Inhibition of miR-223 Expression Using a Sponge Strategy Decreases Restenosis in Rat Injured Carotids.

OBJECTIVE: Restenosis is a frequent complication of angioplasty. It consists of a neointimal hyperplasia resulting from progression and migration of vascular smooth muscle cells (VSMC) into the vessel lumen. microRNA miR-223 has recently been shown to be involved in cardiovascular diseases including atherosclerosis, vascular calcification and arterial thrombosis. In this study, our aim was to assess the impact of miR-223 modulation on restenosis in a rat model of carotid artery after balloon injury. METHODS: The over and down-expression of miR-223 was induced by adenoviral vectors, containing either a pre-miR-223 sequence allowing artificial miR-223 expression or a sponge sequence, trapping the native microRNA, respectively. Restenosis was quantified on stained rat carotid sections. RESULTS: In vitro, three mRNA (Myocyte Enhancer Factor 2C (MEF2C), Ras homolog gene family, member B (RhoB) and Nuclear factor 1 A-type (NFIA)) reported as miR-223 direct targets and known to be implicated in VSMC differentiation and contractility were studied by RT-qPCR. Our findings showed that down-expression of miR-223 significantly reduced neointimal hyperplasia by 44% in carotids, and was associated with a 2-3-fold overexpression of MEF2C, RhoB and NFIA in a murine monocyte macrophage cell line, RAW 264.7 cells. CONCLUSION: Down-regulating miR-223 could be a potential therapeutic approach to prevent restenosis after angioplasty.

Cardiac inflammatory CD11b/c cells exert a protective role in hypertrophied cardiomyocyte by promoting TNFR2- and Orai3- dependent signaling.

Early adaptive cardiac hypertrophy (EACH) is initially a compensatory process to optimize pump function. We reported the emergence of Orai3 activity during EACH. This study aimed to characterize how inflammation regulates store-independent activation of Orai3-calcium influx and to evaluate the functional role of this influx. Isoproterenol infusion or abdominal aortic banding triggered EACH. TNFα or conditioned medium from cardiac CD11b/c cells activated either in vivo [isolated from rats displaying EACH], or in vitro [isolated from normal rats and activated with lipopolysaccharide], were added to adult cardiomyocytes before measuring calcium entry, cell hypertrophy and cell injury. Using intramyocardial injection of siRNA, Orai3 was in vivo knockdown during EACH to evaluate its protective activity in heart failure. Inflammatory CD11b/c cells trigger a store-independent calcium influx in hypertrophied cardiomyocytes, that is mimicked by TNFα. Pharmacological or molecular (siRNA) approaches demonstrate that this calcium influx, depends on TNFR2, is Orai3-driven, and elicits cardiomyocyte hypertrophy and resistance to oxidative stress. Neutralization of Orai3 inhibits protective GSK3ß phosphorylation, impairs EACH and accelerates heart failure. Orai3 exerts a pathophysiological protective impact in EACH promoting hypertrophy and resistance to oxidative stress. We highlight inflammation arising from CD11b/c cells as a potential trigger of TNFR2- and Orai3-dependent signaling pathways.

Effect of intracoronary administration of AAV1/SERCA2a on ventricular remodelling in patients with advanced systolic heart failure: results from the AGENT-HF randomized phase 2 trial.

