©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Growth-related Responses in Arterial Smooth Muscle Cells Are Arrested by Thrombin Receptor Antisense Sequences (*)

(Received for publication, January 10, 1995)

Elliot L. Chaikof (1) Rafael Caban (2) Chang-Ning Yan (3) Gadiparthi N. Rao (3) Marschall S. Runge (2) (3)(§)

From the  (1)Departments of Surgery (Vascular Division) and (2)Medicine (Cardiovascular Division), Emory University School of Medicine, Atlanta, Georgia 30322 and the (3)Department of Internal Medicine, Division of Cardiology, University of Texas Medical Branch, Galveston, Texas 77555-1064

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The capacity of antisense sequences to the thrombin receptor to selectively inhibit thrombin receptor expression and limit mitogenic responses in vascular wall cells was investigated in vitro. Eight phosphorothioate oligodeoxynucleotides based on the sequences of the rat thrombin receptor (including sense, antisense, scrambled, and missense controls) were synthesized, characterized, and purified by high performance liquid chromatography. The antisense oligodeoxynucleotide (ODN 4) inhibitory effect was sequence-specific and both time- and concentration-dependent. A reduction in serum or alpha-thrombin-induced smooth muscle cell (SMC) proliferation was noted as early as 3 days at 30 µM (82%; 6.17 ± 1.01 versus 34.08 ± 3.89 times 10^4 cells/well; p < 0.05) and at a dose as low as 15 µM after 4 days in culture (19%; p < 0.05). Nonspecific effects were enhanced after prolonged exposure of SMC to the antisense oligodeoxynucleotide (geq6 days). A reduction of inositol phosphate generation greater than 50% (p < 0.05) was detected after exposure of SMC to antisense but not to sense or scrambled nucleotide sequences. This was observed after stimulation with both thrombin and SFFLRN (thrombin receptor peptide agonist). Northern blot analysis and enzyme-linked immunosorbent assays revealed 50 and 22% decreases, respectively, in thrombin receptor mRNA and protein (cell surface) levels in antisense oligonucleotide-treated (72 h) SMC as compared to untreated cells, suggesting that thrombin receptor down-regulation occurred at the pretranslational level. Thus, thrombin receptor-specific antisense sequences inhibit growthrelated effects both of serum and thrombin on smooth muscle cells, potentially providing a new strategy for selective inhibition of receptor-mediated arterial injury responses.


INTRODUCTION

Recent investigations have led researchers to postulate that thrombin may play a significant role in normal vessel wall healing under physiological conditions(1, 2) . Hatton et al.(3) have demonstrated that following catheter denudation of the rabbit aorta, thrombin activity remains elevated at the site of smooth muscle cell (SMC) (^1)proliferation for up to 10 days, and Bar-Shavit et al.(4) have confirmed that thrombin bound to the subendothelial extracellular matrix remains functionally active and protected from inactivation by antithrombin III. In support of a role for thrombin in vascular lesion formation, Okazaki et al.(5) have reported that thrombin, but not other vasoactive agonists or growth factors, produces a pattern of transiently increased platelet-derived growth factor-A and decreased platelet-derived growth factor-beta receptor mRNA in vascular smooth muscle cells both in vitro and in vivo, a pattern similar to that observed after vascular injury. Finally, a number of in vitro and in vivo studies have documented smooth muscle proliferation following thrombin exposure(6, 7, 8, 9) , and thrombin inhibitors have been noted to reduce the mitogenic response of cultured smooth muscle cells (10) . Despite these reports and our general knowledge that thrombin mediates procoagulant, mitogenic, vasoactive, and inflammatory effects (all of which may potentially promote a proliferative vascular wall response), we know little of the mechanism by which thrombin initiates the events leading to arterial wall healing.

