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(Circulation Research. 1995;77:503-509.)
© 1995 American Heart Association, Inc.

Articles

Thrombin Regulates Expression of Monocyte Chemoattractant Protein-1 in Vascular Smooth Muscle Cells

Ulrich O. Wenzel, Bruno Fouqueray, Giuseppe Grandaliano, Yong-Soo Kim, Costantinos Karamitsos, Anthony J. Valente, Hanna E. Abboud

From the Departments of Medicine and Pathology, The University of Texas Health Science Center at San Antonio.

Correspondence to Hanna E. Abboud, MD, Department of Medicine, The University of Texas Health Science Center at San Antonio, Floyd Curl Dr, San Antonio, TX 78284-7882.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Thrombin, a serine protease generated at sites of vascular injury, plays a role in the pathogenesis of atherosclerosis and restenosis after angioplasty. Adherence of monocytes to the endothelium and migration into the subendothelial space is an important early event in the pathogenesis of atherosclerosis. Monocyte chemoattractant protein 1 (MCP-1) may be an important mediator of monocyte recruitment to the tissue in this and other diseases. We have characterized the expression of MCP-1 in vascular smooth muscle cells (VSMCs) isolated from human renal artery and studied its regulation by thrombin. Serum-deprived cells release monocyte chemotactic activity that is neutralized (80%) by an MCP-1 antibody. The antibody recognized a 13- and 15-kD protein in smooth muscle cell–conditioned medium. Thrombin stimulates MCP-1 gene expression in a concentration- and time-dependent manner. An increase over basal levels was observed with concentrations of thrombin as low as 0.05 U/mL. The maximal effect occurred at 5 U/mL. The stimulatory effect was detected within 1 hour, reached a maximum at 3 hours, and was still present at 8 to 24 hours after the addition of thrombin. A concentration- and time-dependent effect of thrombin on MCP-1 gene expression was also found in rat VSMCs. The thrombin protease inhibitor hirudin blocked thrombin-induced MCP-1 expression. Thrombin stimulated the release of MCP-1 protein in conditioned medium of human VSMCs as measured by radioimmunoassay and chemotactic assay. Thrombin also increased monocyte chemotactic activity in short-term organ cultures of rat aortic rings and in first passage cells. The effect of thrombin on MCP-1 was mimicked by a thrombin receptor–activating peptide (NH₂-Ser-Phe-Leu-Leu-Arg-Asn-Pro-COOH). These data describe an important biological activity of thrombin in VSMCs and provide a novel mechanism whereby locally released thrombin may contribute to the pathogenesis of atherosclerosis or restenosis after angioplasty.

Key Words: chemotaxis • monocytes • thrombin receptor • thrombin receptor–activating peptide • hirudin

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Thrombin is a multifunctional serine protease that elicits biological responses in a variety of cells. In addition to its critical role in thrombosis and hemostasis, thrombin exhibits multiple cell-activating functions potentially relevant to the biology of the vessel wall. It is a mitogen for numerous cell types, stimulates platelet-derived growth factor production, and increases endothelial cell permeability.^{1 2 3} Thrombin is also a chemotactic factor for monocytes and stimulates monocyte adhesion to endothelial cells.⁴ Accumulating evidence indicates that thrombin may play a critical role in the development of atherosclerosis and in the pathogenesis of restenosis that follows balloon angioplasty.^{5 6} Recent studies identifying a thrombin receptor with a novel form of activation^{7 8 9} and the observation that the thrombin receptor is widely expressed within human atherosclerotic lesions¹⁰ have stimulated interest in the role of thrombin in atherosclerosis. Thus, thrombin as a potent mitogen for cultured VSMCs¹¹ and chemoattractant for monocytes may mediate some of the pathological responses observed in the injured vessel wall. Migration of monocytes may be particularly important in atherogenesis and vascular remodeling, since once in the vessel wall, monocytes represent a reservoir and a source of inflammatory mediators, including arachidonic acid, reactive oxygen metabolites, proteolytic enzymes, nitric oxide, and a variety of cytokines and growth factors promoting activation of resident cells.^{6 12}

