Ellen Bretschneider

Regulation of Thrombomodulin Expression in Human Vascular Smooth Muscle Cells by COX-2–Derived Prostaglandins

There is concern that cyclooxygenase (COX)-2 inhibitors may promote atherothrombosis by inhibiting vascular formation of prostacyclin (PGI2) and an increased thrombotic risk of COX-2 inhibitors has been reported. It is widely accepted that the prothrombotic effects of COX-2 inhibitors can be explained by the removal of platelet-inhibitory PGI2. Using microarray chip technology, we have previously demonstrated that thrombomodulin (TM) mRNA is upregulated in cultured human coronary artery smooth muscle cells by the stable prostacyclin mimetic iloprost. This study is the first to demonstrate a stimulation of the expression of functionally active thrombomodulin in human smooth muscle cells by prostaglandins, endogenously formed via the COX-2 pathway. Because TM is an important inhibitor of blood coagulation, these findings provide a novel platelet-independent mechanism to explain the prothrombotic effects of COX-2 inhibitors. The full text of this article is available online at http://circres.ahajournals.org.

Factor Xa Releases Matrix Metalloproteinase-2 (MMP-2) From Human Vascular Smooth Muscle Cells and Stimulates the Conversion of Pro–MMP-2 to MMP-2: Role of MMP-2 in Factor Xa–Induced DNA Synthesis and Matrix Invasion

Pro–matrix metalloproteinase-2 (pro–MMP-2) is expressed in vascular smooth muscle cells (SMCs). We report that activated coagulation factor X (FXa) induces the release of MMP-2 (65 kDa) from human SMCs. In addition, FXa cleaves pro–MMP-2 (72 kDa) into MMP-2. Pro–MMP-2 and MMP-2 were determined by gelatin zymography. MMP-2 was generated in conditioned medium containing pro–MMP-2 in a concentration-dependent fashion by FXa (3 to 100 nmol/L). FX at concentrations up to 300 nmol/L was ineffective. The conversion of pro–MMP-2 to MMP-2 was inhibited by a selective FXa inhibitor (DX-9065a) at 3 to 10 &mgr;mol/L. There was a concentration-dependent induction of an intermediate MMP-2 form (68 kDa) in lysates of FXa-treated cells. This indicates that cellular mechanisms are involved in FXa-induced conversion of pro–MMP-2. As a possible biological consequence of MMP-2 activation by FXa, DNA synthesis and matrix invasion of SMCs were determined. Both were stimulated by FXa and inhibited by the selective FXa inhibitor DX-9065a and the MMP inhibitor GM 6001 but not by hirudin or aprotinin. It is concluded that stimulation of SMCs by FXa increases the levels of MMP-2 in the extracellular space and that two different mechanisms are involved: release of active MMP-2 and cleavage of secreted pro–MMP-2. Both might contribute to the mitogenic potency of FXa and FXa-stimulated matrix invasion of SMCs.

Evidence for functionally active protease‐activated receptor‐4 (PAR‐4) in human vascular smooth muscle cells

This study investigates, whether in addition to the protease‐activated receptor‐1 (PAR‐1), PAR‐4 is present in vascular smooth muscle cells (SMC) of the human saphenous vein and whether this receptor is functionally active. PAR‐1 and PAR‐4 are stimulated by thrombin and by the synthetic peptides SFLLRN and GYPGQV, respectively. mRNAs for both, PAR‐1 and PAR‐4, were detected in the SMC by using reverse transcriptase polymerase chain reaction (RT – PCR). Treatment of the SMC with GYPGQV (200 μM) resulted in a transient increase in free intracellular calcium. This calcium signal was completely abolished after a preceding challenge with thrombin (10 nM), indicating homologous receptor desensitization. Stimulation of the SMC with 10 nM thrombin or 200 μM SFLLRN caused a time‐dependent activation of the extracellular signal‐regulated kinases‐1/2 (ERK‐1/2) with a maximum at 5 min. In contrast, 100 nM thrombin as well as 200 μM of GYPGQV induced a prolonged phosphorylation of ERK‐1/2 with a maximum at 60 min. These data suggest that PAR‐1 and PAR‐4 are activated by thrombin at distinct concentrations and with distinct kinetics. GYPGQV stimulated [3H]‐thymidine incorporation in SMC. At 500 μM, the peptide increased DNA synthesis 2.5 fold above controls. A comparable mitogenic effect was obtained after stimulation of the SMC by 10 nM thrombin or 100 μM SFLLRN, respectively. These data indicate that a functionally active PAR‐4 is present in SMC and, in addition to PAR‐1, might contribute to thrombin‐induced mitogenesis.