The thrombin receptor has been cloned and classified as a member of the G-protein-coupled family of receptors. This is consistent with observations of phospholipase C-mediated phosphoinositide turnover(11, 12) , phospholipase D activation(13, 14) , protein phosphorylation(15) , and inhibition of adenylate cyclase activity (13, 14) after thrombin stimulation. Furthermore, expression of the thrombin receptor gene has been identified in proliferating arterial smooth muscle cells in vitro(16) , and enhanced expression of this gene after arterial wall injury in vivo has been documented(17) . This suggests that both thrombin receptor gene regulation and receptor activation contribute to vascular wall healing under normal conditions. Likewise, thrombin receptor overexpression could well be a primary event in facilitating maladaptive responses associated with neointimal hyperplasia, both after bypass grafting and in restenosis following balloon catheter angioplasty.

Although several research groups have synthesized oligopeptides and monoclonal antibodies as thrombin receptor antagonists, these attempts have met with limited success. Thus, the importance of thrombin receptor activation on vascular SMC after mechanical vascular wall injury has yet to be determined. Antisense strategies have had some success in the reduction of eukaryotic cell surface receptors, including the reduction by nearly 90% of the epidermal growth factor receptor (18) and dramatic inhibition of the activity of the muscarinic receptor(19) , luteinizing hormone receptor(20) , and acetylcholine receptor subunits(21) . With regard to thrombin, the relative contributions to vascular smooth muscle cell mitogenesis of thrombin receptor activation, thrombin-mediated platelet activation, and fibrin generation, as well as binding or cleavage of other cell surface proteins by this serine protease, are unknown. Thus, the major advantage of using an antisense approach to inhibit thrombin receptor-mediated events in vascular cells is mechanistic, to determine the effect of inhibiting thrombin receptor expression without affecting the role of thrombin in coagulation or the effects of thrombin that result from its binding to other proteins on or near the cell surface, including thrombomodulin(22) , antithrombin III(23) , and protease nexin(24) .

We report that specific reduction of thrombin receptor-mediated events is possible using a receptor-specific antisense DNA sequence. An important determinant of specificity in the system we studied was the purity of the nuclease-resistant phosphorothioate oligodeoxynucleotide sequences. HPLC purification was required to minimize degenerate fragments and the potential of nonspecific gene suppression. The down-regulation of receptor expression in this model is produced by a pretranslational mechanism. Further, we report that when thrombin receptor expression is inhibited using an antisense sequence specific for nucleotides 4-20 of the rat thrombin receptor, there is a significant reduction of smooth muscle cell mitogenesis, not only in response to alpha-thrombin but also in response to stimulation with 10% fetal bovine serum.


EXPERIMENTAL PROCEDURES

Cell Culture

Primary vascular SMC were isolated from the thoracic aortas of 250-300-g male Sprague-Dawley rats using a modification of the method of Travo et al.(25) . Briefly, aortas were dissected, and the adventitia and endothelium were mechanically removed and digested with collagenase and elastase. The identity of cultured cells as SMC was confirmed by staining with an anti-alpha-smooth muscle actin antibody (antibody A2547 from Sigma). Cells were grown in Dulbecco's modified Eagle's medium (DME) supplemented with 10% fetal bovine serum, used in passages 2-5, and maintained in culture at 37 °C in a humidified 5% CO(2) atmosphere.

Oligonucleotide Synthesis

Phosphorothioate oligodeoxynucleotides were synthesized on an Applied Biosystems Inc. (Foster City, CA) 380B DNA synthesizer. Oligodeoxynucleotides (ODNs) were deprotected in saturated aqueous NH(4)OH (12-15 ml/10 µmol of ODNs) for 16-24 h at 55 °C and then dried in a vacuum concentrator. ODNs were then resuspended in 50 mM triethylamine-acetate buffer (TEAbulletAc) (pH 6.5) and purified by reverse-phase HPLC on a Water's Delta Prep 3000 system (Milford, MA). A Vydac C8 column (22 times 250 mm, 10-µm particle size, 300-Å pore size) was utilized and eluted with a linear gradient of acetonitrile buffered in 35-50 mM TEAbulletAc (pH 6.5). Fractions were collected and desiccated. After partial evaporation, an aliquot was run on a microbore reverse-phase HPLC using an ABI (Foster City, CA) VeloSep cartridge (3.4 times 40 mm, C-8 silica, 3-µm particle size) for purity verification.

