A novel function of FoxO transcription factors in thrombin-stimulated vascular smooth muscle cell proliferation.

Thrombin exerts coagulation-independent effects on the proliferation and migration of vascular smooth muscle cells (SMC). Forkhead box-O (FoxO) transcription factors regulate cell proliferation, apoptosis and cell cycle arrest, but a possible functional interaction between thrombin and FoxO factors has not been identified to date. In human cultured vascular SMC, thrombin induced a time-dependent phosphorylation of FoxO1 and FoxO3 but not FoxO4. This effect was mimicked by an activating-peptide (AP) for protease-activated receptor (PAR)-1, and abolished by a PAR-1 antagonist (SCH79797). APs for other PARs were without effect. FoxO1 and FoxO3 phosphorylation were prevented by the PI3 kinase (PI3K) inhibitor LY294002 while inhibitors of ERK1/2 (PD98059) or p38MAPK (SB203580) were ineffective. LY294002 moreover prevented thrombin-stimulated SMC mitogenesis and proliferation. FoxO1 and FoxO3 siRNA augmented basal DNA synthesis and proliferation of SMC. Nuclear content of FoxO proteins decreased time-dependently in response to thrombin, coincided with suppressed expression of the cell cycle regulating genes p21CIP1 and p27kip1 by thrombin. FoxO1 siRNA reduced basal p21CIP1 while FoxO3 siRNA attenuated p27kip1 expression; thrombin did not show additive effects. LY294002 restored p21CIP1 and p27kip1 protein expression. Immunohistochemistry revealed that human native and failed saphenous vein grafts were characterised by the cytosolic presence of p-FoxO factors in co-localisation of p21CIP1 and p27kip1 with SMC. In conclusion, thrombin and FoxO factors functionally interact through PI3K/Akt-dependent FoxO phosphorylation leading to expression of cell cycle regulating genes and ultimately SMC proliferation. This may contribute to remodelling and failure of saphenous vein bypass grafts.

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.

Overexpression of prostaglandin EP3 receptors activates calcineurin and promotes hypertrophy in the murine heart.

AIMS: Prostaglandin E(2) (PGE(2)) has been shown to mediate anti-ischaemic effects and cardiomyocyte hypertrophy and there is evidence for an involvement of the prostaglandin EP(3)-receptor subtype. This study focuses on the EP(3)-mediated hypertrophic action and investigates intracellular signalling pathways of the EP(3)-receptor subtype in the murine heart. METHODS AND RESULTS: Cardiac function was analyzed in vivo by magnetic resonance imaging (MRI) in transgenic (tg) mice with cardio-specific overexpression of the EP(3) receptor in comparison with wild-type (wt) mice. Left ventricular (LV) function was determined in isolated perfused hearts subjected to 60 min of zero-flow ischaemia and 45 min of reperfusion. Calcineurin activity and nuclear activity of nuclear factor of activated T-cells (NFAT) were determined by a modified malachite green assay and ELISA, respectively. Extracellular matrix compounds were analyzed by RT-PCR and histology. MRI indicated a significant increase in end-diastolic and end-systolic volume in tg hearts. LV ejection fraction was severely decreased in tg hearts while the relative LV mass was significantly increased. In Langendorff perfused hearts, EP(3)-receptor overexpression resulted in a marked blunting of the ischaemia-induced increase in LV end-diastolic pressure and creatine kinase release. Analysis of EP(3)-receptor-mediated signalling revealed significantly increased calcineurin activity and nuclear activity of NFAT in tg hearts. Moreover, elevated mRNA levels of collagen types I and III as well as the collagen-binding proteoglycans biglycan and decorin were detected in tg hearts. CONCLUSION: EP(3)-receptor-mediated signalling results in a significant anti-ischaemic action and activation of the pro-hypertrophic calcineurin signalling pathway, suggesting the involvement of the EP(3) subtype in both PGE(2)-mediated cardioprotection as well as cardiac hypertrophy.

Cardiospecific overexpression of the prostaglandin EP3 receptor attenuates ischemia-induced myocardial injury.

