Sirolimus increases tissue factor expression but not activity in cultured human vascular smooth muscle cells

BACKGROUND: Sirolimus-eluting stents (CYPHER stents) demonstrated remarkable efficacy in reducing restenosis rates in patients with coronary artery disease. There is a concern of sub-acute and late stent thrombosis. Tissue factor (TF) is critical in thrombosis. This study investigated the effect of sirolimus on TF expression and activity in cultured human vascular smooth muscle cells (SMCs). METHODS: SMCs were cultured from human saphenous veins and aortas. Quiescent cells were stimulated with sirolimus (0.1 – 20 ng/ml) over 24 hours. Cellular TF expression and activity released into culture medium were measured. The effect of sirolimus on activation of mammalian target of rapamycin (mTOR) was measured by phosphorylation of the substrate p70s6k at T389, and activation of RhoA was measured by pull-down assay. RESULTS: Sirolimus increased TF protein level in cultured human SMCs in a concentration and time-dependent manner (about 2-fold, p < 0.01) reaching maximal effect at 5 ng/ml. The stimulation of TF expression by sirolimus was associated with inhibition of basal activity of mTOR. No effects of sirolimus on RhoA or p38mapk activation that are positive regulators of TF in vascular wall cells were observed. The stimulation of TF expression by sirolimus (20 ng/ml) was prevented by the HMG-CoA reductase inhibitor fluvastatin (1 μmol/L). However, no increase in TF activity released from SMC into culture medium was observed after sirolimus treatment. CONCLUSION: Although sirolimus stimulates TF protein expression in human SMC associated with inhibition of mTOR, it does not enhance TF activity released from the cells, suggesting a relatively safe profile of CYPHER stents. The inhibition of TF expression by fluvastatin favors clinical use of statins in patients undergoing coronary stenting

Endothelial nitric oxide synthase genetransfer restores endothelium-dependentrelaxations and attenuates lesion formationin carotid arteries in apolipoproteinE-deficient mice

Nitric oxide (NO) and monocyte chemoattractant protein-1 (MCP-1) exert partly opposing effects in vascular biology. NO plays pleiotropic vasoprotective roles including vasodilation and inhibition of platelet aggregation, smooth muscle cell proliferation, and endothelial monocyte adhesion, the last effect being mediated by MCP-1 downregulation. Early stages of arteriosclerosis are associated with reduced NO bioactivity and enhanced MCP-1 expression. We have evaluated adenovirus-mediated gene transfer of human endothelial NO synthase (eNOS) and of a N-terminal deletion (8ND) mutant of the MCP-1 gene that acts as a MCP-1 inhibitor in arteriosclerosis-prone, apolipoprotein E-deficient (ApoE-/-) mice. Endotheliumdependent relaxations were impaired in carotid arteries instilled with a noncoding adenoviral vector but were restored by eNOS gene transfer (p < 0.01). A perivascular collar was placed around the common carotid artery to accelerate lesion formation. eNOS gene transfer reduced lesion surface areas, intima/media ratios, and macrophage contents in the media at 5-week follow-up (p < 0.05). In contrast, 8ND-MCP-1 gene transfer did not prevent lesion formation. In conclusion, eNOS gene transfer restores endothelium-dependent vasodilation and inhibits lesion formation in ApoE-/- mouse carotids. Further studies are needed to assess whether vasoprotection is maintained at later disease stages and to evaluate the long-term efficacy of eNOS gene therapy for primary arteriosclerosi

Endothelial Insulin Receptor Restoration Rescues Vascular Function in Male Insulin Receptor Haploinsufficient Mice