MCP-1 is a potent chemotactic factor for monocytes, basophils, and a subset of peripheral blood lymphocytes.¹³ In vitro studies have demonstrated that the expression of MCP-1 is induced by inflammatory cytokines in monocytes and endothelial, mesangial, and smooth muscle cells.^{14 15 16 17} Moreover, MCP-1 is expressed in the vessel wall in vivo.¹⁶

We report that human VSMCs produce MCP-1 and that thrombin enhances the expression of MCP-1 mRNA and protein production in VSMCs by a specific receptor.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Tissue culture reagents were obtained from GIBCO-BRL. All other reagents were analytic grade. Human -thrombin (specific activity, 4000 U/mg) and hirudin were purchased from Sigma Chemical Co. [³²P]CTP (3000 Ci/mmol) was obtained from New England Nuclear, and ¹²⁵I–Bolton-Hunter reagent was from ICN Radio-chemicals. TRAP (NH₂-Ser-Phe-Leu-Leu-Arg-Asn-Pro-COOH) was synthesized by using 9-fluorenylmethoxycarbonyl chemistry on an Applied Biosystems 430A peptide synthesizer at The University of Texas Health Science Center Institutional Protein Core Facility and purified by using reverse-phase high-performance liquid chromatography. The purified peptide was verified by using amino acid composition and mass spectrometry. Thrombin and TRAP were tested for endotoxin content by the limulus amebocyte lysate assay (Association of Cape Cod, Inc) and found to contain <0.1 ng/mL lipopolysaccharide.

VSMC Culture
VSMC cultures were established from small segments of renal arteries from human donor kidneys judged unsuitable for transplantation. The VSMCs were isolated according to the method of Ross.¹⁸ Renal arteries were placed in cold HBSS with calcium and magnesium. Fat and connective tissue were discarded. Thereafter, the vessels were opened longitudinally, the adventitia was stripped off with forceps and gauze, and the luminal side was scraped with a scalpel blade to remove the endothelium. Pieces of 3 to 4 mm² were cut from the vessel and washed, and the explants were placed in DMEM supplemented with 15 mmol/L HEPES and antibiotic/antifungal solution in the presence of 10% FBS. Cells were cultured at 37°C in 5% CO₂/95% air. VSMCs migrated from the explant after 1 to 2 weeks in culture. After the first passage, cells were switched to DMEM supplemented with (mmol/L) HEPES 15, glutamine 2, nonessential amino acids 0.1, and sodium pyruvate 1, along with antibiotic/antifungal solution and 17% FBS (cells isolated from donor kidney 1) or 10% FBS (cells from donor kidneys 2 and 3). No qualitative differences were observed between strains in response to thrombin. Medium was routinely changed every 3 to 4 days. Cells were passaged after brief exposure to HBSS containing trypsin (0.5 mg/mL) and EDTA (0.5 mmol/L) and split at a ratio of 1:2 or 1:3. Cells were used between passages 4 and 12. VSMCs were made quiescent by incubation in serum-free DMEM for 36 hours. The cultured cells were characterized as VSMCs by a "hill-and-valley" growth pattern at confluence and positive immunostaining with monoclonal mouse antihuman smooth muscle -actin antibodies (Sigma). The cells exhibited abundant stress fibers running through the cytoplasm. The rat aortic VSMCs (a kind gift of Dr A. Hahn, Basel, Switzerland) were kept in MEM supplemented with 8 mmol/L HEPES, 8 mmol/L TES, antibiotic/antifungal solution, and 10% FBS as previously described.¹⁹ First-passage cells were isolated from the aortas of male Sprague-Dawley rats (Harlan, Indianapolis, Ind). The rats were killed by decapitation, and the thoracic aortas were removed. Clotted blood, fat, and connective tissue were discarded. Aortic rings were cut, carefully washed, and placed in MEM supplemented with 10% FBS as described above. The outgrowing cells exhibited typical morphological characteristics of VSMCs in vitro. The experiments were performed when the first outgrowth reached confluence. After removing nonadherent aortic pieces, the cell layer was incubated in serum-free medium for 24 hours, followed by the addition of thrombin. For short-term organoid cultures, aortic rings were prepared as described above and placed in 12-well tissue-culture plates containing serum-free MEM. After collection of the conditioned medium, the tissue was dried in a Speed Vac concentrator (Savant), and the dry weight was measured. Differences in dry weight between control and thrombin-treated organoid cultures were <15% in all experiments.

