Anke C. Rosenkranz

Regulation of Functionally Active P2Y12 ADP Receptors by Thrombin in Human Smooth Muscle Cells and the Presence of P2Y12 in Carotid Artery Lesions

Objective—The platelet P2Y12 ADP receptor is a well-known target of thienopyridine-type antiplatelet drugs. This study is the first to describe increased transcriptional expression of a functionally active P2Y12 in response to thrombin in human vascular smooth muscle cells (SMC). Methods and Results—On exposure to thrombin, P2Y12 mRNA was transiently increased, whereas total protein and cell surface expression of P2Y12 were markedly increased within 6 hours and remained elevated over 24 hours. This effect was mediated by activation of nuclear factor &kgr;B. Preincubation with thrombin significantly enhanced the efficacy of the P2Y receptor agonist 2-methylthio-ADP to induce interleukin 6 expression and SMC mitogenesis. Effects induced by 2-methylthio-ADP were prevented by RNA interference-mediated knockdown of P2Y12 and a selective P2Y12-antagonist R-138727, the active metabolite of prasugrel. In addition, positive P2Y12 immunostaining was shown in SMC of human carotid artery plaques and was found to colocalize with tissue factor, the rate-limiting factor of thrombin formation in vivo. Conclusion—These data suggest that the P2Y12 receptor not only is central to ADP-induced platelet activation but also may mediate platelet-independent responses, specifically under conditions of enhanced thrombin formation, such as local vessel injury and atherosclerotic plaque rupture.

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.

Transcriptional Inhibition of Protease-Activated Receptor-1 Expression by Prostacyclin in Human Vascular Smooth Muscle Cells

Objective—Stimulation of protease-activated receptor-1 (PAR-1) by thrombin causes vascular smooth muscle cell (SMC) mitogenesis and has been implicated in the vascular response to injury. Vascular injury is also associated with enhanced formation of PGE2 and PGI2 (prostacyclin). This study investigates whether PGI2 and PGE2 modify the expression of PAR-1 and the cellular response to thrombin in human SMC. Methods and Results—The PGI2-mimetic iloprost (1 to 100 nmol/L) attenuated mRNA, total protein, and cell surface expression of PAR-1. This was associated with inhibition of thrombin-induced mitogenesis and migration. Comparable inhibition of PAR-1 expression was observed with the selective IP-receptor agonist cicaprost, the adenylyl cyclase activator forskolin, the phosphodiesterase inhibitor isobutylmethylxanthine and the PKA activator dibutyryl-cAMP. Similar effects of PGE2 required micromolar concentrations. The specific PKA-inhibitor Myr-PKI prevented PAR-1 downregulation by iloprost. The potential role of Rho family GTPases in PAR-1 regulation was also investigated. Iloprost decreased Rac1 mRNA and the Rac1 inhibitor NSC23766 mimicked the inhibitory effects of iloprost on PAR-1 protein—but not mRNA. The Rho kinase inhibitor Y27632 did not influence PAR-1 expression. Conclusions—IP-receptor agonists may limit the mitogenic actions of thrombin in human SMC by downregulating PAR-1 via modulation of cAMP-/PKA- and Rac1-dependent signaling pathways.

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.

Regulation of protease-activated receptor-1 by vasodilatory prostaglandins via NFAT

AIMSnWe recently reported that prostacyclin suppresses protease-activated receptor-1 (PAR-1) in human vascular smooth muscle cells (VSMC) via cyclic AMP and protein kinase A. This study examines the downstream mechanisms, particularly the role of nuclear factor of activated T-cells (NFAT).nnnMETHODS AND RESULTSnHuman saphenous vein VSMC were exposed to phorbol 12-myristate 13-acetate (PMA) to induce endogenous cyclooxygenase-2-dependent prostaglandin generation. This was found to attenuate PAR-1 expression; similar suppression was seen with the EP2-prostaglandin receptor agonist butaprost. Stimulation of the exchange protein directly activated by cyclic AMP (EPAC) was without effect. The NFAT inhibitor cyclosporin A (CsA) or NFAT2 siRNA both reduced PAR-1 mRNA and protein expression and prevented the stimulatory effects of thrombin or PAR-1 activating peptide (TFLLRN) on ERK1/2 phosphorylation and interleukin-6 expression. CsA or mutation of the NFAT binding motif in the PAR-1 promoter also blunted PAR-1 promoter activity (luciferase reporter assay). These inhibitory actions of CsA were comparable to those of the prostacyclin-mimetic iloprost, and both CsA and iloprost similarly attenuated nuclear NFAT2 localization and binding to the PAR-1 promoter (chromatin immunoprecipitation assay).nnnCONCLUSIONSnThis study provides the first evidence that NFAT2 contributes to the transcriptional control of PAR-1 in human VSMC and that PKA-dependent NFAT2 inhibition represents a mechanism by which vasodilatory prostaglandins regulate the vascular actions of thrombin.

