play a
role in pathogenesis of postangioplasty restenosis. For example, in
the array system, the number of the cyclooxygenase 2 (COX-2) gene
transcripts was almost 10-fold larger in restenosis-resistant
Harlan VSMCs than in restenosis-prone Sasco VSMCs (Table 3). COX-2
produces prostacyclin, which functions through prostacyclin
receptors. Prostacyclin receptor knockout mice have been shown to
exhibit exaggerated restenosis in response to vascular
injury.sup.24, thereby supporting the protective role of
prostacyclin against restenosis. Our array data also showed that
the transcript number of monocyte chemoattractant protein-1 (MCP-1)
gene was 3.36-fold larger in restenosis-prone Sasco VSMCs than in
restenosis-resistant Harlan VSMCs (Table 2). MCP-1 is a chemokine
produced by VSMCs in response to growth stimuli and a potent
chemoattractant of monocytes. Recent studies indicated that higher
plasma MCP-1 levels correlated with restenosis.sup.25. In addition,
our array data indicated that the transcript of VCAM-1.sup.26,
Angiotensin-II receptors.sup.27,28, tissue factor.sup.29-31,
PI3K.sup.32 and tenascin C.sup.33, all of which have been strongly
implicated in the pathogenesis of restenosis in animal studies,
were all upregulated in restenosis-prone Sasco VSMCs (3.3,
9.0-12.8, 4.7, 11.2, and 3.4-fold increase, respectively) (Table
2). Taken together, these observations further support the validity
of the current data set derived from our microarray
experiments.
[0080] Another striking finding derived from the present
investigations is that Harlan VSMCs appear to secrete several
poorly characterized molecules into extracellular space. The four
molecules BTG2, SBF, petraxin, and factalkine (CX.sub.3C) (PARIS-1
through -4, respectively), stand in clear contrast to molecules
secreted by restenosis-prone Sasco VSMCs (Tables 3 and 4). The
secreted molecules from Sasco VSMCs, including TGF-.alpha..sup.34
and MCP-1.sup.35 (Table 2), are well characterized and have been
identified as contributors to restenosis. Just as restenosis-prone
Sasco VSMCs secrete growth-promoting factors into their
microenvironment, it is proposed that restenosis-resistant Harlan
VSMCs secrete growth-limiting factors that negatively regulate the
growth of neighboring VSMCs. These soluble and secreted
growth-inhibitory molecules, named PARISs, are believed to be the
molecules secreted by Harlan VSMCs that were hypothesized to be
identifiable by microarray analyses of genes upregulated in Harlan
VSMCs. The four representative PARISs identified herein are
considered especially valuable for inhibiting cell proliferation.
Further analysis is expected to identify more soluble secreted
PARISs (e.g., PARIS-5, and so on), which may not be as highly
expressed as PARISs 1-4 but nevertheless have useful biological
activity which causes inhibition of cell growth. The term "highly
expressed" means at least a 1.5-fold increase in expression of a
PARIS protein in growing cells from a restenosis-resistant animal
model compared to the level of expression of the same protein in
growing cells from a restenosis prone animal model). TABLE-US-00002
TABLE 2 GENES HIGHLY TRANSCRIBED IN SASCO SD VSMCS (20 OUT OF 32
GENES) Site of action Gene family Gene name Accession Hits Fold
Ref. Gene function and reference Extracellular Secreted TGF-.alpha.
M31076 2 4.32 1500 Treatment with anti-sense oligos to TGF-.alpha.
retarded protein, S cancer cell proliferation. Overexpression of
TGF-.alpha. cytokine like caused increased hepatocyte proliferation
in transgenic mice. Involvement in restenosis likely. MCP-1 X17053
2 3.36 349 MCP-1 is produced by VSMCs in response to PDGF and
recruit monocytes. The role of MCP-1 in postangioplasty restenosis
is clearly shown by multiple studies. Secreted Tenascin C U15550 1
3.42 241 Tenascin C is a matrix glycoprotein that plays protein, S
a role in VSMC survival and proliferation. ECM Involvement in
restenosis likely. F-spondin M88469 1 11.18 9 F-spondin was
initially reported as a secreted matrix protein that S promoted
neural cell adhesion, neurite extension, and neuronal growth. MGP
AI012030 1 3.63 59 MGP is a 10 kDa matrix protein first described
in bone, dentin and cartilage. MGP precursor is recently discovered
in VSMCs. MEGF5 AB011531 2 25.81 5 MEGF5 is a mammalian homolog of
Drosophila slit protein. S Drosophila Slit is a large ECM protein
important in development. Cell Adhesion VCAM-1 M84488 2 3.34 887
VCAM induces PI3K activation and cell proliferation. membrane
molecule and Monoclonal antibody against VCAM inhibits neointimal
analog formation, supporting the role of VCAM in restenosis.
Connexin X51615 1 3.22 707 Connexin is a 21 kDa transmembrane
protein, categorized as a S gap-junction protein. It may have a
potential anti-apoptotic function. Receptor AT-II, type 1 M86912 2
9.0 768 Multiple studies have suggested the role of S type 1 AT-II
receptor in restenosis. AT-II, type 2 X62295 3 12.82 1174 Multiple
studies have suggested the role of type 2 AT-II receptor in
restenosis. Tissue factor U07619 1 4.72 1718 Recombinant tissue
factor inhibitor (rTFPI) prevents neointimal proliferation. Injured
VSMCs express tissue factor on cell surface. Oxidized LDL AB005900
1 8.88 47 The receptor was originally found in endothelial cells
receptor (LOX1) but also later found also in VSMCs and macrophage.
Cytoplasmic House- Guanine AA859837 2 6.15 74 Guanine deaminase is
a 50 kDa protein that keeping deaminase catalyzes the hydrolytic
deamination of guanine. proteins Hydroxysteroid AI105448 1 4.86 366
Hydroxysteroid dehydrogenase converts cortisone dehydrogenase (less
glucocorticoid activity) into cortisol 11.beta.-type 1 (more
glucocorticoid activity). Glycogenin- AA892986 1 13.09 56
Glycogenin is a 42 kDa protein required for homolog the initiation
of glycogen biogenesis. Aldehyde U60063 2 8.06 1354 Lack of
aldehyde dehydrogenase causes the clinical pictures of
dehydrogenase S Sjogren-Larsson syndrome with muscular dystrophy.
VDUP1 AI237654 2 4.38 3 VDUP1 was originally cloned as a protein
interacting with thioredoxin. VDUP1 is upregulated in immortalized
cells but not present in cell-cycle arrested cells Regulatory,
Phosphoinositide- U50412 3 11.24 1903 PI3K plays a critical role in
the proliferation Kinases 3-kinase (PI3K) S of all cell types,
including VSMCs. Regulatory, cAMP-PDE M25350 5 4.72 415 There is a
report that PDE inhibitors, such Signaling S as aminophyline and
amrinone, prevented restenosis. mol Regulatory, NDRG2 AA799560 1
4.26 13 NDRG2 is 47 kDa protein upregulated by N-myc, Survival
related to cell survival and proliferation. Legends: Accession:
Accession number at GenBank; Fold: The signal intensity of a
certain gene is X-fold elevated in Sasco in comparison with Harlan
SD VSMCs; S: Transcript only present in Sasco SD VSMCs; Ref.: The
number of articles appearing in MedLine search; TGF-.alpha.:
Transforming growth factor-.alpha.; MCP-1: Monocyte chemoattractant
protein-1, also known as JE rat immediate-early serum-responsive
gene; ECM: Extracellular matrix; MGP: Matrix Gla protein; MEGF5:
Mammalian homolog of Drosophila slit protein with EGF (epidermal
growth factor) domains; VCAM-1: Vascular cell adhesion molecule-1;
AT-II: Angiotensin-II receptor; VDUP1: Upregulated by
1,25-dihydroxyvitamin D3; PDE: Phosphodiesterase; NDRG2: N-myc
downstream-regulated gene-2 homolog, also known as Bdm1 in rat and
Ndr1-3 in mouse.
[0081] TABLE-US-00003 TABLE 3 GENES HIGHLY TRANSCRIBED IN HARLAN SD
VSMCS (18 OUT OF 34 GENES) Site of action Gene family Gene name
Accession Hits Fold Ref. Gene function and reference Extracellular
Secreted BTG2 M60921 3 4.0 8 Also known as B cell translocation
gene, anti-proliferative and protein, (PARIS3) secreted. Induced by
p53. cytokine like SBF gene AJ011969 1 3.67 8 SBF gene is a poorly
characterized secreted protein whose (MIC-1, H function is unknown.