Monocyte Chemotactic Activity
Monocyte chemotactic activity was determined in modified Boyden chambers by using freshly prepared human peripheral blood mononuclear cells as previously described.^{14 20} VSMCs were grown to confluence, rinsed, and incubated in serum-free medium. Medium was collected at indicated time points, clarified by centrifugation at 10 000g for 2 minutes, and stored at -70°C until analysis. After a 30-minute incubation at 37°C with or without a 1:100 dilution of rabbit antibody to MCP-1, samples were assayed for monocyte chemotactic activity. The antibody has been previously described and extensively characterized.^{20 21} Monocyte chemotactic activity released by human VSMCs, first-passage cells, and organoid cultures in response to thrombin was determined as previously described.²² Cells or organoid cultures were exposed to thrombin (5 U/mL) for 4 hours. The medium containing thrombin was then removed and replaced with fresh serum-free medium without thrombin. Incubation was continued for 20 hours. Conditioned medium was harvested, clarified by centrifugation at 10 000g for 2 minutes, and stored at -70°C until analysis. Chemotactic activity is expressed as the mean number of monocytes migrating per field in 10 high-power fields. Background migration in response to nonconditioned medium varied between 1.3±0.6 and 3.1±0.6 monocytes per high-power field and was subtracted from control and stimulated values, except in Fig 1, left.

View larger version (22K): [in this window] [in a new window]   Figure 1. Left, Inhibition of VSMC-derived monocyte chemotactic activity by MCP-1 antibodies. Serum-free medium conditioned for 48 hours by confluent human VSMCs was incubated with or without a 1:100 dilution of rabbit antiserum to MCP-1 for 30 minutes at 37°C. The conditioned medium was assayed for monocyte chemotactic activity as described. Medium indicates background migration in response to nonconditioned medium. Experiments were carried out in triplicate. Similar inhibition was also found in two other experiments. Right, Characterization of MCP-1 secreted by human VSMCs. Serum-free medium conditioned by VSMCs for 2 days was fractionated by SDS-PAGE. After transfer to nitrocellulose, the blot was incubated with rabbit antiserum to MCP-1, and bands were visualized by luminescence immunodetection as described in "Materials and Methods." Numbers on the right represent sizes in kilodaltons. The figure is representative of two experiments.

Immunoblotting of MCP-1
VSMCs in culture were grown to confluence, rinsed, and incubated in serum-free medium for 48 hours. Conditioned medium was concentrated by using a Maxi-Clean C8 cartridge (Alltech). After loading onto the column and washing with 15% acetonitrile in 0.1% TFA, bound proteins were eluted with 70% acetonitrile in 0.1% TFA and lyophilized. For electrophoresis, the protein was dissolved in SDS sample buffer containing 10% 2-mercaptoethanol, boiled for 2 minutes, and separated on 15% polyacrylamide gels. Separated proteins were transferred electrophoretically to polyvinyl membrane. Blocking was performed in 5% nonfat dry milk prepared in PBS containing 0.1% Tween 20, and MCP-1 protein was identified immunochemically by incubating the membrane sequentially in rabbit antiserum to baboon MCP-1 (1:250 dilution) and horseradish peroxidase–conjugated goat anti-rabbit IgG. The signal was developed with luminescence immunodetection (ECL Western Blotting System, Amersham Co).