Proliferation Assay

SMC were seeded at a density of 25,000 cells/well in six-well plates (Costar, Cambridge, MA). The following day cells were washed twice with phosphate-buffered saline (PBS) and the medium replaced with 0.1% BSA-DME (growth-arrest medium). The cells were maintained in growth-arrest medium for 48 h, after which the media was changed to DME with 10% fetal bovine serum (DME, 10% FBS) and synthetic oligodeoxynucleotides added. The medium and oligodeoxynucleotides were changed every 48 h. Cells were trypsinized and counted manually using a hemocytometer after exposure to ODNs for 3, 4, and 6 days. Each experiment was carried out in triplicate and repeated at least two additional times. Data are expressed as mean ± S.D.

D-myo-Inositol Phosphate Assay

SMC were plated onto 35-mm dishes, allowed to reach 90% confluence, then growth arrested for 48 h. The medium was then changed to DME, 10% FBS, and oligodeoxynucleotides were added for 96 h. During the last 24 h of the 96-h incubation, myo-[^3H]inositol (15 µCi/ml) was added. Unincorporated label was removed by washing cultures in a warm balanced salt solution (termed Na solution): 130 mM NaCl, 5 mM KCl, 1 mM MgCl(2), 1.5 mM CaCl(2), 20 mM HEPES (buffered to pH 7.4 with Tris base), 10 mM dextrose, 10 mM LiCl. SMC were incubated in 1 ml of Na+ solution for 20 min at 37 °C and then stimulated with 140 nM alpha-thrombin, 25 µM SFFLRN, or PBS for 30 min. The reaction was terminated by rapid aspiration of the buffer and addition of 1 ml of chloroform/methanol/HCl (20:40:1, by volume). A second wash with 500 µl of chloroform/methanol/HCl was performed. Organic and aqueous phases were separated by the addition of 900 µl of distilled water and 500 µl of chloroform. After centrifugation (500 times g for 10 min at 4 °C) and two chloroform washes, the organic phases were pooled and evaporated to dryness under N(2). Inositol phosphates in the aqueous phase were analyzed by ion-exchange chromatography on Dowex AG-1X8 resin and quantified by liquid-scintillation spectrometry.

Immunohistochemistry

For bromodeoxyuridine (BrdU) immunohistochemistry, cells were incubated at 37 °C for 2 h with bromodeoxyuridine (30 µg/ml). Cells were then washed in PBS and fixed in acid ethanol. After pretreatment with 10% sheep serum and washes in PBS, cells were incubated with mouse anti-bromodeoxyuridine monoclonal antibody (Amersham Corp.) for a 90-min incubation. This and subsequent steps were performed according to the manufacturer's specifications. The cells were again washed with PBS, incubated for an additional 30 min with peroxidase-conjugated anti-mouse antibody, and stained with diaminobenzidine tetrahydrochloride (Amersham).

Northern Blot Analysis

Growth-arrested SMC were treated for 72 h with DME containing 10% FBS in the presence or absence of antisense (ODN 4) or sense (ODN 3) oligonucleotides, and total cellular RNA was isolated using Tris reagent (Molecular Research Center, Inc., Newark, NJ) using the manufacturer's protocol. Ten µg of RNA were size fractionated by electrophoresis on 1.2% agarose, 2% formaldehyde gel and then transferred to a Nytron filter (Schleicher & Schuell). RNA was cross-linked to the filter using UV irradiation (Stratalinker, Stratagene, La Jolla, CA). After a 4 h prehybridization in 50% (v/v) formamide, 5 times SSC (1 times SSC = 0.15 M NaCl, 0.015 M sodium citrate), 5 times Denhardt's (1 times Denhardt's = 0.02% (w/v) each of Ficoll, polyvinyl pyrrolidone, and bovine serum albumin), 50 mM sodium phosphate (pH 6.5) and 250 µg/ml of sheared salmon sperm DNA at 42 °C, the Nytran filter was hybridized in the above buffer containing 10% (w/v) dextran sulfate and 1 times 10^6 counts/min/ml of P-labeled rat thrombin receptor cDNA probe for 16 h at 42 °C. The filter was washed in 0.1 times SSC containing 0.1% SDS for 60 min at 60 °C (with two changes of solution), and the bands were visualized and quantitated by the use of a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The same filter was rehybridized with P-labeled human rDNA probe to demonstrate equal amounts of RNA in each lane. Labeling of probes with [alpha-P]dCTP was done using a multiprime labeling system kit (Amersham,).