AIMS: Restoration of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2a) activity through gene transfer improved cardiac function in experimental and pilot studies in humans with heart failure. The AGENT-HF (NCT01966887) trial investigated the impact of adeno-associated virus (AAV1)/SERCA2a on ventricular remodelling using multimodality non-invasive cardiac imaging. METHODS AND RESULTS: AGENT-HF was a single centre, randomized, double-blind, placebo-controlled trial in adult patients with NYHA class III-IV ischaemic or non-ischaemic heart failure and left ventricular ejection fraction ≤35%. Eligible patients were randomized to receive a single intracoronary infusion of either 1 × 1013 DNase-resistant particles of AAV1/SERCA2a or placebo. The primary endpoint was change in left ventricular end-systolic volume (LVESV), measured by cardiac computed tomography at 6 month follow-up. We planned to include 40 patients but the trial was terminated prematurely as the sponsor suspended further enrolment following neutral results of the CUPID-2 outcome trial. At the time of termination, nine patients were randomized with five patients infused with AAV1/SERCA2a and four with placebo. At 6 months, LVESV was increased in both groups compared with baseline: median (interquartile range) in AAV1/SERCA2a vs. placebo: 13 (13;14) mL vs. 3.5 (-36;36) mL, P = 0.74, with a mean difference between groups of 11.4 mL in favour of placebo. No safety issues were noted. CONCLUSION: AGENT-HF failed to demonstrate any improvement in ventricular remodelling in response to AAV1/SERCA2a at the dose studied. However, because of premature termination, the study was underpowered to demonstrate an effect of AAV1/SERCA2a and these data should be interpreted with caution.

miR-322 regulates insulin signaling pathway and protects against metabolic syndrome-induced cardiac dysfunction in mice.

We identified murine miR-322, orthologous to human miR-424, as a new regulator of insulin receptor, IGF-1 receptor and sirtuin 4 mRNA in vitro and in vivo in the heart and found that miR-322/424 is highly expressed in the heart of mice. C57Bl/6N mice fed 10weeks of high fat diet (HFD) presented signs of cardiomyopathy and a stable miR-322 cardiac level while cardiac function was slightly affected in 11week-old ob/ob which overexpressed miR-322. We thus hypothesized that mmu-miR-322 could be protective against cardiac consequences of hyperinsulinemia and hyperlipidemia. We overexpressed or knocked-down mmu-miR-322 using AAV9 and monitored cardiac function in wild-type C57Bl/6N mice fed a control diet (CD) or a HFD and in ob/ob mice. The fractional shortening progressively declined while the left ventricle systolic diameter increased in HFD mice infected with an AAVcontrol or with an AAVsponge (decreasing miR-322 bioavailability) but also in ob/ob mice infected with AAVsponge. Similar observations were also found in CD-fed mice infected with AAVsponge. On the contrary over-expressing miR-322 with AAVmiR-322 was efficient in protecting the heart from HFD effects in C57Bl/6N mice. This cardioprotection could be associated with the regulation of identified targets IGF1R, INSR and CD1, a decrease in insulin signaling pathway and an enrichment of genes involved in mitochondrial function and fatty acid oxidation as demonstrated by transcriptome analysis. Altogether, these results emphasize miR-322 as a new potential therapeutic target against cardiac consequences of metabolic syndrome, which represents an increasing burden in the western countries.

Impaired platelet activation and cAMP homeostasis in MRP4-deficient mice.

Molecules that reduce the level of cyclic adenosine 5'-monophosphate (cAMP) in the platelet cytosol, such as adenosine 5'-diphosphate (ADP) secreted from dense granules, trigger platelet activation. Therefore, any change in the distribution and/or availability of cyclic nucleotides or ADP may interfere with platelet reactivity. In this study, we evaluated the role of multidrug resistance protein 4 (MRP4, or ABCC4), a nucleotide transporter, in platelet functions in vivo and in vitro by investigating MRP4-deficient mice. MRP4 deletion resulted in a slight increase in platelet count but had no impact on platelet ultrastructure. In MRP4-deficient mice, the arterial occlusion was delayed and the tail bleeding time was prolonged. In a model of platelet depletion and transfusion mimicking a platelet-specific knockout, mice injected with MRP4(-/-) platelets also showed a significant increase in blood loss compared with mice injected with wild-type platelets. Defective thrombus formation and platelet activation were confirmed in vitro by studying platelet adhesion to collagen in flow conditions, integrin αIIbß3 activation, washed platelet secretion, and aggregation induced by low concentrations of proteinase-activated receptor 4-activating peptide, U46619, or ADP. We found no role of MRP4 in ADP dense-granule storage, but MRP4 redistributed cAMP from the cytosol to dense granules, as confirmed by increased vasodilator-stimulated phosphoprotein phosphorylation in MRP4-deficient platelets. These data suggest that MRP4 promotes platelet aggregation by modulating the cAMP-protein kinase A signaling pathway, suggesting that MRP4 might serve as a target for novel antiplatelet agents.