RNA Isolation and Northern Blotting
Confluent VSMCs were incubated in serum-free medium for 36 hours before exposure to thrombin or other agents. RNA was isolated by one-step guanidinium thiocyanate–phenol–chloroform extraction. RNA quantification was determined by absorbance at 260 nm. RNA quantification and integrity were confirmed by 1% agarose gel electrophoresis and ethidium bromide staining. Total RNA was fractionated by electrophoresis on a 1% agarose-formaldehyde gel and blotted by capillary transfer on a nylon membrane (Gene Screen, New England Nuclear) and cross-linked by UV irradiation (UV Strata-linker 1800, Stratagene). The membrane was probed with baboon MCP-1 cDNA labeled by random priming using a commercial kit (Amersham) and [³²P]dCTP. Blots were prehybridized at 42°C for 2 hours in 50% formamide, 0.1% SDS, 2x Denhardt's solution, 5x SSPE, and 0.1 mg/mL salmon sperm DNA. Probe (10⁶ cpm/mL) was added to the prehybridization solution, and the blot was hybridized for 16 hours at 42°C. Blots were then washed (twice for 15 minutes each at 55°C in 2x standard saline citrate and 0.1% SDS), and autoradiography was performed with Kodak x-ray film and intensifying screen at -70°C. The MCP-1 probe was then removed by boiling, and the same blot was rehybridized to a cDNA probe encoding for the ribosomal protein 36B4. Intensity of the MCP-1 and 36B4 signal was measured in selected experiments by phosphoimage analysis (Ambis).

MCP-1 Protein
To determine whether thrombin and TRAP stimulate MCP-1 protein production, conditioned medium was assayed for MCP-1 protein by a specific and sensitive radioimmunoassay recently described by us.^{14 23 24} Cells were grown to confluence, washed with PBS, and incubated in serum-free medium with thrombin or TRAP for 24 hours. Conditioned medium was clarified by centrifugation at 10 000g for 2 minutes and stored at -70°C until analysis. All determinations were made in duplicate; replicate values varied by <10%.

Statistical Analysis
The data are presented as mean±SEM. Statistical analysis was performed by Student's t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of Monocyte Chemotactic Activity Produced by VSMCs
Confluent cultures of human VSMCs secrete monocyte chemotactic activity into the medium in a time-dependent manner. There was a progressive increase in chemotactic activity up to at least 48 hours of incubation. Chemotactic activity at 8, 24, and 48 hours was 12.0±0.7, 20.7±1.6, and 53.6±1.6 monocytes per high-power field, respectively. Antibody neutralization studies were carried out to determine the contribution of MCP-1 to the chemotactic activity. After incubation with specific rabbit antiserum to MCP-1, chemotactic activity in the medium conditioned by the cells for 48 hours was inhibited by 80% (Fig 1, left). Normal rabbit serum had no inhibitory effect, as shown previously by us.¹⁴ Thus, MCP-1 is responsible for the bulk of the monocyte chemotactic activity produced by the human VSMCs in culture. To further characterize MCP-1 produced by cultured human VSMCs, conditioned medium was analyzed by SDS-PAGE and immunoblotting. Two major protein bands of apparent molecular masses of 13 and 15 kD were identified with antiserum specific to MCP-1 (Fig 1, right). This molecular heterogeneity of MCP-1 has been observed in other cell types. This heterogeneity is most likely due to posttranslational modification of MCP-1 involving the addition of O-linked carbohydrates and sialic acid to the protein core, which has a molecular mass of 9 kD.²¹