PARIS2) Neuronal AI072943 1 6.59 6 Neuronal pentraxin is a 47 kDa
secreted protein, homologous to Pentraxin H C-reactive protein and
amyloid P protein. Overexpression of (PARIS1) pentraxin was
associated with apoptosis in one report. CX3C AF030358 1 5.06 72
CX3C was originally characterized as chemoattractant for T
(Fractalkine; cells and monocytes. One report indicated it to be
induced by PARIS4) p53. Secreted Small proline- AA891911 1 3.46 6
Spr is a 120 kDa proline rich protein, originally described in
protein, ECM rich protein human skin. (spr) Cell Adhesion
H36-alpha7 X65036 2 3.78 1 This is a newly described 120 kDa cell
surface glycoprotein membrane molecule integrin .alpha.- H that
binds lamin. chain Vesicle Caveolin-3 AI043968 2 6.68 43 Caveolin-3
is a muscle specific caveolae also present in transportation H
VSMCs. Channel P2X5 X92069 2 6.82 6 P2X5 is a component of
ligand-gated ion-channel family of H ATP receptor. It makes a
heterodimer with P2X1. MCT2 U62316 1 3.43 1 MCT2 transports
pyruvate and lactate across cellular membrane. Cytoplasmic
Cytoskeleton .gamma.-2 smooth M22323 4 4.26 6 .gamma.-2 actin is
normally present in enteric smooth muscle cells. muscle cell H
actin Cytokeratin-18 AI072634 2 11.24 36 Cytokeratin-18 is a
cytoskeleton and cytosolic protein. Upon H apoptosis, the protein
is cleaved and released into the blood stream, exhibiting a new
epitope detectable by M30 monoclonal antibody. Smooth X16262 2 9.45
189 Smooth muscle MHC-AS is a splicing variant of MHC. muscle, MHC-
H AS b-Nexillin AA799423 2 3.58 1 b-Nexillin is a cytoskeleton
protein that interacts with F-actin and involved in cell-matrix
adhesion. Regulatory Guanylate AA849036 3 10.48 259 Guanylate
cyclase 1 produces cGMP, an intracellular signaling molecules
cyclase 1 H molecule, in response to NO. NO prevents neointimal
proliferation in animal model of restenosis. Cyclooxigenase S67722
3 9.58 655 COX-2 produces prostacyclin. Prostacyclin receptor KO
mice isoform 2 H have aggressive neointima proliferation. Nuclear
Transcriptional HES-1 D13417 3 6.68 25 HES-1 is a helix-loop-helix
protein, functioning as a factor H transcriptional repressor.
Overexpression of HES-1 has been found associated with reduction of
PCNA level and cell cycle arrest. Calbindin- AI102839 2 3.9 205
Calb1 is Notch 3 homolog. Notch 1 function as the enhancer D28k H
of HES-1. Involved in HES-1 pathway. (CCBPS35P) HMG1 X62875 1 3.25
26 HMG1 is a 26 kDa nuclear nonhistone DNA binding protein,
interacting with histone proteins. One group showed HMG1 is a p53
activator. Legends: Accession: Accession number at GenBank; Fold:
The signal intensity of a certain gene is X-fold elevated in Harlan
in comparison with Sasco SD VSMCs; H: Transcript only present in
Harlan SD VSMCs; Ref.: The number of articles appearing in MedLine
search; GdNPF: Glia-derived neurite-promoting factor; EST:
Expressed sequence tag; NGFIAPPSP: NGF-inducible anti-proliferative
putative secreted protein; ECM: Extracellular matrix; MCT2;
Monocarboxylate transporter; MHC-AS; Myosin heavy chain,
alternatively spliced; CCBPS35P; Cerebellar Ca-binding protein spot
35 protein, also known as Calb1; HMG1: High mobility group protein
1.
[0082] TABLE-US-00004 TABLE 4 IDENTIFICATION OF PARISs 1-4 Name
Other name(s) Homology, Family Implicated function Fold-inc. Size
HRI CVSE GenBank SP PARIS-1 Neuronal pentraxin 1 Amyloid P protein
Pro-apoptosis? 6.59 431aa 88% Yes U18772 Yes PARIS-2 SBP (MIC-1,
GDF-15) TGF-.beta. superfamily Anti-inflammatory? 3.67 224aa 56%
Yes NM_019216 Yes PARIS-3 BTG2 Unknown Anti-proliferative? 4.0
158aa 83% Yes M60921 Yes+ PARIS-4 Fractalkine, soluble CX.sub.3C
chemokine Anti-adhesion? 5.06 393aa 63% Yes AF030358 Yes Legend:
HRI: Human-rat identity at the protein level. Size: the number of
amino acids in rat proteins; CVSE: Cardiovascular system
expression, defined by EST database was searched, literature
reviewed. GenBank: GenBank accession number for rat mRNA sequence;
SP: Signal peptide sequence present. This was tested using
SIGFIND.sup.64 an internet based Signal peptide prediction software
that has been described by Reczko et al..sup.62; +Negative by this
server but signal peptide described by Badbury.sup.65 and
others.
[0083] TABLE-US-00005 TABLE 5 PARISs 1-4 SEQUENCES Human PARIS-1
PARIS-2 PARIS-3 PARIS-4 Name Protein Common Neuronal pentraxin I
precursor Prostate differentiation factor BTG2 Fractalkine Others
NPX-1 MIC-1, PLAB, GDF-15 NGF-inducible protein, TIS21,
Neurotactin, small inducible cytokine Nerve growth factor-inducible
subfamily D, CX3CL1 protein PC3 precursor Amino Accession H.
sapiens Q15818 NP_004855 P78543 NP_002987 Acid Number M. musculus
Q62443 Q9Z0J7 Q04211 O35188 R. norvegicus P47971 Q9Z0J6 A40443
O55145 Sequence H. sapiens [SEQ ID NO: 1] [SEQ ID NO: 2] [SEQ ID
NO: 3] [SEQ ID NO: 4] MPAGRARTCALLALCLLGPQD MPGQELRTVNGSQMLLVLLVLS
MSHGKGTDMLPEIAAAVGFLSS MAPISLSWLLRLATFCHLTVLLA
FGPTRFICTSVPVDADMCAAS WLPHGGALSLAEASRASFPGPSE
LLRTRGCVSEQRLKVFSGALQE GQHHGVTKCNITCSKMTSKIPVA
VAAGGAEELRSSNVLQLRETV LHSEDSRFRELRKRYEDLLTRLR ALTEHYKHHWFPEKPSKGSGY
LLIHYQQNQASCGKRAIILETRQH LQQKETILSQKETIRELTAKLG
ANQSWEDSNTDLVPAPAVRILTP RCIRINHKMDPIISRVASQIGLSQ
RLFCADPKEQWVKDAMQHLDR RCESQSTLDPGAGEARAGGGR EVRLGSGGHLHLRISRAALPEGL
PQLHQLLPSELTLWVDPYEVSY QAAALTRNGGTFEKQIGEVKPRT
KQPGSGKNTMGDLSRTPAAET PEASRLHRALFRLSPTASRSWDV
RIGEDGSICVLYEEAPLAASCGL TPAAGGMDESVVLEPEATGESSS
LSQLGQTLQSLKTRLENLEQY TRPLRRQLSLARPQAPALHLRLS LTCKNQVLLGRSSPSKNYVMA
LEPTPSSQEAQRALGTSPELPTGV SRLNSSSQTNSLKDLLQSKIDE
PPPSQSDQLLAESSSARPQLELHL VSS TGSSGTRLPPTPKAQDGGPVGTE
LERQVLSRVNTLEEGKGGPKN RPQAARGRRRARARNGDDCPLG
LFRVPPVSTAATWQSSAPHQPGP DTEERVKIETALTSLHQRISELE
PGRCCRLHTVRASLEDLGWAD SLWAEAKTSEAPSTQDPSTQAST KGQKDNRPGDKFQLTFPLRTN
WVLSPREVQVTMCIGACPSQFR ASSPAPEENAPSEGQRVWGQGQS YMYAKVKKSLPEMYAFTVCM
AANMHAQIKTSLHRLKPDTEPA PRPENSLEREEMGPVPAHTDAFQ
WLKSSATPGVGTPFSYAVPGQ PCCVPASYNPMVLIQKTDTGVSL
DWGPGSMAHVSVVPVSSEGTPSR ANELVLIEWGNNPMEILINDK QTYDDLLAKDCHCI
EPVASGSWTPKAEEPIHATMDPQ VAKLPFVINDGKWHHICVTWT
RLGVLITPVPDAQAATRRQAVGL TRDGVEAYQDGTQGGSGENL
LAFLGLLFCLGVAMFTYQSLQGC APYHPIKPQGVLVLGQEQDTL
PRKMAGEMAEGLRYIPRSCGSNSYVLVPV GGGFDATQAFVGELAHFNIWD
RKLTPGEVYNLATCSTKALSG NVIAWAESHIEIYGGATKWTFEACRQIN mRNA Accession
H. sapiens U61849 NM_004864 U72649 NM_002996 Number M. musculus
NM_008730 NM_011819 AK088976 AF071549 R. norvegicus U18772
NM_019216 M60921 AF030358
EXAMPLE 6
Identification of PARISs 1-4
[0084] To simplify this discussion, the representative molecules
that were identified as described in the foregoing examples through
microarray screening as potential negative regulators of VSMC
growth are called PARISs 1-4. The name "PARIS" is an acronym
derived from the phrase "protein associated with restenosis
inhibition and secreted." Additional identifying information and
properties of these molecules are listed in Tables 4 and 5. In