Adherent Cell ELISA Assay

Growth-arrested SMC were treated as described above and washed with DME/Ham's F-12 (1:1) medium containing 0.1% BSA. Cells were then incubated with rabbit anti-rat thrombin receptor polyclonal antibodies. After incubation of cells with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies, the color was developed with ABST peroxidase substrate according to the manufacturer's protocol (KPL, Gaithersburg, MD) and the ODs were read at 405 nm.


RESULTS

Synthetic Oligonucleotides

Eight DNA sequences were synthesized and are summarized in Table 1. Comparison of sequence 7 with sequence 4 shows four mismatched nucleotides. Sequence 8 is a scrambled version of sequence 4, containing the same nucleotide composition overall but in a random sequence not found in the Genbank data base. The approximate yield following HPLC purification was 50% (Fig. 1).




Figure 1: Oligonucleotide purification. A, pre-HPLC profile. B, post-HPLC profile. Oligonucleotides were purified by reverse-phase HPLC utilizing a linear gradient of acetonitrile buffered in 35-50 mM TEAbulletAc (pH 6.5). Approximate yield following HPLC purification was 50%.



Inhibition of Mitogenic Responses by Antisense DNA

Cell proliferation was specifically inhibited by the use of antisense DNA to the first six codons that follow the initiation codon of the thrombin receptor. Serum-induced mitogenesis was reduced after a 72-h exposure to media containing 30 µM of sequence 4, but was minimally affected by incubation with the remaining seven oligodeoxynucleotide sequences (Fig. 2). This inhibitory effect was both dose- and time-dependent. Inhibition of mitogenic responses was not observed over the dose range of 0.1-5 µM for any sequence. However, a reduction in SMC proliferation was noted beginning at 15 µM after 4 days in culture and at 10 µM after 6 days in culture. At a concentration of 30 µM, SMC proliferative responses were reduced at 3 days. There was almost complete inhibition of SMC proliferation after a 6-day exposure period (Fig. 3), although at this time there was also a significant inhibitory effect by the sense oligodeoxynucleotide. Concentration dependence was noted at both 4 days (Fig. 4) and 6 days (not shown) in culture. Uniformly, at higher oligodeoxynucleotide doses and longer incubation periods, nonspecific inhibition of serum-induced mitogenesis was observed. For confirmation, tritiated thymidine uptake in response to SFFLRN and alpha-thrombin was reduced, and the increase in c-fos mRNA expression, typical of thrombin-induced mitogenesis(26) , did not occur following incubation with sequence 4 (data not shown).


Figure 2: SMC proliferation. Growth-arrested SMC were incubated with DME- 10% FBS alone, DME-BSA alone, or DME-10% FBS plus PBS or the indicated ODN sequences (30 µM). After 72 h of incubation, cells were counted using a hemocytometer. Antisense oligonucleotide, 5`ASTR-2 (30 µM) reduced serum-induced proliferation of SMC (82%; 6.17 ± 1.01 versus 34.08 ± 3.89 times 10^4 cells/well; p < 0.05).




Figure 3: Percent inhibition of serum-induced SMC proliferation: sense (sequence 3) versus antisense (sequence 4) oligonucleotides. At a concentration of 30 µM, sequence 4 reduced the SMC proliferative responses to 10% FBS at 3 days, an effect notably enhanced after a 6-day exposure period. The data are displayed as percent maximal proliferation (no oligonucleotides = 100% proliferation) ± S.D.