Thrombin Stimulates Expression and Secretion of MCP-1
The increased level of MCP-1 gene transcripts in thrombin-treated VSMCs was demonstrated by Northern blot analysis. Unstimulated serum-deprived human VSMCs express low levels of MCP-1 mRNA. When VSMCs were exposed to thrombin (5 U/mL), there was a rapid accumulation of MCP-1 mRNA, which was detected at 1 hour, reached a maximum at 3 hours, and remained elevated for 8 to 24 hours (Fig 2, top left). Induction after 24 hours was not seen in all experiments. As shown in Fig 2, top center, an increase over basal values was observed at 0.05 U/mL thrombin. The maximal effect occurred with 5 U/mL. To determine whether induction of MCP-1 in response to thrombin is specific for VSMC lines isolated from human renal arteries, we examined the effect of thrombin in a rat aortic VSMC line (a kind gift of Dr A. Hahn, Basel, Switzerland). The top right and bottom left panels of Fig 2 show that thrombin induces MCP-1 gene expression in a time- and concentration-dependent manner. Increased MCP-1 mRNA was found 1 hour after treatment with thrombin and persisted for up to 8 hours. An increase over basal value was observed at 0.05 U/mL thrombin, and the maximal effect occurred at 5 U/mL, confirming the results obtained in human VSMCs isolated from the renal artery and indicating that induction of MCP-1 by thrombin is not species specific. Since cultured VSMCs may undergo phenotypic modulation with increasing passage,²⁵ we tested the effect of thrombin on MCP-1 mRNA expression in primary cell outgrowth of aortic rings. Fig 2, bottom center, shows that thrombin also induces MCP-1 mRNA expression in these primary cultures. IL-1 is a potent inducer of MCP-1 in several cell lines. It is at least fourfold more potent than thrombin in stimulating MCP-1 gene expression (Fig 2, bottom right), as reported recently in endothelial cells.²²

View larger version (39K): [in this window] [in a new window]   Figure 2. Top left, Northern blot analysis of MCP-1 gene expression in response to thrombin (Thr) in human VSMCs: time course. Confluent human VSMCs were incubated in serum-free medium for 36 hours, followed by the addition of 5 U/mL Thr. After 1, 3, 8, and 24 hours, the cells were harvested, and RNA was isolated. For Northern blot analysis, 15 µg of total RNA was fractionated on a 1% agarose formaldehyde gel, blotted on a nylon membrane, and hybridized with a baboon cDNA encoding for MCP-1. The probe was removed by boiling, and the blot was rehybridized to a 36B4 cDNA. The figure shows one representative experiment. Similar results were seen in three other experiments. Con indicates control. Top center, Northern blot analysis of MCP-1 gene expression in response to different concentrations of Thr in human VSMCs. Confluent VSMCs were incubated in serum-free medium for 36 hours, followed by the addition of the indicated concentrations of Thr. Cells were harvested after 3 hours, and Northern blot analysis was performed as described for the top left panel. Similar results were observed in two other experiments. Top right, Northern blot analysis of MCP-1 gene expression in response to Thr in rat aortic VSMCs: time course. Confluent rat VSMCs were incubated in serum-free medium for 36 hours, followed by the addition of 5 U/mL Thr. At the indicated time points, the cells were harvested, and Northern blot analysis was performed as described for the top left panel. The figure is representative of three separate experiments. Bottom left, Northern blot analysis of MCP-1 gene expression in response to different concentrations of Thr in rat VSMCs. Confluent VSMCs were incubated in serum-free medium for 36 hours, followed by the addition of the indicated concentrations of Thr. Cells were harvested after 3 hours, and Northern blot analysis was performed as described for the top left panel. The figure is representative of two separate experiments. Bottom center, Northern blot analysis of MCP-1 gene expression in response to Thr in first-passage cells derived from rat aortic rings: time course. Confluent outgrowing cells were incubated in serum-free medium for 24 hours, followed by the addition of 5 U/mL Thr. At the indicated time points, the cells were harvested, and Northern blot analysis was performed as described for the top left panel. The figure is a representative of three experiments. Bottom right, Northern blot analysis of MCP-1 gene expression in response to IL-1 and Thr. Confluent cells were incubated in serum-free medium for 36 hours, followed by the addition of 5 U/mL IL-1 or 5 U/mL Thr. Cells were harvested after 3 hours, and Northern blot analysis was performed as described for the top left panel.