Table 4 the homology or protein family of each of the four proteins
is identified, along with its implicated function. The fold
increase in expression, number of amino acids and percent human-rat
identity are shown. The GenBank accession number for the rat mRNA
sequence of each PARIS 1-4 are also indicated in Table 4. The
GenBank accession numbers of the amino acid and mRNA sequences
PARISs 1-4 from human, mouse and rat are given in Table 5. The
sequences referenced by those accession numbers are hereby
incorporated herein by reference. The amino acid sequences for
human PARISs 1-4 are also set out in the attached Sequence Listing
as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
[0085] The identification of PARISs represents a new paradigm
because a negative growth-regulatory mechanism of VSMCs by secreted
molecules has never been well elucidated and because none of the
molecules mentioned above has ever been implicated before in the
growth regulation of VSMCs. It should be appreciated that the
elucidation of secreted negative regulators of VSMC growth
constitutes a significant advance in the prevention and treatment
of restenosis. Although it is possible to treat restenosis by
blocking the effect of soluble growth-promoting molecules (such as
MCP-1, TGF-.alpha., PDGF, and others) by abolishing their binding
to their receptors or by inhibiting their intracellular signal
transduction pathways, using small molecules or antibodies, these
approaches are complex and time-consuming. In contrast, when fully
characterized and validated, the secreted negative regulators of
VSMC growth, as represented by those molecules identified herein,
are expected to be suitable for simple parenteral or local
administration to prevent restenosis. Advantageously, there should
be little or no toxicity because they are naturally occurring
molecules.
[0086] The four representative PARISs range in size from 17 to 47.4
kDa (predicted). As indicated in Table 4, the human PARISs 1-4 are
orthologs of the rat PARISs, having very close amino acid sequences
(i.e., human/rat identities ranging from 56-88%.) Additional
exemplary orthologs are identified in Table 7. Since PARISs 1-4
were originally identified in transcripts from pure cultured VSMCs,
the inventors considered their presence in the cardiovascular
system is likely. In fact, recent Northern and Western blot
analyses confirmed the presence of PARIS-4 in cardiovascular
tissue. It was also concluded in this study that putative signal
peptide sequences were present. In the following paragraphs, the
properties of PARIS 1-4 are described.
PARIS-1
[0087] The sequence originally identified as AI072943 (EST
sequence) represents a 364-nucleotide cDNA fragment. A BLAST search
showed a 100% match with the 3-terminus untranslated region of
neuronal pentraxin-1 (GenBank Acc. No. U18772). The rat neuronal
pentraxin-1 was originally identified as a 47-kDa protein that
binds to the snake venom toxin taipoxin. Structurally, neuronal
pentraxin-1 is homologous to the acute-phase proteins serum amyloid
P protein and C-reactive protein of the pentraxin family.sup.36.
Neuronal pentraxin-2, which has 54% amino acid identity to neuronal
pentraxin-1, has been cloned by library screening using neuronal
pentraxin-1 as a probe.sup.37. Both proteins are apparently
secreted, since they can both be detected in conditioned media by
Western blot analysis.sup.38. Recently, neuronal pentraxin-1 was
shown to be upregulated, both at the transcript and protein levels,
in cerebellar granule cells undergoing potassium
deprivation-induced cell death.sup.39. When the cells were treated
with antisense oligonucleotides directed against neuronal
pentraxin-1, more cells survived upon potassium deprivation,
further supporting the protein's role in negative growth regulation
and cell death.sup.39. It appears that no other functional study of
this molecule using either recombinant protein or overexpression
strategies has ever been performed. A Medline search revealed only
6 articles that used neuronal pentraxin in their titles, however
none of which indicates negative growth regulatory function.
Overall, this protein has not previously been well
characterized.
PARIS-2
[0088] The sequence originally identified as AJ011969 (EST
sequence) represents the cDNA of the rat MIC-1 protein. This
protein is also known as SBP and GDF-15 (GenBank Acc. No.
NM.sub.--019216). MIC-1 was originally identified by subtraction
cloning as a molecule that is upregulated in
phorbol-12-myristate-13-acetate-(PMA)-stimulated U937 cells as
opposed to retinoic acid (RA)-differentiated U937 cells.sup.40.
Structurally, MIC-1 is remotely homologous to TGF-.beta..sup.40.
This protein is apparently secreted, since
FLAG-epitope-(DYKDDDDK)-tagged MIC-1, when overexpressed in CHO
cells, is successfully immunoprecipitated from conditioned media by
anti-FLAG antibody.sup.40. Although the processing, secretion, and
degradation pathway of MIC-1 has been fairly well
investigated.sup.41, there has been no scientifically sound
functional study done on this molecule. TGF-.beta.1-knockout mice
die of severe widespread inflammation.sup.42, suggesting that one
of the major functions of the TGF-.beta. family is the negative
regulation of inflammation. Taken together, these data suggest that
the function of MIC-1 is also anti-inflammatory. A Medline search
revealed only 10 articles that used MIC-1, SBP, or GDF-15 in their
titles, none of which clearly shows its growth inhibitory function.
Overall, this protein is very poorly characterized.
PARIS-3
[0089] The sequence identified as M60921 represents the cDNA of the
rat BTG-2 protein. This protein is also known as TIS21, PC3, and
NGF-inducible anti-proliferative putative secreted protein. BTG-2
was originally cloned as a molecule whose transcription is induced
by nerve growth factor (NGF) stimulation of PC12 pheochromocytoma
cells.sup.43. Overexpression of this protein has never been
performed in tissue culture cell system. BTG-2 protein reportedly
interacts with proteins of various functions, including
protein-kinase-C.alpha.-binding protein (rPICK1).sup.44,
protein-arginine N-methyltransferase.sup.45, and CCR4-associated
factor 1 (CAF1).sup.46. In addition, intracellular localization of
BTG-2 has never been clearly shown, despite the presence of signal
peptide sequence. Its function in negative growth regulation is
vaguely implied by the fact that BTG-depleted cells are less
susceptible to Adriamycin challenge and the fact that
BTG-overexpressing cells are growth-suppressed.sup.47. A Medline
search revealed only 23 articles that used BTG-2, TIS, PC3 or
NGF-inducible anti-proliferative putative secreted protein in their
titles. No previous studies definitively show its role in negative
growth regulation. Overall, this protein is poorly
characterized.