Figure 4: SMC proliferation: dose-response effect of sequence 4. Inhibition of mitogenic responses was not observed over the dose range of 0.1-5 µM. A reduction in SMC proliferation was noted beginning at 15 µM after 4 days in culture (19%; p < 0.05), and concentration dependence at higher doses was noted.



Inositol Phosphate Analysis

Following exposure of SMC to either alpha-thrombin or SFFLRN, an increase in inositol phosphate production occurs. Depending on the timing of the assay, this increase represents an approximate 30-150% increase over base-line total inositol phosphate levels. As a measure of the effect of inhibiting thrombin receptor expression using antisense oligodeoxynucleotides, we measured total inositol phosphates in cultured SMC following stimulation with either alpha-thrombin or SFFLRN. Total inositol phosphates did not increase following stimulation with either alpha-thrombin or SFFLRN of arterial smooth muscle cells treated with antisense sequence 4 (Table 2). The response of SMC treated with either the scrambled or missense sequence was not significantly different from that of SMC treated with PBS.



Immunohistochemistry

Proliferating vascular smooth muscle cells were detected in vitro using BrdU immunohistochemistry. Representative micrographs of BrdU uptake under various culture conditions are presented in Fig. 5. Treatment of cells with antisense sequence 4, but not sense or missense sequences, reduced BrdU uptake in a manner which paralleled the reduction of SMC proliferation noted by either direct cell counting or tritiated thymidine uptake.


Figure 5: Anti-bromodeoxyuridine immunoperoxidase staining. Detection of proliferating vascular smooth muscle cells using bromodeoxyuridine immunohistochemistry after a 3-day exposure to: A, DME, 10% FBS; B, DME, 0.1% BSA; C, DME, 10% FBS plus 5`-STR-2; D, DME, 10% FBS plus 5`MSTR-2; E, DME, 10% FBS plus 5`-ASTR-2. Oligonucleotide sequences were used at a final concentration of 30 µM.



Northern Blot Analysis

To determine whether the growth-inhibitory effect of sequence 4 is due to a decreased synthesis of thrombin receptor, we first analyzed its effects on thrombin receptor mRNA levels. Growth-arrested SMC were treated with DME, 10% FBS for 72 h in the presence or absence of 30 µM antisense (ODN 4) or sense (ODN 3) oligonucleotides and total cellular RNA was isolated. An equal amount of RNA (10 µg) from each condition was then analyzed for thrombin receptor transcripts as described under ``Experimental Procedures.'' Incubation of SMC with antisense oligonucleotide (ODN 4) caused a 50% decrease in thrombin receptor mRNA levels as compared with the amounts in untreated cells (Fig. 6). Neither antisense nor sense oligonucleotides affected rRNA levels, suggesting that the effect of antisense oligonucleotide on thrombin receptor mRNA is specific.


Figure 6: Down-regulation of thrombin receptor mRNA by antisense oligonucleotides. SMC were treated for 72 h with 30 µM of indicated antisense or sense oligonucleotides and total cellular RNA was isolated. Ten µg of RNA from each treatment were analyzed for thrombin receptor transcripts by Northern blotting using the respective P-labeled cDNA probe. Similar results were obtained in three independent experiments.



Adherent Cell ELISA Assay

To further prove that the decrease in thrombin receptor mRNA levels in SMC treated with antisense sequence also resulted in a comparable reduction in the protein, cell surface thrombin receptor protein levels were quantitated in cells treated identically to those described above using adherent cell ELISA assay. As evident from Fig. 7, SMC treated with antisense oligonucleotide (ODN 4) had significantly lower levels of thrombin receptor antigen compared to untreated cells.


Figure 7: Decreased thrombin receptor protein in antisense oligonucleotide-treated SMC. Cells were treated for 72 h with 30 µM of antisense (5`ASTR-2) or sense (5`-STR-2) oligonucleotides and cell surface associated thrombin receptor protein content was measured by adherent cell ELISA assay. Similar results were obtained in three separate experiments.