Regulation of Human Vascular Protease-Activated Receptor-3 through mRNA Stabilization and the Transcription Factor Nuclear Factor of Activated T Cells (NFAT)

Thrombin promotes vascular smooth muscle cell (SMC) proliferation and inflammation via protease-activated receptor (PAR)-1. A further thrombin receptor, PAR-3, acts as a PAR-1 cofactor in some cell-types. Unlike PAR-1, PAR-3 is dynamically regulated at the mRNA level in thrombin-stimulated SMC. This study investigated the mechanisms controlling PAR-3 expression. In human vascular SMC, PAR-3 siRNA attenuated thrombin-stimulated interleukin-6 expression and extracellular signal-regulated kinases 1/2 phosphorylation, indicating PAR-3 contributes to net thrombin responses in these cells. Thrombin slowed the decay of PAR-3 but not PAR-1 mRNA in the presence of actinomycin D and induced cytosolic shuttling and PAR-3 mRNA binding of the mRNA-stabilizing protein human antigen R (HuR). HuR siRNA prevented thrombin-induced PAR-3 expression. By contrast, forskolin inhibited HuR shuttling and destabilized PAR-3 mRNA, thus reducing PAR-3 mRNA and protein expression. Other cAMP-elevating agents, including the prostacyclin-mimetic iloprost, also down-regulated PAR-3, accompanied by decreased HuR/PAR-3 mRNA binding. Iloprost-induced suppression of PAR-3 was reversed with a myristoylated inhibitor of protein kinase A and mimicked by phorbol ester, an inducer of cyclooxygenase-2. In separate studies, iloprost attenuated PAR-3 promoter activity and prevented binding of nuclear factor of activated T cells (NFAT2) to the human PAR-3 promoter in a chromatin immunoprecipitation assay. Accordingly, PAR-3 expression was suppressed by the NFAT inhibitor cyclosporine A or NFAT2 siRNA. Thus human PAR-3, unlike PAR-1, is regulated post-transcriptionally via the mRNA-stabilizing factor HuR, whereas transcriptional control involves NFAT2. Through modulation of PAR-3 expression, prostacyclin and NFAT inhibitors may limit proliferative and inflammatory responses to thrombin after vessel injury.

Redox regulation of human protease-activated receptor-2 by activated factor X

Activated factor X (FXa) exerts coagulation-independent actions such as proliferation of vascular smooth muscle cells (SMCs) through the protease-activated receptors PAR-1 and PAR-2. Both receptors are upregulated upon vascular injury but the underlying mechanisms have not been defined. We examined if FXa regulates PAR-1 and PAR-2 in human vascular SMCs. FXa increased PAR-2 mRNA, protein, and cell-surface expression and augmented PAR-2-mediated mitogenesis. PAR-1 was not influenced. The regulatory action of FXa on PAR-2 was concentration-dependent and mimicked by a PAR-2-selective activating peptide. PAR-2 regulation was not influenced by the thrombin inhibitor argatroban or PAR-1 siRNA. FXa increased dichlorofluorescein diacetate fluorescence and 8-isoprostane formation and induced expression of the NADPH oxidase subunit NOX-1. NOX-1 siRNA prevented FXa-stimulated PAR-2 regulation, as did ebselen and cell-permeative and impermeative forms of catalase. Exogenous H(2)O(2) increased PAR-2 expression and mitogenic activity. FXa promoted nuclear translocation and PAR-2/DNA binding of nuclear factor κB (NF-κB); NF-κB inhibition prevented PAR-2 regulation by FXa. FXa also promoted PAR-2 mRNA stabilization through increased human antigen R (HuR)/PAR-2 mRNA binding and cytoplasmic shuttling. HuR siRNA abolished FXa-stimulated PAR-2 expression. Thus FXa induces functional expression of PAR-2 but not of PAR-1 in human SMCs, independent of thrombin formation, via a mechanism involving NOX-1-containing NADPH oxidase, H(2)O(2), NF-κB, and HuR.

Direct inhibitors of thrombin and factor Xa attenuate clot-induced mitogenesis and inflammatory gene expression in human vascular smooth muscle cells

Direct inhibitors of thrombin and factor Xa attenuate clot-induced mitogenesis and inflammatory gene expression in human vascular smooth muscle cells -

Cholesterol induces apoptosis-associated loss of the activated leukocyte cell adhesion molecule (ALCAM) in human monocytes.

The activated leukocyte cell adhesion molecule (ALCAM/CD166) is associated with cell migration and leukocyte invasion into the vessel wall. This study investigates the impact of cholesterol loading on the expression of ALCAM, as compared with P-selectin glycoprotein ligand-1 (PSGL-1), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in monocytic U937 cells and human primary monocytes. Cells were enriched with cholesterol by incubation with a cyclodextrin-cholesterol complex. Expression of adhesion molecules and apoptosis was determined by flow cytometry. Migration was quantified by chemotaxis toward serum. Incubation with cholesterol (10-100 μg/ml) for 16 h caused a concentration-dependent increase in apoptosis. Enhanced apoptosis was associated with reduction of ALCAM by >70%. While PSGL-1 was affected similarly, expression of VCAM-1 was markedly increased by cholesterol and ICAM-1 levels were not regulated. The nonselective caspase/apoptosis inhibitor Q-VD-OPh partially prevented cholesterol-modulated alteration of adhesion molecule expression. Migration of cholesterol-rich monocytic cells toward serum was greatly reduced. This effect was partially restored by Q-VD-OPh and was dependent on ALCAM as shown by ALCAM-neutralizing antibodies. In conclusion, cholesterol-induced apoptosis in monocytes is accompanied by reduced expression of ALCAM and attenuated monocyte migration. This may restrain monocytes at cholesterol-rich sites and thereby expedite vascular lesion formation.