To test whether the increase in MCP-1 mRNA levels in response to thrombin is associated with an increase in protein production and secretion, MCP-1 protein levels were measured by a specific and sensitive radioimmunoassay. As shown in Fig 3, top left, secretion of MCP-1 was significantly increased after stimulation with thrombin. The basal secretion of MCP-1 is consistent with a low but detectable MCP-1 mRNA in VSMCs cultured under serum-free conditions and is in agreement with the release of chemotactic activity by serum-deprived cells. The relatively modest increase in MCP-1 in response to thrombin when compared with the marked elevation of mRNA encoding MCP-1 implies that the synthesis of the protein is not tightly linked to mRNA accumulation. We find this dissociation not only in response to thrombin but also with other inducers of MCP-1, such as IL-1, tumor necrosis factor-, and interferon gamma, in at least two cell types, human mesangial cells and fat-storing cells.^{14 23} To test whether the increase in secretion of immunoreactive MCP-1 in response to thrombin is associated with an increase in monocyte chemotactic activity, we measured the chemotactic activity present in the conditioned medium. Conditioned medium from thrombin-stimulated human VSMCs expressed significantly higher levels of monocyte chemotactic activity than medium from control cells (Fig 3, top right). Similar results were obtained in the first-passage rat cells. As shown in Fig 3, bottom left, conditioned medium from thrombin-stimulated first-passage cells had significantly more monocyte chemotactic activity than control medium (P<.05). We also measured chemotactic activity released from short-term organoid cultures of freshly isolated blood vessels after treatment with thrombin. The short-term organoid culture approach used here permits study of vascular tissue in the context of its normal matrix at a state much closer to the in vivo condition than the cultured cells. Thrombin significantly increased the release of monocyte chemotactic activity in rat aortic rings, as shown in Fig 3, bottom right (P<.03). Antibody neutralization studies were carried out to determine the contribution of MCP-1 to the increased chemotactic activity in response to thrombin. After incubation with rabbit antiserum to MCP-1, chemotactic activity in the medium conditioned by the human VSMCs was inhibited by 70% (25.8±1.8, 47.6±3.1, and 15.4±1.3 monocytes per high-power field for control, thrombin, and thrombin+antibody, respectively). Thus, MCP-1 is responsible for most of the monocyte chemotactic activity produced by human VSMCs in response to thrombin.

View larger version (21K): [in this window] [in a new window]   Figure 3. Top left, MCP-1 production. VSMCs were grown to confluence in multiwell plates. The cells were rinsed and incubated in serum-free medium with and without thrombin (5 U/mL). After 24 hours, the medium was collected and assayed for MCP-1 by radioimmunoassay as described. Values are mean of four independent experiments performed in duplicate or triplicate wells. *P<.002. Top right, Monocyte chemotactic activity of human VSMCs in response to thrombin. Confluent VSMCs were washed and incubated in serum-free medium. Cells were exposed to thrombin (5 U/mL) for 4 hours. The medium was changed, and the incubation was continued for 20 hours. Conditioned medium was harvested, clarified by centrifugation at 10 000g for 2 minutes, and stored at -70°C until analysis. Human monocyte chemotactic activity was determined in chemotaxis chambers. Chemotactic activity is expressed as the mean number of monocytes migrating per field in 10 high-power fields. Values are mean of three experiments carried out in duplicate. *P<.05. Bottom left, Monocyte chemotactic activity of first-passage cells in response to thrombin. Confluent first passage cells were washed and incubated in serum-free medium. Cells were exposed to thrombin (5 U/mL) for 4 hours. The medium was changed, and the incubation was continued for 20 hours. Chemotactic activity was determined as described for the top right panel. Values are mean of four experiments performed in duplicate samples. *P<.05. Bottom right, Monocyte chemotactic activity of short-term organoid cultures in response to thrombin. Freshly isolated rat aortic rings were washed and incubated in serum-free medium. Cells were exposed to thrombin (5 U/mL) for 4 hours. The medium was changed, and the incubation was continued for 20 hours. Chemotactic activity was determined as described for the top right panel. Values are mean of four experiments performed in duplicate samples. *P<.03.

Hirudin and TRAP
The thrombin protease inhibitor hirudin binds to the thrombin anion exosite and also blocks its catalytic site. Incubation of thrombin with hirudin before treating the VSMCs blocked thrombin-induced increase in MCP-1 expression (Fig 4, left). This suggests that the anion-binding exosite and catalytic activity of thrombin is required for the induction of the MCP-1 gene. This result agrees with other studies showing that chemical modification of thrombin that reduces binding or proteolytic activity also inhibits many but not all of its biological effects on cells.