Evidence for functionally active protease-activated receptor-3 (PAR-3) in human vascular smooth muscle cells

The present study investigates whether vascular smooth muscle cells of the human saphenous vein (SMC) express a functionally active protease-activated receptor-3 (PAR-3). PAR-3 mRNA was detected by RT-PCR. In the presence of thrombin, a rapid and transient increase in PAR-3 mRNA was observed. Stimulation of SMC with thrombin or the synthetic PAR-3-activating peptide, TFRGAP, resulted in transient mobilization of intracellular calcium. After a preceding challenge with thrombin, the calcium signal to TFRGAP was abolished, suggesting cleavage and subsequent desensitization of PAR-3 by thrombin. Activation of PAR-3 by TFRGAP elicited a time-dependent activation of the extracellular-signal-regulated kinase (ERK)-1/2 with a maximum response 10-20 min after stimulation. At 200 microM, TFRGAP increased [3H]-thymidine incorporation into cellular DNA about two-fold. These data indicate that PAR-3 is expressed in human SMC and triggers intracellular signaling. Thus, in the SMC PAR-3 might contribute to thrombin-induced responses.

Evidence for proteinase-activated receptor-2 (PAR-2)-mediated mitogenesis in coronary artery smooth muscle cells.

This study investigates, whether in addition to the thrombin receptor (PAR‐1), the proteinase‐activated receptor‐2 (PAR‐2) is present in vascular smooth muscle cells (SMC) and mediates mitogenesis. PAR‐2 is activated by low concentrations of trypsin and the synthetic peptide SLIGRL. Stimulation of bovine coronary artery SMC by trypsin (2 nM) caused a 3 fold increase in DNLA‐synthesis. A similar effect was observed with 10 nM thrombin. Trypsin‐induced mitogenesis was inhibited by soybean trypsin inhibitor, indicating that the proteolytic activity of the enzyme was required for its mitogenic effect. The specific PAR‐2‐activating peptide SLIGRL or the PAR1‐activating peptide SFFLRN did not elicit mitogenesis. When the SMC were exposed to SLIGRL (40 nM), a homologous desensitization of cytosolic Ca2+ mobilization was found after subsequent stimulation with trypsin (40 nM) but not thrombin (15 nM). Trypsin (2 nM) as well as SLIGRL (100 μM) activated the nuclear factor κB (NFκB) with a maximum response 2 h after stimulation of the SMC. This suggests that both agonists acted via a common receptor, PAR‐2. Maximum activation of NFκB by thrombin (10 nM) was detected after 4–5 h. These data suggest that PAR‐2 is present in coronary SMC and mediates a mitogenic response. Activation of NFκB via either PAR‐1 or PAR‐2 does not predict mitogenesis.

Thrombin-Induced Mitogenesis in Coronary Artery Smooth Muscle Cells Is Potentiated by Thromboxane A2 and Involves Upregulation of Thromboxane Receptor mRNA

BACKGROUND Previous studies have shown that thrombin is a potent though slow-acting mitogen for vascular smooth muscle cells (SMC). Because thrombin generation in vivo is accompanied by platelet activation, it has been suggested that platelet-derived factors might enhance thrombin-induced SMC proliferation. No information is available so far on the possible role of thromboxane A2. METHODS AND RESULTS Thrombin (1 U/mL) caused a threefold to fourfold increase of DNA synthesis in cultured bovine coronary artery SMC as assessed from [3H]thymidine incorporation. U 46619, a stable thromboxane A2 mimetic, had only a minor stimulating effect on its own but potentiated the thrombin effect sixfold to sevenfold above control (P<.05). These findings were paralleled by a 52+/-5% (P<.05) increase in cell number at 48 hours after addition of both mitogens as compared with 24+/-5% with thrombin alone and no change with U 46619 alone. Thromboxane A2 receptor mRNA was found to be upregulated sixfold 20 minutes after thrombin stimulation. Pretreatment of SMC with thrombin for 4 hours markedly increased U 46619-induced mitogen-activated protein kinase activity, indicating thrombin-induced upregulation of functional thromboxane receptors in SMC. CONCLUSIONS Thrombin-induced proliferation of SMC is markedly enhanced by thromboxane A2. This might result in an enhancement of SMC proliferation by platelet-derived thromboxane A2 in vivo.

Human Vascular Smooth Muscle Cells Express Functionally Active Endothelial Cell Protein C Receptor

The endothelial cell protein C receptor (EPCR) is expressed on endothelial cells and regulates the protein C anticoagulant pathway via the thrombin-thrombomodulin complex. Independent of its anticoagulant activity, activated protein C (APC) can directly signal to endothelial cells and upregulate antiapoptotic and antiinflammatory genes. Here we show that vascular smooth muscle cells (SMCs) also express EPCR. EPCR protein on SMCs was detected by flow cytometry and Western blotting. EPCR mRNA was identified by quantitative RT-PCR. To examine the functionality of EPCR, intracellular signaling in APC-stimulated SMCs was analyzed by determination of intracellular free calcium transients using confocal laser scanning microscopy. Phosphorylation of extracellular signal–regulated kinases 1 and 2 (ERK-1/2) was detected by immunoblotting. APC-induced ERK-1/2 phosphorylation was inhibited by an anti-EPCR antibody and by a cleavage site blocking anti–PAR-1 antibody, indicating that binding of APC to EPCR and cleavage of protease-activated receptor-1 (PAR-1) were involved. APC elicited an increase in [3H]-thymidine incorporation. The mitogenic effect of APC was significantly enhanced in the presence of thrombin. EPCR expression was also detected in SMCs in the fibrous cap of human carotid artery plaques. The present data demonstrate functionally active EPCR in SMCs and suggest that EPCR-bound APC might modulate PAR-1-mediated responses of SMCs to vascular injury.