Inhalable delivery of AAV-based MRP4/ABCC4 silencing RNA prevents monocrotaline-induced pulmonary hypertension.

UNLABELLED: The ATP-binding cassette transporter MRP4 (encoded by ABCC4) regulates membrane cyclic nucleotides concentrations in arterial cells including smooth muscle cells. MRP4/ABCC4 deficient mice display a reduction in smooth muscle cells proliferation and a prevention of pulmonary hypertension in response to hypoxia. We aimed to study gene transfer of a MRP4/ABCC4 silencing RNA via intratracheal delivery of aerosolized adeno-associated virus 1 (AAV1.shMRP4 or AAV1.control) in a monocrotaline-induced model of pulmonary hypertension in rats. Gene transfer was performed at the time of monocrotaline administration and the effect on the development of pulmonary vascular remodeling was assessed 35 days later. AAV1.shMRP4 dose-dependently reduced right ventricular systolic pressure and hypertrophy with a significant reduction with the higher doses (i.e., >10(11) DRP/animal) as compared to AAV1. CONTROL: The higher dose of AAV1.shMRP4 was also associated with a significant reduction in distal pulmonary arteries remodeling. AAV1.shMRP4 was finally associated with a reduction in the expression of ANF, a marker of cardiac hypertrophy. Collectively, these results support a therapeutic potential for downregulation of MRP4 for the treatment of pulmonary artery hypertension.

Emergence of Orai3 activity during cardiac hypertrophy.

AIMS: Stromal interaction molecule 1 (STIM1) has been shown to control a calcium (Ca(2+)) influx pathway that emerges during the hypertrophic remodelling of cardiomyocytes. Our aim was to determine the interaction of Orai1 and Orai3 with STIM1 and their role in the constitutive store-independent and the store-operated, STIM1-dependent, Ca(2+) influx in cardiomyocytes. METHODS AND RESULTS: We characterized the expression profile of Orai proteins and their interaction with STIM1 in both normal and hypertrophied adult rat ventricular cardiomyocytes. Orai1 and 3 protein levels were unaltered during the hypertrophic process and both proteins co-immunoprecipitated with STIM1. The level of STIM1 and Orai1 were significantly greater in the macromolecular complex precipitated by the Orai3 antibody in hypertrophied cardiomyocytes. We then used a non-viral method to deliver Cy3-tagged siRNAs in vivo to adult ventricular cardiomyocytes and silence Orai channel candidates. Cardiomyocytes were subsequently isolated then the voltage-independent, i.e. store-independent and store-operated Ca(2+) entries were measured on Fura-2 AM loaded Cy3-labelled and control isolated cardiomyocytes. The whole cell patch-clamp technique was used to measure Orai-mediated currents. Specific Orai1 and Orai3 knockdown established Orai3, but not Orai1, as the critical partner of STIM1 carrying these voltage-independent Ca(2+) entries in the adult hypertrophied cardiomyocytes. Orai3 also drove an arachidonic acid-activated inward current. CONCLUSION: Cardiac Orai3 is the essential partner of STIM1 and drives voltage-independent Ca(2+) entries in adult cardiomyocytes. Arachidonic acid-activated currents, which are supported by Orai3, are present in adult cardiomyocytes and increased during hypertrophy.

Synergistic role of protein phosphatase inhibitor 1 and sarco/endoplasmic reticulum Ca2+ -ATPase in the acquisition of the contractile phenotype of arterial smooth muscle cells.