Reduced systemic insulin signaling promotes endothelial dysfunction and diminished endogenous vascular repair. We investigated whether restoration of endothelial insulin receptor expression could rescue this phenotype. Insulin receptor knockout (IRKO) mice were crossed with mice expressing a human insulin receptor endothelial cell–specific overexpression (hIRECO) to produce IRKO-hIRECO progeny. No metabolic differences were noted between IRKO and IRKO-hIRECO mice in glucose and insulin tolerance tests. In contrast with control IRKO littermates, IRKO-hIRECO mice exhibited normal blood pressure and aortic vasodilatation in response to acetylcholine, comparable to parameters noted in wild type littermates. These phenotypic changes were associated with increased basal- and insulin-stimulated nitric oxide production. IRKO-hIRECO mice also demonstrated normalized endothelial repair after denuding arterial injury, which was associated with rescued endothelial cell migration in vitro but not with changes in circulating progenitor populations or culture-derived myeloid angiogenic cells. These data show that restoration of endothelial insulin receptor expression alone is sufficient to prevent the vascular dysfunction caused by systemically reduced insulin signaling

Regulation of vascular endothelial function by RhoA/Rho-kinase pathway: implications for cardiovascular diseases

Das Gefässendothel spielt eine wichtige Rolle bei der Aufrechterhaltung normaler kardiovaskulärer Funktionen. Es verhindert die Kontraktion glatter Muskulatur, hemmt die Proliferation und/oder Migration der glatten Muskelzellen und übt eine antithrombotische Wirkung aus. Einer der wichtigsten Faktoren endothelialer Herkunft ist Stickstoffmonooxid (NO), das durch Aktivierung der endothelialen NO-Synthase (eNOS) produziert wird. eNOS setzt als Substrat L-Arginin unter physiologischen Bedingungen und in Anwesenheit von Cofaktoren zu NO um. Im Verlauf bestimmter Krankheiten, z.B. bei Arteriosklerose, Diabetes und Bluthochdruck, oder im Alter ist die biologische Verfügbarkeit von NO jedoch verringert, was mit der Entstehung und dem Fortschreiten dieser Prozesse in Verbindung gebracht wird. Diese verringerte biologische Verfügbarkeit von NO wird entweder auf eine geringere eNOSExpression oder einen Rückgang der Enzymaktivität bzw. eine Inaktivierung von aktivem NO durch oxidativen Stress zurückgeführt. Die RhoGTPase ist ein Mitglied der Ras-Superfamilie kleiner GTP-bindender Proteine. Sie ist nicht nur an der Regulation mit Cytoskelettreorganisation verbundener zellulärer Prozesse wie Blutplättchenaggregation, Kontraktion glatter Muskulatur und Zellmigration, sondern auch an den Regulation der Genexpression beteiligt. In den letzten Jahren haben Studien ergeben, dass RhoA eNOS-Expression in endothelialen Zellen durch Destabilisierung der eNOS-mRNA unterdrückt. Hemmung von RhoA durch HMG-CoAReduktaseinhibitoren oder Statine erhöht eNOS-Expression. Dies kann zu den positiven Effekten der Medikation bei Patienten mit koronarer Herzkrankheit beitragen. Im ersten Teil des Projekts haben wir die Rolle der RhoGTPase RhoA bei der Herunterregulierung der eNOS-Expression in menschlichen Endothelzellen bestätigt. Diese Wirkung wird durch den Effektor Rho-Kinase (ROCK) vermittelt. Insbesondere haben wir eine Beteiligung des RhoA/ROCK-Signalwegs an der Hemmung der Akt/eNOS-Kaskade nachgewiesen, d.h., die Aktivierung von RhoA/ROCK hemmt PKB/Akt-Aktivität und verhindert infolgedessen Aktivierung oder Phosphorylierung von eNOS an S-1177. Im zweiten Teil des Projekts haben wir eine weitere wichtige Funktion des RhoA/ROCK-Signalwegs in der Regulation der eNOS-Aktivität, die Stimulation des Enzyms Arginase in Endothelzellen, untersucht. Die Arginase setzt L-Arginin zu Harnstoff um, wodurch intrazelluläres L-Arginin, das für die NO-Bildung eingesetzt wird, abgezogen wird. Wir konnten zeigen, dass die Ueberexpression aktiver Formen von RhoA oder ROCK die Arginaseaktivität in Endothelzellen erhöht. Interessanterweise war die RhoA-Expression in den Aortae arteriosklerotischer ApoEP -/- P- Mäuse deutlich höher als in denen der Wildtyp- Mäusen. Dies korrelierte mit einer erhöhten Arginaseaktivität in den ApoE-/- Mäusen. In den Aortae der arteriosklerotischen Mäuse rief L-Arginin eine stärkere Kontraktion als in denen der Wildtyp-Mäuse hervor. Diese Reaktion wurde bei Behandlung mit dem Arginaseinhibitor L-Norvalin in eine stärkere Relaxation der ApoEP -/- P-Aortae gegenüber den Wildtypgefässen umgekehrt. Diese Ergebnisse zeigen, dass die durch den RhoA/ROCK-Signalweg