PARIS-4
[0090] The sequence identified as AF030358 represents the cDNA of
the rat fractalkine protein. Fractalkine is also known as the
CX.sub.3C chemokine. This protein is unique because even though it
is expressed on the cell surface, its N-terminus chemokine head can
be cleaved from a mucin stalk.sup.48. Fractalkine is expressed on
various cells including endothelial cells, VSMCs, and dendritic
cells, while its receptor (CX.sub.3CR1) has been so far
demonstrated on T cells, monocytes, macrophages, and natural killer
cells.sup.49. Its expression is upregulated by TNF-.alpha. and
IL-.beta..sup.50. Fractalkine has different functions, depending on
its form: membrane-bound fractalkine is implicated in
integrin-independent leukocyte migration.sup.48, while soluble
fractalkine is an anti-inflammatory agent that interferes with the
ligation of membrane-bound fractalkine to its receptor on the
leukocyte surface.sup.51,52. It is especially interesting to note
that a group of investigators has been able to show the attenuation
of THP-1 cell adhesion to activated VSMCs by soluble fractalkine
(50 nM).sup.53. It is now proposed that fractalkine expressed on
VSMCs may be cleaved under certain circumstances and play a role in
the attenuation of inflammation. A Medline search revealed 72
articles that used fractalkine or CX.sub.3C in their titles. Only
one paper investigated VSMCs and fractalkine.sup.53. Overall, the
role of fractalkine in VSMC growth is poorly characterized and no
previous work clearly demonstrated its negative regulatory function
with respect to cell growth. For the sake of simplicity and because
of the functional difference between soluble and membrane-bound
fractalkine, it is the extracellular domain of fractalkine (i.e.
soluble fractalkine) that is defined herein as PARIS-4.
EXAMPLE 7
Initial Validation and Characterization of PARISs
[0091] After identifying the first four PARISs, as described in the
foregoing Examples, one was chosen as a representative for
examination in a series of experiments designed to test the
hypothesis that proliferating VSMCs secrete PARISs, chemokine-like
molecules that negatively regulate the growth of neighboring VSMCs
could be supported in that PARIS. Because some of the key reagents
needed to test the hypothesis were already available in the
inventors' laboratory for PARIS-4, it was decided to begin with
PARIS-4. In brief, the rationale was that, if PARIS-4 produced by
VSMCs did inhibit VSMC growth under carefully defined assay
conditions, then it could be concluded that the other PARISs
discovered using the same methods will very likely behave similarly
in accordance the general hypothesis. The experimental data
establishes that PARIS-4 is, in fact, produced by VSMCs, and that
PARIS-4 causes VSMCs to grow more slowly than in the absence of
PARIS-4.
[0092] Accordingly, the following discussion and experimental data
focus primarily on PARIS-4 (soluble fractalkine), and is considered
to be representative of PARISs 1-3 as well as any as yet
unidentified soluble secreted proteins that are also highly
expressed in growing vascular smooth muscle cells. In brief, it was
determined that PARIS-4 is in fact secreted from vascular smooth
muscle cells (FIGS. 8 and 14). In addition, it was found that
PARIS-4 production is much more robust in vascular smooth muscle
cells from restenosis resistant rat substrains (Harlan SD rats)
(FIGS. 15A-C and 16). Furthermore, it was demonstrated that the
addition of recombinant PARIS-4 to vascular smooth muscle cell
culture will slow the growth rate of vascular smooth muscle cells
(FIG. 17). In ongoing work, a large amount of PARIS-4 is currently
being produced and additional in vivo confirmation that this
molecule will prevent post-angioplasty restenosis in a rat
carotid-artery model of restenosis (FIGS. 18A-D) is planned.
Real-time RT-PCR Analysis
[0093] A real-time RT-PCR analysis was developed for the
quantitation of PARIS-4 messages in Harlan and Sasco carotid artery
VSMCs. The results are shown in FIG. 11. PARIS-4 (Y-axis)
represents PARIS-4 index, which is PARIS-4 mRNA (ng) normalized to
18S eukaryotic RNA (ng). Asterisk denotes P<0.05.
[0094] The experimental protocol was as follows: VSMCs from the
carotid arteries of 4 Sasco and 4 Harlan rats were used. When VSMCs
on 10-cm dishes were 80% confluent, cells were harvested. The total
RNA were extracted using a RNeasy Midi kit (Qiagen). The real time
RT-PCR was performed according to the instructions from Applied
Biosystems (Foster City, Calif.), using the following primer and
probe sets for the detection of PARIS-4 (rat fractalkine)
transcripts: TABLE-US-00006 forward primer,
5'-TACTCTGCTGGCGGGTCAG-3'; reverse primer,
5'-ATCTTGTGGCACGTGATGTTG-3'; probe, 5'-ACCTCGGCATGACGAA-3'.
The probe was labeled at the 5'-end with 6-carboxyl-fluorescein
(FAM.TM.) and at the 3'-end with a 6-carboxytetramethylrhodamine
(TAMRA.TM.). For the detection of eukaryotic 18S RNA for
normalization, the pre-developed assay mixture for 18S, consisting
of appropriate primers and probe labeled by VIC.TM. and
non-fluorescent quencher (PDAR, ABI) was used. The quantitative
real time RT-PCRs were performed in quadruplicate, using the TaqMan
RT-PCR kit (ABI) in the 7900 HT Sequence Detector system. Both
PARIS-4 and 18S critical thresholds were determined and converted
to weight (ng) using a standard curve constructed on serially
diluted rat normal total RNA. PARIS-4 index was then calculated as
above. It is readily apparent in FIG. 11 that there is
up-regulation of PARIS-4 message in Harlan VSMCs. The PARIS-4
message in Harlan VSMCs was 4 times more abundant than in Sasco
VSMCs. Northern Blot Analysis of Rat PARIS-4
[0095] Referring now to FIG. 12, the results of a Northern blot
analysis of rat PARIS-4 (Fractalkine) are shown. The method was as
follows: Rat PolyA+ RNA blot was purchased from OriGene
Technologies (Rockville, Md.). PARIS-4 cDNA was cloned by RT-PCR
from total RNA isolated from Harlan VSMCs and ligated in frame to a
pBlueBac4.5 vector (Invitrogen) and subjected to automated DNA
sequencing. Labeling of probes (PARIS-4 and .beta.-actin) were
performed using Random Prime kit (Roche) with gel purified PCR
products of PARIS-4 and .beta.-actin as templates and with
.sup.32-.alpha.-dCTP as a labeling agent. Generated probes were
purified using a Qiagen nucleotide clean up kit. Hybridization was
performed at 60.degree. C. for 16 hrs in ExpressHyb solution. The
membrane was then washed 5 times with 2.times.SSC with 0.1% SDS at
room temperature and twice with 1.times.SSC with 0.1% SDS at
50.degree. C. before it was exposed to a phosphoimager screen for
8-12 hrs. Signals were detected by BioRad Molecular Scanner FX and
Quantity One software system. The Northern blot analysis showed
PARIS-4 to be abundant in brain, kidney and heart. In FIG. 12 kbp
refers to kilo-base pairs. The rich presence of PARIS-4 in the
heart is consistent with the hypothesis that PARIS-4 is involved in
the growth regulation of cells in cardiovascular tissue and, in
particular, VSMCs.
Western Blot Analysis of PARIS-4
[0096] A commercially available antibody suited for PARIS-4 Western
blot was identified and used to evaluate the expression of PARIS-4
protein in various tissues. Results are shown in FIG. 13, in which
kDa denotes kilo Dalton, and r-PARIS-4 indicates recombinant
PARIS-4. Western blot analysis of rat PARIS-4 (Fractalkine) shows
its abundant presence in brain, heart, liver and testis.
[0097] The assay protocol was as follows: A ready-made rat tissue
blot, containing 10 .mu.g per lane of different rat tissue lysates
were purchased from Imgenex (San Diego, Calif.). Western blot
analysis was performed using a standard technique with mouse
anti-rat fractalkine antibody (Clone 96834) from R&D Systems,
Inc. (1:500 dilutions). Bound antibodies were detected using
anti-mouse IgG-horse-raddish-peroxidase (HRP)-conjugates and West
Pico HRP substrates (Pierce, Rockford, Ill.). In order to evaluate
the loading condition of the samples, the same membrane was probed
with anti-actin antibody (Chemicon, Temecula, Calif.). Recombinant
PARIS-4 was used as positive control (r-PARIS-4, FIG. 13).
Evaluation of the signals of PARIS-4 and actin (including
normalization to the total actin amounts) suggests that PARIS-4 is
highly expressed in brain, heart, testis and liver. Of particular
note, brain and heart are the only organs that showed high levels
of PARIS-4 both transcripts and proteins. In the kidney, PARIS-4
protein expression was only modest while its transcript was
abundant. In the testis and liver, PARIS-4 protein expression was
large in quantity while its transcript was minimally present. The
concordant presence of PARIS-4 transcript and protein in the heart
further supports the proposition that PARIS-4 plays an important
regulatory role in cardiovascular cells.
ELISA Analysis of PARIS-4
[0098] Next, an ELISA system for PARIS-4 was developed and
optimized using commercially available reagents (R&D Systems).