BACKGROUND: Phenotypic modulation or switching of vascular smooth muscle cells from a contractile/quiescent to a proliferative/synthetic phenotype plays a key role in vascular proliferative disorders such as atherosclerosis and restenosis. Although several calcium handling proteins that control differentiation of smooth muscle cells have been identified, the role of protein phosphatase inhibitor 1 (I-1) in the acquisition or maintenance of the contractile phenotype modulation remains unknown. METHODS AND RESULTS: In human coronary arteries, I-1 and sarco/endoplasmic reticulum Ca2+ -ATPase expression is specific to contractile vascular smooth muscle cells. In synthetic cultured human coronary artery smooth muscle cells, protein phosphatase inhibitor 1 (I-1 target) is highly expressed, leading to a decrease in phospholamban phosphorylation, sarco/endoplasmic reticulum Ca2+ -ATPase, and cAMP-responsive element binding activity. I-1 knockout mice lack phospholamban phosphorylation and exhibit vascular smooth muscle cell arrest in the synthetic state with excessive neointimal proliferation after carotid injury, as well as significant modifications of contractile properties and relaxant response to acetylcholine of femoral artery in vivo. Constitutively active I-1 gene transfer decreased neointimal formation in an angioplasty rat model by preventing vascular smooth muscle cell contractile to synthetic phenotype change. CONCLUSIONS: I-1 and sarco/endoplasmic reticulum Ca2+ -ATPase synergistically induce the vascular smooth muscle cell contractile phenotype. Gene transfer of constitutively active I-1 is a promising therapeutic strategy for preventing vascular proliferative disorders.

miR-424/322 regulates vascular smooth muscle cell phenotype and neointimal formation in the rat.

AIMS: Our aim was to identify new microRNAs (miRNAs) implicated in pathological vascular smooth muscle cells (VSMCs) proliferation and characterize their mechanism of action. METHODS AND RESULTS: MicroRNAs microarray and qRT-PCR results lead us to focus on miR-424 or its rat ortholog miR-322 (miR-424/322). In vitro mir-424/322 level was decreased shortly after the induction of proliferation and increased in a time-dependent manner later on. In vivo its expression increased in the rat carotid artery from Day 4 up to Day 30 after injury. miR-424/322 overexpression in vitro inhibited proliferation and migration without affecting apoptosis and prevented VSMC dedifferentiation. Furthermore, miR-424/322 overexpression resulted in decreased expression of its predicted targets: cyclin D1 and Ca(2+)-regulating proteins calumenin and stromal-interacting molecule 1 (STIM1). Using reporter luciferase assays, we confirmed that cyclin D1 and calumenin mRNAs were direct targets of miR-322, whereas miR-322 effect on STIM1 was indirect. Nevertheless, consistent with the decreased STIM1 level, the store-operated Ca(2+) entry was reduced. We hypothesized that miR-424/322 could be a negative regulator of proliferation overridden in pathological situations. Thus, we overexpressed miR-424/322 in injured rat carotid arteries using an adenovirus, and demonstrated a protective effect against restenosis. CONCLUSION: Our results demonstrate that miR-424/322 is up-regulated after vascular injury. This is likely an adaptive response to counteract proliferation, although this mechanism is overwhelmed in pathological situations such as injury-induced restenosis.

STIM1 and Orai in cardiac hypertrophy and vascular proliferative diseases.

Cardiac hypertrophy and vascular proliferative diseases are associated with major alterations in calcium homeostasis and calcium signaling. The recent discovery of STIM and Orai has generated great enthusiasm concerning the role of these proteins in the cardiovascular system. We will review the major results concerning the existence and the role of these proteins in the cardiovascular system in normal and pathological situations and their implication in cardiovascular remodeling.

Plasticity-related gene-1 inhibits lysophosphatidic acid-induced vascular smooth muscle cell migration and proliferation and prevents neointima formation.

Plasticity-related gene-1 (PRG-1) protects neuronal cells from lysophosphatidic acid (LPA) effects. In vascular smooth muscle cells (VSMCs), LPA was shown to induce phenotypic modulation in vitro and vascular remodeling in vivo. Thus we explored the role of PRG-1 in modulating VSMC response to LPA. PCR, Western blot, and immunofluorescence experiments showed that PRG-1 is expressed in rat and human vascular media. PRG-1 expression was strongly inhibited in proliferating compared with quiescent VSMCs both in vitro and in vivo (medial vs. neointimal VSMCs), suggesting that PRG-1 expression is dependent on the cell phenotype. In vitro, adenovirus-mediated overexpression of PRG-1 specifically inhibited LPA-induced rat VSMC proliferation and migration but not platelet-derived growth factor-induced proliferation. This effect was abolished by mutation of a conserved histidine in the lipid phosphate phosphatase family that is essential for interaction with lipid phosphates. In vivo, balloon-induced neointimal formation in rat carotid was significantly decreased in vessels infected with PRG-1 adenovirus compared with ß-galactosidase adenovirus (-71%; P < 0.05). PRG-1 overexpression abolished the activation of the p42/p44 signaling pathway in LPA-stimulated rat VSMCs in culture and in balloon-injured rat carotids. Taken together, these findings provide the first evidence of a protective role of PRG-1 in the vascular media under pathophysiological conditions.