BACKGROUND: The generation of prostaglandin E2 (PGE2) is significantly increased in acute myocardial ischemia and reperfusion. PGE2, in addition to other prostaglandins, protects the reperfused ischemic myocardium. It has been hypothesized that this cardioprotection is mediated by E-type prostaglandin receptors of the Gi-coupled EP3 subtype. METHODS AND RESULTS: We tested this hypothesis by generating transgenic (tg) mice with cardiospecific overexpression of the EP3 receptor. According to ligand binding, a 40-fold overexpression of the EP3 receptor was achieved in membranes prepared from tg hearts compared with wild-type (wt) littermates. In isolated cardiomyocytes from tg mice, the forskolin-induced rise in cAMP was markedly attenuated, indicating coupling of the overexpressed EP3 receptor to inhibitory G proteins (Gi) with constitutive receptor activity. There was no evidence for EP3 receptor coupling to Gq-mediated protein kinase C signaling. Isolated hearts from tg and wt mice were subjected to 60 minutes of no-flow ischemia and 45 minutes of reperfusion. In tg hearts, ischemic contracture was markedly delayed compared with wt hearts, and the ischemia-induced increase in left ventricular end-diastolic pressure was reduced by 55%. Creatine kinase and lactate dehydrogenase release was significantly decreased by 85% and 73%, respectively, compared with wt hearts. CONCLUSIONS: Constitutive prostaglandin EP3 receptor signaling exerts a protective effect on cardiomyocytes, which is probably Gi mediated and results in a remarkable attenuation of myocardial injury during ischemia and reperfusion. Cardioprotective actions of E-type prostaglandins may be mediated by this receptor subtype.

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.

Cyclooxygenase COX-2a, a novel COX-2 mRNA variant, in platelets from patients after coronary artery bypass grafting.

There are two principal cyclooxygenase isoforms referred to as COX-1 and COX-2. Recently, COX-3 has been identified. We have demonstrated the expression of COX-2 in platelets from patients after coronary artery bypass grafting (CABG). Careful biochemical analysis revealed that, when compared to recombinant COX-2, platelet COX-2 had a slightly higher electrophoretic mobility. Two COX-2 sequences (approximately 1.8 kb, approximately 1.7 kb) were cloned from platelet mRNA. The approximately 1.7 kb sequence, designated COX-2a, differed from the human COX-2 sequence only in a deletion from position +458 to +567. Similar to the human COX-3, there is a frame shift in the COX-2a sequence resulting in a TAA stop codon at position +490. Thus, the expression of a COX-2a protein corresponding to the 67 kDa COX-2 protein is not clear. However, the marked shifting from COX-2 to COX-2a in platelets from some patients after CABG is a striking finding.

Gene expression profile of the Gs-coupled prostacyclin receptor in human vascular smooth muscle cells.

Migration and proliferation of medial smooth muscle cells (SMC) in the arterial intima contributes to the development of atherosclerotic plaques and restenotic processes after coronary angioplasty. Prostacyclin (PGI2)-mediated stimulation of cyclic adenosine 3'5'-monophosphate (cAMP) signaling is believed to be important for maintaining SMC in a quiescent state. In order to identify new cellular targets of PGI2/cAMP action, we have used microarray screening to examine changes in the transcriptional profile in human vascular SMC in response to exposure to the stable PGI2 mimetic iloprost. We have identified 83 genes with significantly altered expression after iloprost (100 nM) exposure for 6 hr. Fifty-one genes were upregulated, among them stanniocalcin precursor (18.8+/-2.7), zinc finger transcription factor (7.8+/-2.0), hyaluronan synthase 2 (6.8+/-1.8), cyclooxygenase 2 (4.7+/-0.8), dual specific phosphatase (3.9+/-0.5) and vascular endothelial growth factor (2.3+/-0.4). Thirty-two genes were reduced, among them cystein-rich angiogenic protein (-14.9+/-1.3), monocyte chemotactic protein 1 (-7.4+/-1.1) and plasminogen activator inhibitor PAI-1 (-4.5+/-0.5). By means of semi-quantitative RT-PCR, time-courses of gene expression were established. The present study identified genes not hitherto recognized to be targets of PGI2 action, providing further insight into its cAMP-mediated effects on SMC growth, migration and matrix secretion.

Iloprost down-regulates the expression of the growth regulatory gene Cyr61 in human vascular smooth muscle cells.

Prostacyclin and its mimetics have repeatedly been shown to act antiatherogenic and to inhibit neointima formation in several animal models of vascular injury. Treatment of human vascular smooth muscle cells with the prostacyclin mimetic iloprost (100 nm) drastically reduces expression of Cyr61, encoding the growth-regulatory cystein-rich angiogenic protein, without affecting the degradation rate of Cyr61 mRNA. Thrombin-induced Cyr61 expression was inhibited completely in the presence of iloprost. It is concluded that vasoprotective actions of prostacyclin in vivo may in part be due to inhibition of expression of the growth regulatory gene Cyr61 at sites of vascular lesions.

Long-term-desensitization of prostacyclin receptors is independent of the C-terminal tail.