View larger version (28K): [in this window] [in a new window]   Figure 4. Left, Inhibition of thrombin (Thr)–induced MCP-1 gene expression by the Thr antagonist hirudin (Hiru). Confluent VSMCs were incubated in serum-free medium for 36 hours, followed by the addition of Thr (5 U/mL) or Thr (5 U/mL) preincubated with the inhibitor Hiru at a ratio of 3 Thr-inactivating units to 1 U Thr or Hiru alone. All three preparations were incubated for 30 minutes at 37°C before addition to the cells. Cells were harvested after 3 hours, and Northern blot analysis was performed as described for Fig 2, top left panel. Similar results were observed in two separate experiments. Center, Northern blot analysis of MCP-1 gene expression in response to TRAP. Confluent VSMCs were made quiescent in serum-free medium for 36 hours and incubated with Thr (5 U/mL) or TRAP (100 µmol/L). Cells were harvested after 3 hours, and Northern blot analysis was performed as described for Fig 2, top left panel. This blot is representative of three experiments. Right, Hiru does not inhibit induction of MCP-1 by TRAP. Confluent VSMCs were incubated in serum-free medium for 36 hours, followed by the addition of TRAP (100 µmol/L) or TRAP preincubated with Hiru (15 U/mL). Preparations were incubated for 30 minutes at 37°C before addition to the cells. Cells were harvested after 3 hours, and Northern blot analysis was performed as described for Fig 2, top left panel.

We next tested whether the induction of MCP-1 by thrombin is mediated by a receptor similar to that described by Vu et al.⁷ Addition of the thrombin receptor agonist peptide TRAP increased MCP-1 mRNA levels in human VSMCs, as shown in Fig 4, center. The increase in MCP-1 mRNA levels was also associated with a significant increase in protein secretion (control, 3.0±0.2 ng/mL; TRAP, 6.5±1.2 ng/mL; mean of two experiments performed in triplicate; P<.02). These data suggest that thrombin's effect on MCP-1 production is mediated by a receptor similar to the recently cloned thrombin receptor. As expected, preincubation of TRAP with hirudin before the addition to VSMCs did not interfere with the induction of MCP-1 by TRAP (Fig 4, right).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Thrombin acts on a variety of cell types to orchestrate hemostatic, inflammatory, and proliferative or reparative response to injury.²⁶ In the present study, we characterize a major monocyte chemotactic activity in human VSMCs and describe a novel biological activity of thrombin in these cells. VSMCs release monocyte chemotactic activity that exhibits the physiochemical properties of MCP-1 and which can be inhibited >80% by an MCP-1 antibody. Thrombin, acting via a specific receptor, induces MCP-1 expression in human VSMCs at both the mRNA and the protein level.

One of the important early events in the pathogenesis of atherosclerosis is the adherence of monocytes to the endothelium, followed by their migration into the subendothelial space. The entry between endothelial cells is presumably in response to a gradient of one or more chemotactic factors derived from cells present within the arterial wall.^{27 28} MCP-1 is a potent chemotactic factor for monocytes, basophils, and a subset of lymphocytes.¹³ MCP-1 mRNA and protein have been identified in vivo within atherosclerotic lesions in humans^{16 29} and hypercholesterolemic primates,³⁰ in transplant arteriosclerosis,³¹ and within the proliferative intimal lesions associated with balloon injury.^{29 32} Interestingly, by use of immunohistochemistry, macrophages and smooth muscle cells were identified as the predominant MCP-1 antigen–positive cells in human atherosclerotic lesions and abdominal aneurysms.³³ Our in vitro data show that the majority of the monocyte chemotactic activity produced by human VSMCs can be attributed to MCP-1.