erhöhte Arginaseaktivität an arteriosklerotischer Fehlfunktion des Endothels beteiligt ist. Im dritten Teil des Projekts konnten wir nachweisen, dass der RhoA/ROCKSignalweg zusammen mit p38P mapk P die Expression des endothelialen Tissue-Faktors erhöht. Tissue-Faktor ist kritisch für die Gerrinselbildung und spielt eine wichtige Rollen bei akuten koronaren Symptomen. In diesem Teil des Projekts zeigten unsere Resultate, dass das High- Density-Lipoprotein (HDL) einerseits den PI3K/Akt/eNOS-Signalweg aktiviert und andererseits die Expression des Tissue-Faktors hemmt. Die Hemmung der Expression des endothelialen Tissue-Faktors durch HDL ist auf zwei Mechanismen zurückzuführen: Hemmung von RhoA und Aktivierung der PI3K, wobei die weiter abwärts im PI3KSignalweg liegenden Enzyme Akt und eNOS nicht involviert sind.The vascular endothelium plays an important role in the maintenance of normal cardiovascular functions. It prevents smooth muscle contraction, inhibits smooth muscle cell proliferation and/or migration and exerts anti-thrombotic effects. One of the most important factors derived from endothelial cells is nitric oxide (NO) which is produced via activation of endothelial NO synthase (eNOS). eNOS metabolizes the substrate L–arginine to NO in the presence of co-factors under physiological conditions. However, under disease conditions, such as atherosclerosis, diabetes, hypertension, aging, etc., NO bioavailability is decreased, which is implicated in initiation and evolution of the disease process. The decreased NO bioavailability is either attributed to a decreased eNOS gene expression or decrease in enzyme activity or inactivation of bioactive NO by oxidative stress. RhoGTPase is a member of the Ras superfamily of small GTP-binding proteins which is not only involved in regulation of cellular functions associated with cytoskeleton reorganisation such as platelet aggregation, smooth muscle contraction and cell migration, but also in regulation of gene expression. Studies in recent years demonstrate that RhoA suppresses eNOS gene expression in endothelial cells by destabilizing eNOS mRNA. Inhibition of RhoA by the HMG-CoA reductase inhibitors or statins upregulates eNOS expression. These may importantly contribute to the beneficial effects of the drugs in patients with coronary heart diseases. In the first part of the project we further confirmed the role of the RhoGTPase, RhoA in down-regulation of eNOS gene expression in human endothelial cells. This effect is mediated by down-stream effector Rho-kinase (ROCK). Most importantly, we established a role of RhoA/ROCK pathway in inhibition of Akt/eNOS cascade, i.e. activation of RhoA/ROCK inhibits PKB/Akt activity and subsequently activation or phosphorylation of eNOS at S-1177. In the second part of the project we explored another important role of Rho/ROCK pathway in regulation of eNOS activity namely stimulation of arginase in endothelial cells. Arginase competes with eNOS for the substrate L-arginine. It metabolizes L-arginine to urea, thus depletes intracellular L-arginine for NO production. We showed that overexpression of RhoA or ROCK active mutants enhances arginase activity in endothelial cells. Interestingly, in ApoEP -/- P atherosclerotic mice aorta, RhoA expression was much more pronounced as compared to the wild type animals, which is correlated with a higher arginase activity in ApoEP -/- P mice aortas. In atherosclerotic ApoEP -/- P mice aortas, L-arginine caused a more pronounced contraction as compared to wild type mice. This response was reversed to a greater relaxation by an arginase inhibitor L-norvaline in ApoEP -/- Pmice compared to the wild type animals. The results demonstrate that an increased arginase activity mediated by Rho/ROCK pathway is involved atherosclerotic endothelial dysfunction. In the third part of the project, we demonstrated that Rho/ROCK pathway together with p38P mapk P increases expression of endothelial tissue factor which is critical in thrombus formation and acute coronary syndromes. In this part of the project, we showed that high density lipoprotein (HDL) on one hand activates PI3K/Akt/eNOS pathway and on the other hand inhibits tissue factor expression. The inhibition of endothelial tissue factor expression by HDL is mediated through inhibition of RhoA and activation of PI3K, independent of the downstream enzymes, Akt/eNOS. Taken together, our results demonstrate multi-functional roles of Rho/ROCK pathway in endothelial dysfunction, which may be importantly involved in the pathogenesis of cardiovascular disease