DISCUSSION

Increasing evidence suggests that thrombin generation may contribute to normal vessel wall healing following arterial injury, as well as to those maladaptive responses which lead to atherosclerosis, neointimal hyperplasia, or restenosis. For example, Sarembock et al.(27, 28) and, more recently, Gorog et al.(29) have reported that antithrombin treatment with recombinant hirudin or [sca]d-phenylalanine-proline-arginine-chloromethyl ketone, an irreversible active site thrombin inhibitor) reduces restenosis after in vivo balloon catheter angioplasty in a rabbit model. Nonetheless, no means exist to block thrombin receptor activation selectively in order to delineate precisely the role of receptor-mediated events in this complex pathway. Indeed, although it is tempting to speculate that thrombin initiates neointimal hyperplasia via a direct effect on smooth muscle mitogenesis, other thrombin receptor-mediated phenomena may well hold as great or greater significance, including: (a) the initiation of extracellular matrix production by SMC(30, 31) ; (b) the activation of platelet aggregation and degranulation(32, 33) ; (c) the stimulation of endothelial cells(34, 35, 36) ; or (d) the generation and release of cytokines by neutrophils, monocytes, and T cells(37, 38) . Further, the functional relevance of other known cell surface thrombin-binding proteins is unknown. These questions cannot be adequately addressed until selective blocking of the thrombin receptor is achieved. To date, both small peptide sequences and antibodies have been investigated as possible thrombin receptor antagonists without significant success in vivo. (^2)

We have observed that an antisense oligodeoxynucleotide which follows the initiation codon of the thrombin receptor mRNA selectively inhibits the mitogenic responses of vascular smooth muscle cells to fetal bovine serum, alpha-thrombin, and the thrombin receptor agonist peptide, SFFLRN. The observed nonspecific inhibitory effects could be due to the presence of contaminating short nucleic acid sequences following ODN synthesis because removal of these sequences by HPLC minimized the nonspecific effects. A significant and specific reduction in proliferation, whether measured by direct cell counting or tritiated thymidine and BrdU incorporation, was noted following a 72-96-h exposure of vascular SMC to ODN sequence 4, but not to sense, missense, or scrambled nucleotide sequences. However, nonspecific inhibition of cell proliferation by phosphorothioate oligonucleotides was observed under conditions of either high concentration or prolonged incubation.

Based on our data, the specific inhibition of mitogenic responses at lower concentrations and at early time points is most likely a consequence of a direct decrease in receptor expression. Indeed, as determined by adherent cell ELISA assay, thrombin receptor protein was decreased significantly in antisense oligonucleotide-treated cells. The effect of the antisense ODN appears to be at the level of pretranslation as it affected the thrombin receptor mRNA levels. A similar effect of antisense oligonucleotides on the down-regulation of the respective mRNA was reported for protein kinase Calpha in human A549 lung carcinoma cells(39) . Thrombin receptor-dependent signal transduction, as measured by inositol phosphate generation and mitogenesis, is decreased when SMC are exposed to antisense sequence 4. These findings thus show a correlation between thrombin receptor down-regulation and decreased growth in antisense oligonucleotide-treated SMC.

The ability of an antisense sequence to the thrombin receptor to inhibit serum-induced SMC proliferation probably reflects significant levels of alpha-thrombin in the serum. This hypothesis may be supported further by the fact that hirudin, an inhibitor of thrombin, reduced serum-induced growth of SMC by only 30%, yet hirudin completely blocked alpha-thrombin-induced SMC proliferation (data not shown). In addition, Melzig et al.(10) documented that heparin and synthetic inhibitors of thrombin decreased the rate of division of porcine vascular smooth muscle cells in culture supplemented with 10% calf serum. Our own studies have shown low levels of natural thrombin inhibitors, including antithrombin III, in commercial serum. (^3)The inability to completely abolish the serum-induced SMC proliferation no doubt reflects the presence of other well-known smooth muscle cell mitogens in the serum which are unaffected by the presence of antisense sequences.

We have demonstrated selective inhibition of thrombin receptor expression using an antisense approach. Admittedly, the flexibility of this approach is limited by the requirement for ODN supplementation to culture media and the inevitable nonspecific reduction in cell proliferation after prolonged incubation. Nonetheless, this work provides an important first step by which the role of these receptor-mediated events in atherosclerosis and injury-induced vascular restenosis can be precisely defined.