The protocol was as follows: 96-well plates were coated with 0.8
.mu.g/mL of goat anti-rat fractalkine antibody (R&D systems)
overnight. After wash, plates were blocked with PBS supplemented
with 1% BSA and 5% sucrose for 1 hr. After wash, 100 .mu.L of
samples or standards were added in quadruplicate and incubated for
2 hrs at room temperature (RT). After extensive wash, 100 .mu.L of
biotinylated goat anti-rat fractalkine antibody (0.3 .mu.g/mL) was
added and incubated for 2 hrs at RT. After wash, 100 .mu.L of
streptavidin-HRP solution was added and incubated for 20 min at RT.
After wash, 100 .mu.L of substrate solution (H.sub.2O.sub.2 plus
tetramethylbenzidine) was added and incubated for 20 min. Stop
solution was then added (2N H.sub.2SO.sub.4). And plates were read
using a micro-plate reader set to 450 nm with a reference at 570
nm. Experiments were performed at least 3 times and results were
essentially identical. The results are shown in FIG. 14, in which
** indicates P<0.001 in comparison with RPTECs; + indicates
P<0.001 in comparison with Sasco; RPTECs indicates renal
proximal tubule epithelial cells; and FCS indicates fetal calf
serum. Notably, PARIS-4 concentration of media, determined by this
system, was zero. In addition, PARIS-4 was not detectable in the
conditioned media from human aorta vascular smooth muscle cells,
suggesting the anti-rat primary antibody did not cross react with
human fractalkine (i.e., the anti-rat antibody was capable of
differentiating human from rat fractalkine). It was also found that
fetal calf serum (FCS) did not contain detectable PARIS-4, as shown
in FIG. 14. Rat Harlan and Sasco VSMCs secreted, respectively,
approximately 10 fold and 3 fold as much PARIS-4 as rat renal
proximal tubule epithelial cells (RPTECs), consistent with the data
presented in FIG. 13.
EXAMPLE 8
Demonstration of More Rapid Production of PARIS-4 in Harlan
VSMCs
[0099] Using the above-described ELISA system, it was found that
Harlan conditioned media had two times more PARIS-4 than did Sasco
conditioned media, as shown in FIGS. 15A-C. FIG. 15A shows the
PARIS-4 concentration from condition media of 24 hr incubation;
FIG. 15B: PARIS-4 from conditioned media of 48 hr incubation; FIG.
15C: PARIS-4 secretion rate (/hr). Asterisk denotes P<0.05. #:
P=0.060, %: P=0.054. PARIS-4 index was obtained by normalizing
concentration of PARIS-4 (ng/mL) to cell number (million). The
ELISA protocol was as described in Example 7.
[0100] Referring now to FIG. 16, the results of a cell growth time
course experiment are shown. The protocol was as follows: 0.5
million Harlan and Sasco VSMCs were seeded on each well of 6 well
dishes with 3 mL Media 231 with growth supplements. Time 0, 24, 48,
72, 96, 120 and 144 hrs after the exchange of media, 350 .mu.L of
media was harvested and the same volume of fresh media added. ELISA
of PARIS-4 was performed in triplicate as described above. The time
course experiment revealed more abundant and more rapid production
of PARIS-4 by Harlan VSMCs than by Sasco SD VSMCs under the same
conditions. PARIS-4 concentrations (ng/mL/million cells) were
significantly higher for times 48, 72, 122, and 144 hrs (P<0.05,
2 Sample-T test) with strong trends for times 24 and 96 hrs
(P=0.094 and 0.063, respectively). Overall, PARIS-4 concentration
was significantly higher in Harlan than in Sasco (F=50.0, ANOVA,
P<0.0001). PARIS-4 production was more rapid in the first 24-48
hrs than later hours. In addition, time course experiments showed
that VSMCs produced PARIS-4 more rapidly in the first 24 hrs than
later hours (FIG. 16) and that Harlan VSMCs secreted more PARIS-4
per hour than did Sasco VSMCs (FIGS. 15-C and 16). These data
suggest that PARIS-4 protein is more rapidly and more abundantly
secreted by Harlan VSMCs. Taken together, they very strongly
support the validity of microarray and real-time RT-PCR data (for
the PARIS-4 transcripts) at the protein level.
[0101] As discussed above, VSMCs grow much more slowly in Harlan
conditioned media than in Sasco conditioned media. VSMCs from Sasco
SD rats grew much more slowly in Harlan conditioned media rich in
PARIS-4, as shown in FIG. 8A. Sasco, not Harlan VSMCs, were chosen
for this assay because Harlan VSMCs rapidly produced PARIS-4 and
would suppress their own growth, as made clear in FIGS. 15 and 16.
This fact explains the lack of significant difference in growth
rates of Harlan VSMCs in the exact same experiments (data not
shown).
EXAMPLE 9
Inhibition of VSMC Growth by Recombinant PARIS-4
[0102] Cell growth assays were carried out to evaluate the effect
of addition of PARIS-4 to growing VSMCs. The protocol was as
follows: 1.times.10.sup.4 cells (either Sasco VSMCs or A7r5 VSMCs
[ATCC]) were seeded on 6-well plates in duplicate. Next day, media
were exchanged for media either containing recombinant rat PARIS-4
(fractalkine) at the final concentration of 10000 ng/mL (140 nM) or
the same volume of PBS. The number of cells in each well was
determined every 48 hours for 6 days. Graphs of the results are
shown in FIGS. 17A-B. Double asterisks denote P<0.001 by ANOVA
(General linear model). Error bars denote standard deviations (SD).
Hr denotes hours after the addition of r-PARIS-4. FIG. 17A shows
Sasco VSMC cell number after 48, 96 and 144 hours culture, with or
without recombinant rat PARIS-4. FIG. 17B shows the results
obtained for rat A7r5 VSMCs. It was found that the recombinant rat
PARIS-4 suppressed the growth of VSMCs in both Sasco and A7r5
VSMCs. This constitutes direct proof of causality between PARIS-4
and growth suppression of VSMCs. In additional tests, 14 nM of
PARIS-4 was found to inhibit VSMC growth (data how shown).
EXAMPLE 10
Immunohistochemical Detection of PARIS-4
[0103] Next, a new immunohistochemical (IHC) method was developed
using tyramide signal amplification (TSA) to more effectively
detect PARIS-4 in paraffin-embedded tissues. The results are shown
in FIGS. 18A-D. The principle of TSA methods is illustrated in FIG.
18A. The open circles denote insoluble DNP-tyramide molecules;
Solid circles denote 3,3'-diaminobenzidine (DAB). TSA staining was
performed according to the manufacturer's instructions (NEN.RTM.
Life Science Products; Boston, Mass.) with optimization for
PARIS-4, as indicated. The following steps is conceptually
illustrated in FIG. 18A.
[0104] (1)(2) After standard steps of deparaffinization,
rehydration, and quenching of tissue peroxidase, tissue sections
were incubated with biotinylated goat anti-rat PARIS-4 antibody
(R&D Systems).
[0105] (3) After wash, tissue sections were incubated with
streptavidin-horse radish peroxidase (HRP), followed by
[0106] (4) application of dinitrophenyl (DNP) labeled tyramide.
DNP-labeled tyramide was catalyzed to by HRP to form insoluble DNP
depositions immediately adjacent to the immobilized HRP enzyme.
[0107] (5) These insolubly deposited DNP labels were detected by
anti-DNP antibody conjugated to HRP.
[0108] (6) Finally, DAB, a substrate of HRP, was added. Because the
added labels are deposited proximal to the initial immobilized HRP
enzyme site, there is minimal loss in resolution.
[0109] FIG. 18B shows photographically (X100) the TSA-enhanced
immunohistochemistry of restenotic tissue. The restenotic tissue
was stained using the TSA staining procedure described above. Ab(-)
indicates no anti-PARIS-4 antibody (left panel). Ab(+) indicates
Anti-PARIS-4 antibody present (right panel). M denotes media. In
these photomicrographs it can be readily seen that the PARIS-4
signals are most intense in the neointima.
[0110] A comparison of PARIS-4 expression in Harlan and Sasco
restenotic tissues is shown in FIG. 18C. Balloon-injured carotid
arteries from Harlan (right panel) and Sasco (left panel) were
stained using the methods above and presented in the lower
magnification (X40). An asterisk denotes the clot formation within
the lumen of the artery. Open arrows indicate the absence of
PARIS-4 signal in Sasco neointima (left panel); closed arrows
indicate the strong PARIS-4 signals seen in Harlan neointima (right
panel).