Persistent stimulation of the G(s) protein-coupled prostacyclin receptor (IP-R) causes its slow desensitization in a variety of cell types, a significant desensitization requiring several hours. To evaluate the role of the human IP-R C-terminus in desensitization and agonist-induced internalization, a C-terminally truncated hIP-receptor was generated. The C-terminal 68 amino acid residues were deleted by introduction of a stop codon for exchange of the original S319 codon (termed D318 mutant). Wild-type (WT) and truncated receptor were expressed in COS1 cells. Pretreatment of cells with the stable prostacyclin mimetic cicaprost (200 nM) desensitized cAMP production via WT and D318 receptors to similar extents. The cAMP response of WT and D318, respectively, was reduced by approximately 50% of maximal cAMP formation after 8 hr of continuous agonist stimulation, indicating significant long-term desensitization. Moreover, agonist-promoted sequestration of WT and D318 C-terminally tagged with green fluorescent protein was demonstrated, indicating that receptor internalization was not prevented by truncation of the C-terminus. These results demonstrated that long-term desensitization and sequestration of hIP-R did not depend on structures located in the hIP-R C-terminus.

Regulation of cyclooxygenase-2 expression by iloprost in human vascular smooth muscle cells. Role of transcription factors CREB and ICER.

Prostaglandin-endoperoxide synthase-2 (PGH-synthase) or cyclooxygenase-2 (COX-2) is inducible by a variety of stimuli, e.g. inflammatory mediators, growth factors and hormones and is believed to be responsible for the majority of inflammatory prostanoid production. Moreover, it has been demonstrated that COX-2 contributes substantially to prostacyclin-synthesis in patients with atherosclerosis. In this study, we demonstrate an up-regulation of COX-2 mRNA, protein and product formation by the prostacyclin-mimetic iloprost in human vascular smooth muscle cells (hSMC). COX-2 mRNA expression was induced transiently between 1 and 6 hr and returned to basal levels after 16 hr of iloprost stimulation. COX-2 protein was induced concomitantly between 3 and 6 hr of iloprost stimulation. This was accompanied by an increase in PGI(2) formation. Forskolin, a direct activator of adenylyl cyclase, and dibutyryl cAMP, a cell-permeable cAMP analogue-induced COX-2 mRNA, suggesting a cAMP-dependent COX-2 expression in hSMC. Iloprost-induced COX-2 protein expression and PGI(2) formation was synergistically elevated by co-stimulation with the phorbolester PMA (phorbol-12-myristate-13-acetate). It is concluded, that the observed up-regulation of COX-2 with subsequent release of newly synthesized PGI(2) and the synergistic effect of iloprost and phorbolester on PGI(2) formation provide a positive feedback of prostaglandins on their own synthesizing enzyme. This might be important for control of hSMC proliferation, migration and differentiation as well as inhibition of platelet aggregation.

Selective cyclooxygenase-2 inhibition upregulates renal cortical alpha V integrin expression.

BACKGROUND: Cyclooxygenase-2 (COX-2), the inducible isoform of the cyclooxygenases, is upregulated in various inflammatory renal diseases and responsible for prostaglandin formation. As prostaglandins are known to influence cell adhesion processes, we investigated the effect of COX-2 inhibition on the expression of alpha(v) integrins, which are also enhanced in renal diseases and control the adherence between the endothelium and the extracellular matrix (ECM) in the glomerulus. METHODS: Healthy female Wistar rats and animals with previously induced passive Heymann nephritis (PHN) received either 5 mg/kg body weight/day celecoxib or a placebo. After 28 days, renal cortical mRNA expression of COX-2 and alpha(v) integrin subunits was determined. RESULTS: Rats with PHN showed a significant 1.7-fold increase in renal cortical mRNA expression of alpha(v) integrin subunits. Treatment with celecoxib increased cortical alpha(v) integrin mRNA expression 2.2-fold (p < 0.05) in healthy animals and 4.0-fold (p < 0.05) in rats with PHN, but lowered COX-2 mRNA expression in rats with PHN to 0.8-fold (p < 0.05). An inverse correlation between the expression of COX-2 and alpha(v) integrins in rats with PHN was demonstrated. CONCLUSIONS: It is suggested that COX-2-derived prostaglandins suppress the expression of alpha(v) integrins. This implies a previously unknown role for COX-2 in chronic inflammation in the kidney.

Activation of IP and EP(3) receptors alters cAMP-dependent cell migration.