FOOTNOTES

*
This study was supported in part by National Institutes of Health Grants HL-48667 and HL-02414.

This work was presented in abstract form at the 1993 American Heart Association Scientific Conference on the Molecular Biology of the Vascular Wall, Boston.

§
To whom correspondence should be addressed: Div. of Cardiology and Sealy Center for Molecular Cardiology, University of Texas Medical Branch, 9.138 Medical Research Bldg., Rt. 1064, 301 University Blvd., Galveston, TX 77666-1064.

(^1)
The abbreviations used are: SMC, smooth muscle cells; HPLC, high performance liquid chromatography; DME, Dulbecco's modified Eagle's medium; ODNs, oligodeoxynucleotides; PBS, phosphate-buffered saline; BSA, bovine serum albumin; BrdU, bromodeoxyuridine; ELISA, enzyme-linked immunosorbent assay.

(^2)
S. Hanson and L. Harker, personal communication.

(^3)
K. Wick and M. Runge, unpublished data.


REFERENCES

  1. Wilcox, J. N. (1991) Circulation 84, 432-435 [Medline] [Order article via Infotrieve]
  2. Carney, D. H., Mann, R., Redin, W. R., Pernia, S. D., Berry, D., Heggers, J. P., Hayward, P. G., Robson, M. C., Christie, J., Annable, C., and Fenton, J. W. I. (1992) J. Clin. Invest. 89, 1469-1477 [Medline] [Order article via Infotrieve]
  3. Hatton, M. W., Moar, S. L., and Richardson, M. (1989) Am. J. Pathol. 135, 499-508 [Abstract]
  4. Bar-Shavit, R., Benezra, M., Sabbah, V., Bode, W., and Vlodavsky, I. (1992) Am. J. Respir. Cell Mol. Biol. 6, 123-130 [Medline] [Order article via Infotrieve]
  5. Okazaki, H., Majesky, M. W., Harker, L. A., and Schwartz, S. M. (1992) Circ. Res. 71, 1285-1293 [Abstract]
  6. Graham, D. J., and Alexander, J. J. (1990) J. Vasc. Surg. 11, 307-312 [CrossRef][Medline] [Order article via Infotrieve]
  7. Herbert, J. M., Lamarche, I., and Dol, F. (1992) FEBS Lett. 301, 155-158 [CrossRef][Medline] [Order article via Infotrieve]
  8. Weiss, R. H., and Ives, H. E. (1991) Biochem. Biophys. Res. Commun. 181, 617-622 [Medline] [Order article via Infotrieve]
  9. Weiss, R. H., and Nuccitelli, R. (1992) J. Biol. Chem. 267, 5608-5613 [Abstract/Free Full Text]
  10. Melzig, M., Harms, C., Teuscher, E., Voigt, B., and Wagner, G. (1986) Biomed. Biochim. Acta 45, 1199-1202 [Medline] [Order article via Infotrieve]
  11. Paris, S., and Pouyssegur, J. (1987) J. Biol. Chem. 262, 1970-1976 [Abstract/Free Full Text]
  12. Huang, E. M., and Detwiler, T. C. (1987) Biochem. J. 242, 11-18 [Medline] [Order article via Infotrieve]
  13. Brass, L. F., Woolkalis, M. J., and Manning, D. R. (1988) J. Biol. Chem. 263, 5348-5355 [Abstract/Free Full Text]
  14. Brass, L. F., Manning, D. R., Williams, A. G., Woolkalis, M. J., and Poncz, M. (1991) J. Biol. Chem. 266, 958-965 [Abstract/Free Full Text]
  15. Levin, E. G., and Santell, L. (1991) J. Biol. Chem. 266, 174-181 [Abstract/Free Full Text]
  16. Zhong, C., Hayzer, D. J., Corson, M. A., and Runge, M. S. (1992) J. Biol. Chem. 267, 16975-16979 [Abstract/Free Full Text]