[0111] FIG. 18D shows the signal quantification. Using NIH "ImageJ"
Software (NIH, Bethesda, Md.), DAB signal intensities were
quantified and expressed in an arbitrary unit. DAB signal of Harlan
neointima (right panel) was significantly higher (at least 2-fold)
than that of Sasco neointima (left panel). Although a standard IHC
method had yielded poor signal intensities in the past (data not
shown) in these tissues, the present immunohistochemistry study
using tyramide signal amplification (TSA) revealed definitively
that PARIS-4 is overexpressed in the neointimal of balloon-injured
rat carotid arteries in Harlan rats. Taken together with the data
above (Examples 8 and 9), it is likely that PARIS-4, expressed more
abundantly in Harlan neointimal VSMCs, is protective against
neointimal proliferation and restenosis because PARIS-4 negatively
regulates VSMC growth.
EXAMPLE 11
Quantification of PARIS-4 in Sera from Harlan and Sasco Rats
[0112] ELISA assays were next carried out on sera harvested from
Harlan and Sasco SD rats (N=12), as described in Example 7.
Intriguingly, Harlan sera contained a slightly higher but
statistically not significant level of PARIS-4 than did Sasco sera
(1102.+-.302 and 1053.+-.153 [ng/mL] for Harlan and Sasco,
respectively, NS). In light of the fact that serum PARIS-4
concentrations are not different between Harlan and Sasco rats, it
is suggested that PARIS-4 functions as a chemokine in local
microenvironment rather than as a hormone in systemic
environment.
EXAMPLE 12
Large-scale Production of PARIS-4
[0113] The feasibility of a large scale PARIS-4 protein production
using baculovirus-Sf9 cell system was investigated. As illustrated
in FIG. 19, a Baculovirus-Sf9 cell system yield a large amount of
recombinant proteins. FIG. 19A is a schematic showing the
production of recombinant AcMNPV-PARIS-4 and AcMNPV-luciferase
(LUC). Asterisk denotes the detailed structure of the insert. Note
that proteins are double-tagged with HA- and His.sub.x6 tags.
AcMNPV: Autographa californica nuclear polyhedrosis virus
(baculovirus). The procedure was as follows: PARIS-4 cDNA was
obtained by reverse transcription PCR (RT-PCR) using rat mRNA as a
template with appropriate primer sets. The cDNAs of PARIS-4 and
Luciferase were cloned into the pBlueBac4.5 vector, which had been
modified in the inventors' laboratory to include HA- and His.sub.x6
tags. These baculovirus transfer vectors were co-transfected with
Invitrogen's linear Bac-N-Blue.TM. DNA into Sf21 insect cells to
produce recombinant AcMNPVs. The recombinant viruses were
plaque-purified and propagated. A small scale production and
purification by Ni-NTA beads (Qiagen) were performed, using the
lysates from Sf-9 cells infected with either AcMNPV-PARIS-4 or
AcMNPV-LUC. The purified proteins were evaluated by Western blot
analysis. Following antibodies were used; anti-HA (Roche),
anti-His6 (Roche), anti-PARIS-4 (R&D Systems), and
anti-luciferase (CalBiochem, San Diego, Calif.). FIG. 19B shows the
Western blot analysis of proteins produced by recombinant
AcMNPV-PARIS-4 and -LUC. In the figure, "r" indicates recombinant.
FIG. 19C is a Coomassie blue stained gel electrophoresis of
Ni-NTA.TM. (Quiagen, Inc., Valencia, Calif.) purified PARIS-4 and
Luciferase (control) proteins. MWM: molecular weight marker, CL:
cell lysate, FT: flow through, WSH: wash, ET1-5: elutions 1-5. The
results suggest the successful production and purification of
recombinant proteins. In ongoing work directed at scaling up
protein production, it has been demonstrated that 645 .mu.g of
r-luciferase can be produced from 10.sup.8/100 mL of Sf-9
cells/media. More than 6 mg of protein production from 1000 mL of
suspension culture is expected.
[0114] In light of all of the evidence presented in the foregoing
Examples, it is concluded that PARIS-4 is one of the proteins that
are produced by VSMCs and are more abundantly produced by Harlan
VSMCs (restenosis resistant), and which are present in Harlan
conditioned media (FIGS. 15A-C and 16). It is these proteins that
are thought to inhibit the growth of VSMCs in vitro (FIGS. 8 and
17), and which inhibit the formation of neointima after balloon
injury in vivo (FIGS. 18A-D). Because these data for PARIS-4
clearly validate the microarray analyses identifying the soluble
negative growth inhibitors produced by the proliferating VSMCs, it
is believed that PARISs 1-3, which were identified by the exact
same microarray analyses, are also true negative growth regulators
of VSMCs.
[0115] It is expected that additional PARIS proteins will be
identified that share at least 24% amino acid identity with the
above-identified rat PARISs, preferably sharing at least 40%
identity, and still more preferably sharing about 60-100% amino
acid identity. The counterpart proteins to the representative
PARISs, in all mammals, are intended to be within the scope of the
present invention. Furthermore, proteins having at least 40%
homology to the above-identified rat amino acid sequences are also
expected to provide at least some measure of cell growth inhibitory
properties similar to those exemplified herein. Accordingly, all
such proteins or polypeptides are considered to be PARISs. For
example, homologous proteins may include a number of amino acid
substitutions in which the differing amino acids have similar
R-group substituents in terms of size, electrophilic character,
charge, and the like. Some exemplary substitutions are listed in
Table 6. TABLE-US-00007 TABLE 6 AMINO ACID SUBSTITUENTS FOR PARIS
HOMOLOGS NATIVE AMINO ACID AMINO ACID SUBSTITUTIONS alanine
glycine; serine arginine lysine asparagine glutamine; histidine
aspartic acid glutamic acid cysteine serine glutamine asparagine
glycine alanine histidine asparagine; glutamine isoleucine leucine;
valine leucine isoleucine; valine lysine arginine; glutamine;
glutamic acid methionine leucine; tyrosine serine threonine
threonine serine tryptophan tyrosine tyrosine tryptophan;
phenylalanine valine isoleucine; leucine
[0116] Some highly preferred PARIS proteins have the amino acid
sequences of SEQ ID NOs.: 1-4 (human PARISs 1-4), and correspond to
GenBank Accession No. Q15818 (PARIS-1), GenBank Accession No.
NP.sub.--004855 (PARIS-2), GenBank Accession No. P78543 (PARIS-3),
and GenBank Accession No. NP.sub.--002987 (PARIS-4), respectively,
are listed in Table 7, along with all of their orthologs from
representative animal models. The percent identity of orthologs of
the rat PARISs 1-4 was estimated using the UniGene system of the
National Center for Biotechnology Information of the National
Institutes of Health. The UniGene system automatically partitions
GenBank sequences into a non-redundant set of gene-oriented
clusters. Each UniGene cluster contains sequences that represent a
unique gene, as well as related information such as the tissue
types in which the gene has been expressed and map location..sup.63
TABLE-US-00008 TABLE 7 SELECTED MODEL ORGANISM PARIS PROTEIN
SIMILARITIES PARIS-1 PARIS-2 PARIS-3 PARIS-4 GenBank Acc. GenBank
GenBank Acc. GenBank No. Identity.sup.1 Acc. No. Identity.sup.2 No.
Identity.sup.3 Acc. No. Identity.sup.4 H. sapiens Q15818 95%/432 aa
NP_004855.1 100%/308 aa P78543 92%/158 aa NP_002987.1 67%/393 aa M.
musculus Q62443 99%/432 aa Q9Z0J7 58%/308 aa Q04211 97%/158 aa
O35188 85%/393 aa R. norvegicus P47971 100%/432 aa Q9Z0J7 59%/294
aa A40443 100%/158 aa O55145 100%/393 aa D. P27091 31%/120 aa
melanogaster C. elegans NP_504709.1 29%/138 aa NP_505150.1 24%/210
aa .sup.1UniGene Cluster Rn.54707 R. norvegicus .sup.2UniGene
Cluster Hs.296638 H. sapiens .sup.3UniGene Cluster Rn.27923 R.
norvegicus .sup.4UniGene Cluster Rn.4106 R. norvegicus
EXAMPLE 13
Deterrence or Prevention of Post-Angioplasty Restenosis
[0117] PARISs produced in sterile, endotoxin-free environment using
a standard CHO cell culture system will be administered to patients
undergoing angioplasty procedures in order to suppress the growth
of vascular smooth muscle cells and restenosis. It is believed that
use of PARISs will be less expensive and more inclusive (i.e., they
may be administered without special instruments or personnel) than
conventional post-angioplasty restenosis treatments and
preventatives.