Thrombin receptors in vascular smooth muscle cells - function and regulation by vasodilatory prostaglandins

The vast majority of thrombin (>95%) is generated after clotting is completed, suggesting that thrombin formation serves purposes beyond coagulation, such as tissue repair after vessel injury. Two types of vascular thrombin binding sites exist: protease-activated receptors (PARs) and thrombomodulin (TM). Their expression is low in contractile vascular smooth muscle cells (SMC), the dominating subendothelial cell population, but becomes markedly up-regulated upon injury. In human SMC, PAR-1, PAR-3, and PAR-4 mediate thrombin-induced proliferation, migration and matrix biosynthesis as well as generation of inflammatory and growth-promoting mediators. Thrombin-responsive PARs are transcriptionally down-regulated in human vascular SMC by vasodilatory prostaglandins (PGI2/PGE2). For PAR-1 and PAR-3 this mechanism involves cAMP-dependent inactivation of the transcription factor NFAT. The human PAR-4 promoter does not possess NFAT recognition motifs suggesting involvement of other cAMP-regulated effectors. Unlike PARs, TM is induced in SMC exposed to vasodilatory prostaglandins. Enhanced thrombin binding to TM might ameliorate PAR-mediated SMC stimulation. Also expressed in human SMC is the endothelial protein C receptor (EPCR), which serves as an anchor to facilitate generation of activated protein C (aPC) by TM-bound thrombin. Whether prostaglandins affect aPC-generation is not known. In SMC, thrombin and aPC act synergistically via PAR-1 to modify tissue remodelling, in contrast to their antagonistic interaction in the coagulation pathways. Overall, this will contribute to plaque stability and wound healing. The processes outlined here are likely to become clinically relevant after up-regulation of vascular cyclooxygenase2, the rate limiting step in vascular PGE2/PGI2 biosynthesis, such as in advanced atherosclerosis and acute coronary syndromes.

ADP-, PAF- and adrenaline-induced platelet aggregation and thromboxane formation are not affected by a thromboxane receptor antagonist at physiological external Ca++ concentrations

Thromboxane (TX) receptor antagonists are of considerable clinical interest in prevention of acute thrombembolic vessel occlusion. This study demonstrates that the selective TX receptor antagonist, daltroban, at a concentration (10 microM) that does not inhibit TX synthesis, markedly inhibits ADP-, PAF- and adrenaline-induced platelet secretion and TX formation. With the exception of ADP-induced platelet secretion, these actions are only detectable in citrated platelet-rich plasma but not in plasma anticoagulated by hirudin. Since TX antagonists are supposed to act at physiological external Ca++ concentrations in the clinics, it is questionable whether in vitro studies in Ca(++)-deprived media are the optimum model to evaluate the clinical potential of these compounds.

High Glucose Enhances Thrombin Responses via Protease-Activated Receptor-4 in Human Vascular Smooth Muscle Cells

Objective—Diabetes is associated with vascular remodeling and increased thrombin generation. Thrombin promotes vascular smooth muscle cell (SMC) mitogenesis and migration via protease-activated receptors (PAR)-1, PAR-3, and PAR-4. We investigated the effect of high glucose on expression and function of vascular thrombin receptors. Methods and Results—In human vascular SMCs, high glucose (25 versus 5.5 mmol/L) induced a rapid and sustained increase in PAR-4 mRNA, protein, and cell surface expression. PAR-1 and PAR-3 expression were not changed. High glucose pretreatment (48 hours) enhanced thrombin or PAR-4-activating peptide but not PAR-1-activating peptide evoked intracellular calcium mobilization, migration, and tumor necrosis factor &agr; gene expression. This enhancement of thrombin-stimulated migration and gene expression by high glucose was abolished by endogenous PAR-4 knockdown.PAR-4 regulation was prevented by inhibition of protein kinase (PK)C-&bgr; and -&dgr; isoforms or nuclear factor (NF)&kgr;B. Nuclear translocation of NF&kgr;B in high glucose-stimulated SMCs led to PKC-dependent NF&kgr;B binding to the PAR-4 promoter in a chromatin immunoprecipitation assay. Furthermore, in situ hybridization and immunohistochemistry confirmed high abundance of PAR-4 in human diabetic vessels as compared with nondiabetic vessels. Conclusion—High glucose enhances SMC responsiveness to thrombin through transcriptional upregulation of PAR-4, mediated via PKC-&bgr;, -&dgr;, and NF&kgr;B. This may play an important role in the vascular complications of diabetes.