  17. Coughlin, S. R., Vu, T. K., Hung, D. T., and Wheaton, V. I. (1992) J. Clin. Invest. 89, 351-355 [Medline] [Order article via Infotrieve]
  18. Moroni, M. C., Willingham, M. C., and Beguinot, L. (1992) J. Biol. Chem. 267, 2714-2722 [Abstract/Free Full Text]
  19. Davidson, A., Mengod, G., Matus-Leibovitch, N., and Oron, Y. (1991) FEBS Lett. 284, 252-256 [CrossRef][Medline] [Order article via Infotrieve]
  20. West, A. P., and Cooke, B. A. (1991) Mol. Cell. Endocrinol. 79, R9-R14
  21. Listerud, M., Brussaard, A. B., Devay, P., Colman, D. R., and Role, L. W. (1991) Science 254, 1518-1521 [Medline] [Order article via Infotrieve]
  22. Soff, G. A., Jackman, R. W., and Rosenberg, R. D. (1991) Blood 77, 515-518 [Abstract]
  23. Knoller, S., and Savion, N. (1991) Eur. J. Biochem. 195, 801-806 [Abstract]
  24. Low, D. A., Scott, R. W., Baker, J. B., and Cunningham, D. D. (1982) Nature 298, 476-478 [Medline] [Order article via Infotrieve]
  25. Travo, P., Barrett, G., and Burnstock, G. (1980) Blood Vessels 17, 110-116 [Medline] [Order article via Infotrieve]
  26. Gibbons, G. H., Yang, Z., McDonald, T., and Patel, S. (1993) Clin. Res. 41, 143 (abstr.)
  27. Gimple, L. W., Gertz, S. D., Haber, H. L., Ragosta, M., Powers, E. R., Roberts, W. C., and Sarembock, I. J. (1992) Circulation 86, 1536-1546 [Abstract]
  28. Sarembock, I. J., Gertz, S. D., Gimple, L. W., Owen, R. M., Powers, E. R., and Roberts, W. C. (1991) Circulation 84, 232-243 [Abstract]
  29. Gorog, P. G., Semeria, F. J., and Gorog, D. A. (1993) Thromb. Hemostasis 69, 804
  30. Bar-Shavit, R., Benezra, M., Eldor, A., Hy-Am, E., Fenton, J. W. I., Wilner, G. D., and Vlodavsky, I. (1990) Cell Regul. 1, 453-463 [Medline] [Order article via Infotrieve]
  31. Zwaginga, J. J., de-Boer, H. C., IJsseldijk, M. J., Kerkhof, A., Muller-Berghaus, G., Gruhlichhenn, J., Sixma, J. J., and de-Groot, P. G. (1990) Arteriosclerosis 10, 437-448 [Abstract]
  32. Hung, D. T., Vu, T. K., Wheaton, V. I., Ishii, K., and Coughlin, S. R. (1992) J. Clin. Invest. 89, 1350-1353
  33. Detwiler, T. C., Chang, A. C., Speziale, M. V., Browne, P. C., Miller, J. J., and Chen, K. (1992) Semin. Thromb. Hemostasis 18, 60-66
  34. Schwartz, S. M., and Liaw, L. (1993) J. Cardiovasc. Pharmacol. 1, S31-S49 [Medline] [Order article via Infotrieve]
  35. Minter, A. J., Dawes, J., and Chesterman, C. N. (1992) Thromb. Hemostasis 67, 718-723
  36. Joseph-Silverstein, J., and Rifkin, D. B. (1987) Semin. Thromb. Hemostasis 13, 504-513
  37. Libby, P., Salomon, R. N., Payne, D. D., Schoen, F. J., and Pober, J. S. (1989) Transplant Proc. 21, 3677-3684 [Medline] [Order article via Infotrieve]
  38. Faulk, W. P., Labarrere, C. A., Pitts, D., and Halbrook, H. (1993) J. Heart Lung Transplant. 12, 219-229
  39. Dean, N. M., McKay, R., Condon, T. P., and Bennett, C. F. (1994) J. Biol. Chem. 269, 16416-16424

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