Sirolimus increases tissue factor expression but not activity in cultured human vascular smooth muscle cells

Background Sirolimus-eluting stents (CYPHER stents) demonstrated remarkable efficacy in reducing restenosis rates in patients with coronary artery disease. There is a concern of sub-acute and late stent thrombosis. Tissue factor (TF) is critical in thrombosis. This study investigated the effect of sirolimus on TF expression and activity in cultured human vascular smooth muscle cells (SMCs).MethodsSMCs were cultured from human saphenous veins and aortas. Quiescent cells were stimulated with sirolimus (0.1 – 20 ng/ml) over 24 hours. Cellular TF expression and activity released into culture medium were measured. The effect of sirolimus on activation of mammalian target of rapamycin (mTOR) was measured by phosphorylation of the substrate p70s6k at T389, and activation of RhoA was measured by pull-down assay.ResultsSirolimus increased TF protein level in cultured human SMCs in a concentration and time-dependent manner (about 2-fold, p < 0.01) reaching maximal effect at 5 ng/ml. The stimulation of TF expression by sirolimus was associated with inhibition of basal activity of mTOR. No effects of sirolimus on RhoA or p38mapk activation that are positive regulators of TF in vascular wall cells were observed. The stimulation of TF expression by sirolimus (20 ng/ml) was prevented by the HMG-CoA reductase inhibitor fluvastatin (1 μmol/L). However, no increase in TF activity released from SMC into culture medium was observed after sirolimus treatment.ConclusionAlthough sirolimus stimulates TF protein expression in human SMC associated with inhibition of mTOR, it does not enhance TF activity released from the cells, suggesting a relatively safe profile of CYPHER stents. The inhibition of TF expression by fluvastatin favors clinical use of statins in patients undergoing coronary stenting

Rho GTPase/Rho Kinase Negatively Regulates Endothelial Nitric Oxide Synthase Phosphorylation through the Inhibition of Protein Kinase B/Akt in Human Endothelial Cells

Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB

Chronic vasopeptidase inhibition restores endothelin‐converting enzyme activity and normalizes endothelin levels in salt‐induced hypertension