[0118] Production of the PARIS protein will be carried out as
follows: A PARIS-cDNA will be ligated into mammalian expression
vector with the neomycin resistant gene that contains the sequence
to allow the addition of polyhistidine tags at the C-terminus of
the PARIS protein. CHO cells will be stably transfected with the
vector and selected using G418. The clones that express PARIS most
abundantly will be selected. Cells will then be adjusted to
serum-free medium system. PARIS will be secreted into the media
since it contains a secretion marker. PARIS will then be purified
using the metal ion chromatography with Ni-NTA beads to near
homogeneity. To achieve the further purity, the purified protein
will further be purified by ion-exchange chromatography. All the
procedures will be completed under sterile and endotoxin-free
conditions. Purified proteins will be tested for the endotoxin
contents.
[0119] An appropriate PARIS protein dose will be determined as
follows: Various amounts of proteins will be parenterally
administered first to animals (then after completion of full animal
studies to human) and multiple blood samplings will be performed
over time (ex. 1, 2, 4, 8, and 24 hrs). The samples will be then
evaluated by ELISA methods described earlier. The ideal dosing of
the PARIS would be such that plasma concentration of PARIS is 5-10
fold higher than normal serum concentrations of PARIS. The ideal
methods of parenteral administration will be determined using
animals first and then validated in human. Delivery of the purified
PARIS(s) will be achieved by one of following methods: intravenous
injection, subcutaneous injection, intraperitoneal injection,
transcutaneous delivery, local delivery using PARIS-coated stents
or infusion catheters; with/without liposomal or nanomolecular
delivery systems. Suitable carriers for protein drugs are well
known in the art. The PARISs may be coated onto stents using known
techniques that have been previously employed with other
drug-eluting stents.
[0120] It is also envisioned that in a certain circumstance, the
combination of two or more PARISs will be administered to enhance
the anti-proliferative effect of PARISs. PARISs can be used
together with drug-coated stents, plain stents, and any other
interventional methods used in current and future percutaneous
coronary interventions.
EXAMPLE 14
Treatment of Post-Angioplasty Restenosis
[0121] For the treatment of post-angioplasty or in stent
restenosis, PARISs will be delivered through routes in Example 13,
before, during or after the PCI to address the restenosis,
including stent (coated or noncoated) implantation, brachytherapy,
and any other current and future PCI methods appropriate for the
condition. Advantageously, a stent deployed in conjunction with
conventional PCI methods to address restenosis may be readily
coated with PARISs using substantially known techniques.
EXAMPLE 15
Delaying or Arresting Progression of Atherosclerosis
[0122] Purified PARISs are administered to patients with
atherosclerosis in order to suppress the growth of vascular smooth
muscle cells at the site of an atherosclerotic lesion. PARISs can
be subcutaneously injected into patients who are at high risk for
developing premature atherosclerosis, injected into patients who
have already had atherosclerosis with or without its long term
complications (e.g., CAD, MI, angina, etc.). Patients who are
expected to benefit from long term PARIS treatment include those
with cardiac transplantation (for deterring or preventing
transplantation atherosclerosis) and those patients receiving
coronary artery bypass grafts (CABG), for deterrence or prevention
of graft failure. Systemic or subcutaneous administration of PARISs
is expected to offer advantages for treatment of small vessels,
where conventional drug-eluting stents are not appropriate.
EXAMPLE 16
Deterrence, Prevention and Treatment of Other Smooth Muscle
Cell-Related Proliferative Disorders
[0123] This unique group of proteins (PARISs) are also believed to
hold promise for treating a variety of other proliferative
disorders. Although PARISs were originally identified as the
proteins produced by VSMCs for inhibiting the growth of VSMCs, it
is likely that PARISs have similar biological activity (e.g., cell
growth inhibitory effects) on normal and abnormal cells other than
vascular smooth muscle cells. For example, postsurgical keloid
formation involves not only VSMCs but also fibroblasts and other
cell types. PARISs may block keloid formation through the negative
growth regulation over all of these cells. Furthermore,
proliferative diabetic retinopathy represents the formation of
neo-arteries in the retina, which are fragile, and tends to bleed.
PARISs may be used to prevent such neo-artery formation. Certain
tumors, such as hemangioma, rhabdomyoma, rhabdomyosarcoma,
fibromyoma of the uterus, and other vascular and muscular tumors,
either benign or malignant, may be effectively treated by PARISs.
Furthermore, tumors and cancers that do not contain smooth muscle
components may well respond to PARISs in a higher dose. In summary,
PARISs may prove useful in any type of malignancy in humans.
[0124] While the preferred embodiments of the invention have been
shown and described, modifications thereof can be made by one
skilled in the art without departing from the spirit and teachings
of the invention. The embodiments described herein are exemplary
only, and are not intended to be limiting. Many variations and
modifications of the invention disclosed herein are possible and
are within the scope of the invention. For example, biologically
active portions of the above-described PARIS proteins are also
contemplated as part of the present invention. Such bioactive
polypeptides may serve as receptor ligation regions for a native
PARIS, or may correspond to a region of a PARIS that participates
in protein-protein interaction with another protein, which
regulates the activity of the PARIS. Accordingly, the scope of
protection is not limited by the description set out above and is
intended to include all equivalents of the subject matter described
herein. The disclosures of all patents, patent applications and
publications cited herein are hereby specifically incorporated
herein by reference, to the extent that they provide materials,
methods or other details supplementary to those set forth
herein.
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Sequence CWU 1