VSMCs in the vessel wall are separated from the lumen by the endothelial cell layer. Thrombin is produced at sites of endothelial injury. In early stages of atherosclerosis or after balloon angioplasty, endothelial injury may facilitate contact between thrombin and the VSMC layer of the arterial wall. Atherosclerotic lesions are areas of increased permeability with increased passage of plasma proteins into the vessel wall. Moreover, thrombin has been shown to induce gap formation between adjacent endothelial cells³⁴ and may thus migrate through the intact endothelial layer and reach subendothelial structures. Bar Shavit et al³⁵ recently demonstrated that thrombin can be immobilized onto the subendothelial extracellular matrix in a manner that leaves the molecule functionally active, indicating that thrombin may be present within the vessel wall exhibiting local activity. Accumulating evidence indicates that in atherosclerosis, activation of VSMCs can occur within the vessel wall under conditions in which the endothelium appears to be intact.¹² It is noteworthy that the amounts of thrombin, which induce MCP-1 in vitro in the present study, compare well with concentrations found in vivo. Vascular injury by balloon angioplasty results in activation of the coagulation cascade, and locally produced thrombin can achieve a concentration of 140 nmol/L (14 U/mL).³⁶

The cloned thrombin receptor belongs to the seven-transmembrane-spanning class of receptors linked to G proteins. Receptor activation occurs by cleavage of the N-terminal extracellular domain of the receptor by thrombin. The new exposed N terminus acts as a tethered peptide ligand and activates the receptor. Accordingly, receptor activation can be mimicked by synthetic peptides corresponding to the new N terminus. Whether the cloned functional receptor is responsible for all the cellular effects of thrombin remains controversial.³⁷ TRAP is as effective as thrombin in inducing MCP-1 in VSMCs, indicating that this effect is mediated by a receptor similar to the recently cloned thrombin receptor.^{7 8 9 10} Further confirmation was provided by Northern analysis, which demonstrated that cultured human VSMCs used in the present study expressed a single mRNA species of 3.5 kb when hybridized to a cDNA of the cloned human thrombin receptor (data not shown). In this connection, it is of interest that Soifer et al³⁸ found robust thrombin receptor expression during embryonal development before prothrombin mRNA was detected. This raises the question whether other proteases or peptide ligands can activate the thrombin receptor.

Our results are at variance with a recent study by Taubmann et al,³² who found no effect of human thrombin on JE mRNA expression in rat aortic VSMCs. This gene encodes a glycoprotein whose amino acid sequence is homologous to that of MCP-1. This discrepancy cannot be attributed to species specificity, since we found that thrombin also induced MCP-1 in rat VSMCs. Our studies were performed in serum-free medium, whereas those by Taubmann et al were carried out in the presence of 0.5% calf serum. Serum is known to contain thrombin inhibitors, such as antithrombin III,³⁹ which may have inactivated thrombin.

Injury to the vessel wall results in changes that are mediated by both inflammatory and coagulation pathways. Our in vitro data suggest that thrombin, in addition to its critical role in coagulation, could contribute to the inflammatory component of vascular response to injury by induction of the potent and monocyte-specific chemoattractant MCP-1 in the cells of the vessel wall. That induction of MCP-1 by thrombin may play a role in the inflammatory response to tissue injury in general and not only in atherosclerosis is suggested by observations that thrombin induces MCP-1 in mesangial and endothelial cells also.^{22 40} Additional studies are needed to determine the role of MCP-1 in mediating the effects of thrombin in vivo. In vivo intervention studies to block the biological activity of thrombin, ie, with hirudin, will be needed to evaluate the significance of thrombin-induced MCP-1 expression in atherogenesis.

These data describe a novel biological activity of thrombin in human and rat VSMCs and provide a new mechanism whereby locally released thrombin may contribute to atherosclerosis or restenosis after angioplasty.

	Selected Abbreviations and Acronyms

 

FBS = fetal bovine serum IL-1 = interleukin-1 MCP-1 = monocyte chemoattractant protein-1 TFA = trifluoroacetic acid TRAP = thrombin receptor–activating protein VSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was supported in part by the VA Medical Research Service and National Institutes of Health grants DK-33665, DK-43988, and HL-26890. Dr Wenzel is supported by the German Research Foundation. Dr Fouqueray was supported by a grant from Foundation Pour la Recherche Medicale, France. The authors also acknowledge Kathleen Woodruff and Sergio Garcia for their expert technical help.

Received March 20, 1995; accepted May 19, 1995.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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