Background. Vasopeptidase inhibition (VPI) represents a new therapeutic principle including both inhibition of angiotensin‐converting enzyme (ACE) and neutral endopeptidase (NEP). The present study investigated the effect of the vasopeptidase inhibitor omapatrilat on endothelin‐1 (ET‐1)‐mediated vascular function in salt‐induced hypertension. Methods. Dahl salt‐sensitive rats (n=6/group) on standard or salt‐enriched (4% NaCl) chow were treated for 8 weeks with either omapatrilat (36±4 mg/kg/day), captopril (94±2 mg/kg/day) or placebo. Aortic and renal artery segments were isolated and suspended in organ chambers for isometric tension recording. Functional endothelin‐converting enzyme (ECE) activity was assessed in native segments and after preincubation with omapatrilat. Furthermore, vascular ECE protein levels as well as plasma and tissue ET‐1 levels were determined. Results. The increase in systolic blood pressure of salt‐fed rats was prevented by omapatrilat and captopril to a comparable degree. In salt‐induced hypertension, functional ECE activity (calculated as the ratio of the contraction to big ET‐1 divided by the contraction to ET‐1) in renal arteries (0.46±0.05) and in aorta (0.68±0.05) was reduced as compared with control animals (0.9±0.05 and 0.99±0.04, respectively; P<0.05). While omapatrilat in vitro blunted the response to big endothelin‐1 (big ET‐1) and diminished ECE activity further (P<0.01 vs native segments), chronic treatment with omapatrilat in vivo restored contractions to ET‐1 (120±6%) and big ET‐1 (98±9%) in renal arteries, and therefore normalized renovascular ECE activity. In addition, omapatrilat normalized plasma ET‐1 concentrations (12.9±1.2 vs 16.6±1.4 pg/ml on high salt diet; P<0.05) and renovascular ECE protein levels. Conclusions. In salt‐induced hypertension, vasopeptidase inhibition restores alterations in the endothelin system, such as renovascular ECE activity and responsiveness to ET‐1 and big ET‐1 with chronic but not acute in vitro application. Thus, the beneficial effects of vasopeptidase inhibition may reflect a resetting of cardiovascular control systems and therefore may be particularly suited to treat hypertension and heart failur

Chronic vasopeptidase inhibition restores endothelin‐converting enzyme activity and normalizes endothelin levels in salt‐induced hypertension

Background. Vasopeptidase inhibition (VPI) represents a new therapeutic principle including both inhibition of angiotensin‐converting enzyme (ACE) and neutral endopeptidase (NEP). The present study investigated the effect of the vasopeptidase inhibitor omapatrilat on endothelin‐1 (ET‐1)‐mediated vascular function in salt‐induced hypertension. Methods. Dahl salt‐sensitive rats (n=6/group) on standard or salt‐enriched (4% NaCl) chow were treated for 8 weeks with either omapatrilat (36±4 mg/kg/day), captopril (94±2 mg/kg/day) or placebo. Aortic and renal artery segments were isolated and suspended in organ chambers for isometric tension recording. Functional endothelin‐converting enzyme (ECE) activity was assessed in native segments and after preincubation with omapatrilat. Furthermore, vascular ECE protein levels as well as plasma and tissue ET‐1 levels were determined. Results. The increase in systolic blood pressure of salt‐fed rats was prevented by omapatrilat and captopril to a comparable degree. In salt‐induced hypertension, functional ECE activity (calculated as the ratio of the contraction to big ET‐1 divided by the contraction to ET‐1) in renal arteries (0.46±0.05) and in aorta (0.68±0.05) was reduced as compared with control animals (0.9±0.05 and 0.99±0.04, respectively; P<0.05). While omapatrilat in vitro blunted the response to big endothelin‐1 (big ET‐1) and diminished ECE activity further (P<0.01 vs native segments), chronic treatment with omapatrilat in vivo restored contractions to ET‐1 (120±6%) and big ET‐1 (98±9%) in renal arteries, and therefore normalized renovascular ECE activity. In addition, omapatrilat normalized plasma ET‐1 concentrations (12.9±1.2 vs 16.6±1.4 pg/ml on high salt diet; P<0.05) and renovascular ECE protein levels. Conclusions. In salt‐induced hypertension, vasopeptidase inhibition restores alterations in the endothelin system, such as renovascular ECE activity and responsiveness to ET‐1 and big ET‐1 with chronic but not acute in vitro application. Thus, the beneficial effects of vasopeptidase inhibition may reflect a resetting of cardiovascular control systems and therefore may be particularly suited to treat hypertension and heart failur