Migration of vascular smooth cells from the media to the intima essentially contributes to neointima formation after percutaneous transluminal angioplasty and stent implantation. The stable prostacyclin mimetic iloprost has been shown to inhibit neointima formation in experimental restenosis, but it is currently unknown whether this may be caused by an antimigratory effect. Hence, the present study analyses (i) the influence of G(s)-coupled prostacyclin (IP) receptors on cell migration and (ii) verifies whether EP(3) receptors with opposite (i.e., G(i)) coupling may conversely stimulate cell migration. In a modified Boyden chamber model, it was shown that iloprost dose-dependently inhibits the migration of primary human arterial smooth muscle cells, which constitutively express the IP receptor. On the other hand, human arterial smooth muscle cell migration was stimulated by the EP(3) receptor agonist M&B 28.767. To independently study the effects of these receptors, IP or EP(3) receptors were stably overexpressed in chinese hamster ovary cells (CHO-IP and CHO-EP(3)). Chemotaxis of CHO cells transfected with G(s)-coupled IP receptors was concentration-dependently inhibited by iloprost (2-100 nM), while there was no effect of iloprost on mock-transfected CHO. By contrast, CHO-cells that overexpressed EP(3) receptors showed a significant, concentration dependent (1-100 nM) increase of cell migration in presence of the selective EP(3) agonist M&B 28.767. It is concluded that the prostacyclin mimetic iloprost inhibits vascular cell migration, which probably depends on a G(s)-mediated increase of intracellular cAMP. EP(3) receptors conversely stimulate CHO migration.

Prostacyclin enhances the expression of LPS/INF-gamma-induced nitric oxide synthase in human monocytes.

BACKGROUND: Nitric oxide (NO) is an important mediator of inflammatory processes, including macrophage-mediated cellular host defense, and is found to be increased in peritonitis. The ability of human mononuclear cells to contribute to the NO production by expression of active inducible NO synthase (iNOS) is still discussed controversely. AIMS: This study was designed to investigate the influence of prostacyclin receptor (IP receptor) activation on iNOS expression and NO formation in human peripheral blood monocytes. METHOD AND RESULTS: Using reverse transcriptase-polymerase chain reaction, we demonstrated that human monocytes express high levels of IP receptor mRNA. Stimulation of monocytes with the IP receptor selective agonist cicaprost (100 nM) significantly induced cellular cyclic adenosine monophosphate formation, indicating functional coupling of the receptor to G(s). Treatment of cells with lipopolysaccharide (LPS)/interferon gamma (IFN-gamma) further enhanced the IP receptor mRNA expression 2.7 +/- 0.1-fold above basal levels (n = 6). Analysis of iNOS expression revealed barely detectable mRNA levels in unstimulated monocytes which were increased 3.75 +/- 0.3-fold (n = 5) after costimulation with 1 microg/ml LPS and 250 U/ml INF-gamma for 16 h. Further increases of iNOS mRNA expression (9.4 +/- 0.9-fold above basal, n = 5) were obtained, if the monocytes were costimulated with 1 microg/ml LPS, 250 U/ml INF-gamma, and 100 nM cicaprost for 16 h. Measurement of the NO generation correlated with the polymerase chain reaction data: treatment of cells with 1 microg/ml LPS plus 250 U/ml INF-gamma increased the NO(2) production to 2.6 microM, being above the basal level of 2.0 microM, as determined in the cell culture medium. Additional treatment with 100 nM cicaprost further significantly increased the NO(2) production to 3.43 microM. CONCLUSIONS: An IP receptor mediated increase in cyclic adenosine monophosphate formation plays an important role in enhancing LPS/IFN-gamma-induced iNOS expression in human monocytes/macrophages and may, therefore, contribute to the increased production of NO during peritonitis.

Obligatory role of cyclic adenosine monophosphate response element in cyclooxygenase-2 promoter induction and feedback regulation by inflammatory mediators.

BACKGROUND: Cyclooxygenase-2 (COX-2) plays a key role in human inflammatory disorders such as vascular inflammation. COX-2 promoter activity is induced by proinflammatory mediators, but the role of cyclic adenosine monophosphate response element (CRE) in promoter stimulation remains unclear. METHODS AND RESULTS: Transient transfection of a 0.9-kb COX-2 promoter fragment bearing CRE mutation abrogated COX-2 promoter activity induced by proinflammatory mediators in human endothelial cells and fibroblasts. Dual mutations of CRE and an upstream CCAAT/enhancer binding protein (C/EBP) site did not have an additional effect. Binding of CREB-2, ATF-2, USF-2, and c-Jun transactivators to a wild-type and CRE-mutated oligonucleotide was analyzed by a novel DNA-binding assay. CREB-2 and ATF-2 in nuclear extracts of unstimulated endothelial cells bound to CRE, whereas USF-2 and c-Jun or c-Fos bound to non-CRE sites. CREB-2 and c-Fos binding was increased by phorbol 12-myristate 13-acetate but not tumor necrosis factor-alpha. The binding assay and chromatin immunoprecipitation revealed binding of P300 coactivator to the COX-2 promoter region. CONCLUSIONS: CRE plays an obligatory role in COX-2 promoter activation by diverse stimuli. CREB-2 and ATF-2 bound to CRE serve as an anchor for P300 interaction with upstream transactivators and downstream transcription machinery.