1
4 1 429 PRT Homo sapiens GenBank / Q15818 2001-10-16 (1)..(429) 1
Met Pro Ala Gly Arg Ala Arg Thr Cys Ala Leu Leu Ala Leu Cys Leu 1 5
10 15 Leu Gly Pro Gln Asp Phe Gly Pro Thr Arg Phe Ile Cys Thr Ser
Val 20 25 30 Pro Val Asp Ala Asp Met Cys Ala Ala Ser Val Ala Ala
Gly Gly Ala 35 40 45 Glu Glu Leu Arg Ser Ser Asn Val Leu Gln Leu
Arg Glu Thr Val Leu 50 55 60 Gln Gln Lys Glu Thr Ile Leu Ser Gln
Lys Glu Thr Ile Arg Glu Leu 65 70 75 80 Thr Ala Lys Leu Gly Arg Cys
Glu Ser Gln Ser Thr Leu Asp Pro Gly 85 90 95 Ala Gly Glu Ala Arg
Ala Gly Gly Gly Arg Lys Gln Pro Gly Ser Gly 100 105 110 Lys Asn Thr
Met Gly Asp Leu Ser Arg Thr Pro Ala Ala Glu Thr Leu 115 120 125 Ser
Gln Leu Gly Gln Thr Leu Gln Ser Leu Lys Thr Arg Leu Glu Asn 130 135
140 Leu Glu Gln Tyr Ser Arg Leu Asn Ser Ser Ser Gln Thr Asn Ser Leu
145 150 155 160 Lys Asp Leu Leu Gln Ser Lys Ile Asp Glu Leu Glu Arg
Gln Val Leu 165 170 175 Ser Arg Val Asn Thr Leu Glu Glu Gly Lys Gly
Gly Pro Lys Asn Asp 180 185 190 Thr Glu Glu Arg Val Lys Ile Glu Thr
Ala Leu Thr Ser Leu His Gln 195 200 205 Arg Ile Ser Glu Leu Glu Lys
Gly Gln Lys Asp Asn Arg Pro Gly Asp 210 215 220 Lys Phe Gln Leu Thr
Phe Pro Leu Arg Thr Asn Tyr Met Tyr Ala Lys 225 230 235 240 Val Lys
Lys Ser Leu Pro Glu Met Tyr Ala Phe Thr Val Cys Met Trp 245 250 255
Leu Lys Ser Ser Ala Thr Pro Gly Val Gly Thr Pro Phe Ser Tyr Ala 260
265 270 Val Pro Gly Gln Ala Asn Glu Leu Val Leu Ile Glu Trp Gly Asn
Asn 275 280 285 Pro Met Glu Ile Leu Ile Asn Asp Lys Val Ala Lys Leu
Pro Phe Val 290 295 300 Ile Asn Asp Gly Lys Trp His His Ile Cys Val
Thr Trp Thr Thr Arg 305 310 315 320 Asp Gly Val Glu Ala Tyr Gln Asp
Gly Thr Gln Gly Gly Ser Gly Glu 325 330 335 Asn Leu Ala Pro Tyr His
Pro Ile Lys Pro Gln Gly Val Leu Val Leu 340 345 350 Gly Gln Glu Gln
Asp Thr Leu Gly Gly Gly Phe Asp Ala Thr Gln Ala 355 360 365 Phe Val
Gly Glu Leu Ala His Phe Asn Ile Trp Asp Arg Lys Leu Thr 370 375 380
Pro Gly Glu Val Tyr Asn Leu Ala Thr Cys Ser Thr Lys Ala Leu Ser 385
390 395 400 Gly Asn Val Ile Ala Trp Ala Glu Ser His Ile Glu Ile Tyr
Gly Gly 405 410 415 Ala Thr Lys Trp Thr Phe Glu Ala Cys Arg Gln Ile
Asn 420 425 2 308 PRT Homo sapiens GenBank / NP_004855 2003-04-07
(1)..(308) 2 Met Pro Gly Gln Glu Leu Arg Thr Val Asn Gly Ser Gln
Met Leu Leu 1 5 10 15 Val Leu Leu Val Leu Ser Trp Leu Pro His Gly
Gly Ala Leu Ser Leu 20 25 30 Ala Glu Ala Ser Arg Ala Ser Phe Pro
Gly Pro Ser Glu Leu His Ser 35 40 45 Glu Asp Ser Arg Phe Arg Glu
Leu Arg Lys Arg Tyr Glu Asp Leu Leu 50 55 60 Thr Arg Leu Arg Ala
Asn Gln Ser Trp Glu Asp Ser Asn Thr Asp Leu 65 70 75 80 Val Pro Ala
Pro Ala Val Arg Ile Leu Thr Pro Glu Val Arg Leu Gly 85 90 95 Ser
Gly Gly His Leu His Leu Arg Ile Ser Arg Ala Ala Leu Pro Glu 100 105
110 Gly Leu Pro Glu Ala Ser Arg Leu His Arg Ala Leu Phe Arg Leu Ser
115 120 125 Pro Thr Ala Ser Arg Ser Trp Asp Val Thr Arg Pro Leu Arg
Arg Gln 130 135 140 Leu Ser Leu Ala Arg Pro Gln Ala Pro Ala Leu His
Leu Arg Leu Ser 145 150 155 160 Pro Pro Pro Ser Gln Ser Asp Gln Leu
Leu Ala Glu Ser Ser Ser Ala 165 170 175 Arg Pro Gln Leu Glu Leu His
Leu Arg Pro Gln Ala Ala Arg Gly Arg 180 185 190 Arg Arg Ala Arg Ala
Arg Asn Gly Asp Asp Cys Pro Leu Gly Pro Gly 195 200 205 Arg Cys Cys
Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly 210 215 220 Trp
Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys 225 230
235 240 Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala
Gln 245 250 255 Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp Thr Glu
Pro Ala Pro 260 265 270 Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val
Leu Ile Gln Lys Thr 275 280 285 Asp Thr Gly Val Ser Leu Gln Thr Tyr
Asp Asp Leu Leu Ala Lys Asp 290 295 300 Cys His Cys Ile 305 3 158
PRT Homo sapiens GenBank / P78543 2002-06-15 (1)..(158) 3 Met Ser
His Gly Lys Gly Thr Asp Met Leu Pro Glu Ile Ala Ala Ala 1 5 10 15
Val Gly Phe Leu Ser Ser Leu Leu Arg Thr Arg Gly Cys Val Ser Glu 20
25 30 Gln Arg Leu Lys Val Phe Ser Gly Ala Leu Gln Glu Ala Leu Thr
Glu 35 40 45 His Tyr Lys His His Trp Phe Pro Glu Lys Pro Ser Lys
Gly Ser Gly 50 55 60 Tyr Arg Cys Ile Arg Ile Asn His Lys Met Asp
Pro Ile Ile Ser Arg 65 70 75 80 Val Ala Ser Gln Ile Gly Leu Ser Gln
Pro Gln Leu His Gln Leu Leu 85 90 95 Pro Ser Glu Leu Thr Leu Trp
Val Asp Pro Tyr Glu Val Ser Tyr Arg 100 105 110 Ile Gly Glu Asp Gly
Ser Ile Cys Val Leu Tyr Glu Glu Ala Pro Leu 115 120 125 Ala Ala Ser
Cys Gly Leu Leu Thr Cys Lys Asn Gln Val Leu Leu Gly 130 135 140 Arg
Ser Ser Pro Ser Lys Asn Tyr Val Met Ala Val Ser Ser 145 150 155 4
397 PRT Homo sapiens GenBank / NP_002987 2003-04-07 (1)..(397) 4
Met Ala Pro Ile Ser Leu Ser Trp Leu Leu Arg Leu Ala Thr Phe Cys 1 5
10 15 His Leu Thr Val Leu Leu Ala Gly Gln His His Gly Val Thr Lys
Cys 20 25 30 Asn Ile Thr Cys Ser Lys Met Thr Ser Lys Ile Pro Val
Ala Leu Leu 35 40 45 Ile His Tyr Gln Gln Asn Gln Ala Ser Cys Gly
Lys Arg Ala Ile Ile 50 55 60 Leu Glu Thr Arg Gln His Arg Leu Phe
Cys Ala Asp Pro Lys Glu Gln 65 70 75 80 Trp Val Lys Asp Ala Met Gln
His Leu Asp Arg Gln Ala Ala Ala Leu 85 90 95 Thr Arg Asn Gly Gly
Thr Phe Glu Lys Gln Ile Gly Glu Val Lys Pro 100 105 110 Arg Thr Thr
Pro Ala Ala Gly Gly Met Asp Glu Ser Val Val Leu Glu 115 120 125 Pro
Glu Ala Thr Gly Glu Ser Ser Ser Leu Glu Pro Thr Pro Ser Ser 130 135
140 Gln Glu Ala Gln Arg Ala Leu Gly Thr Ser Pro Glu Leu Pro Thr Gly
145 150 155 160 Val Thr Gly Ser Ser Gly Thr Arg Leu Pro Pro Thr Pro
Lys Ala Gln 165 170 175 Asp Gly Gly Pro Val Gly Thr Glu Leu Phe Arg
Val Pro Pro Val Ser 180 185 190 Thr Ala Ala Thr Trp Gln Ser Ser Ala
Pro His Gln Pro Gly Pro Ser 195 200 205 Leu Trp Ala Glu Ala Lys Thr
Ser Glu Ala Pro Ser Thr Gln Asp Pro 210 215 220 Ser Thr Gln Ala Ser
Thr Ala Ser Ser Pro Ala Pro Glu Glu Asn Ala 225 230 235 240 Pro Ser
Glu Gly Gln Arg Val Trp Gly Gln Gly Gln Ser Pro Arg Pro 245 250 255
Glu Asn Ser Leu Glu Arg Glu Glu Met Gly Pro Val Pro Ala His Thr 260
265 270 Asp Ala Phe Gln Asp Trp Gly Pro Gly Ser Met Ala His Val Ser
Val 275 280 285 Val Pro Val Ser Ser Glu Gly Thr Pro Ser Arg Glu Pro
Val Ala Ser 290 295 300 Gly Ser Trp Thr Pro Lys Ala Glu Glu Pro Ile
His Ala Thr Met Asp 305 310 315 320 Pro Gln Arg Leu Gly Val Leu Ile
Thr Pro Val Pro Asp Ala Gln Ala 325 330 335 Ala Thr Arg Arg Gln Ala
Val Gly Leu Leu Ala Phe Leu Gly Leu Leu 340 345 350 Phe Cys Leu Gly
Val Ala Met Phe Thr Tyr Gln Ser Leu Gln Gly Cys 355 360 365 Pro Arg
Lys Met Ala Gly Glu Met Ala Glu Gly Leu Arg Tyr Ile Pro 370 375 380
Arg Ser Cys Gly Ser Asn Ser Tyr Val Leu Val Pro Val 385 390 395
* * * * *