miR-421 and miR-30c inhibit SERPINE 1 gene expression in human endothelial cells.

In this work, we assessed whether SERPINE1 expression could be under the influence of microRNAs (miRNAs) predicted to bind the SERPINE1 3'UTR region. We specifically focused on the 3'UTR region harboring a common polymorphism, rs1050955, that have been found associated to SERPINE1 monocyte expression, and investigated whether the presence of different alleles at rs1050955 could modify the miRNAs binding efficiency and affect PAI-1 protein levels. We demonstrated that, in human umbilical vein endothelial cells, both miR-421 and miR-30c directly interacted with PAI-1 mRNA to inhibit the expression of the associated protein. However, these inhibitory mechanisms were independent on the allele present at the rs1050955 locus. We further showed that miR-421 levels correlated with PAI-1 activity in the plasma sample of 40 patients with venous thrombosis. Our results strongly suggest that the regulation of PAI-1 molecule could be under the influence of several miRNAs whose measurement in the plasma of patients could be envisaged as a biomarker for inflammatory and thrombotic disorders.

A new method of ultrasonic nonviral gene delivery to the adult myocardium.

Cardiac gene transfer is a powerful molecular tool to improve our understanding of the role of new proteins and mutants in cardiac pathophysiology. There is a need for a simple efficient myocardial gene delivery technique in order to study the physiological role of proteins in their native environment. Here we tested a new method of myocardial nonviral gene delivery, by using the combination of ultrasound energy (USE), liposomes and high pressure injections to the rat heart. Wistar rats were subjected to intra-myocardial injections of liposomes-DNA or siRNA mix. The heart was exposed after an inter-costal incision, and then injections were conducted between two sets of USE heart exposure. Ultrasound application resulted in much higher transfection efficiency (2% of left ventricle) than the liposomes-DNA alone (0.12% of left ventricle) as shown by the beta-galactosidase staining. The ultrasonic based liposomes-DNA delivery resulted in low inflammatory response, as well as in low cardiac fibrosis as shown by total collagen staining. Quantitative real time polymerase chain reaction (PCR) showed that the ultrasonic delivery resulted in cardiac specific transduction. Moreover, 23,906±2197 and 71,883±4065 calcium tolerant transfected cardiac myocytes were isolated following the delivery of a GFP plasmid or tagged siRNA, respectively. This was sufficient to perform single cell physiological measurements and biochemical experiments on homogenates. We developed an interesting safe method for local gene transfer in the heart using ultrasound and liposomes gene delivery. This method is particularly useful to study the effect of gene transfer on cardiac myocytes maintained in their normal environment in animal models.

Calcium signaling in vascular smooth muscle cells: from physiology to pathology.

Cyclic variations in calcium (Ca(2+)) concentrations, through a process called excitation-contraction coupling, allow regulation of vascular smooth muscle cells contractility and thus modulation of vascular tone and blood pressure. As a second messenger, Ca(2+) also activates signaling cascades leading to transcription factors activation in a process called excitation-transcription coupling. Furthermore, recent evidences indicate an interaction between post-transcriptional regulation by microRNAs (miRNAs) and Ca(2+) signaling. All these actors, which are frequently altered in vascular diseases, will be reviewed here.

Regulation of cAMP homeostasis by the efflux protein MRP4 in cardiac myocytes.

Recent studies indicate that members of the multidrug-resistance protein (MRP) family belonging to ATP binding cassette type C (ABCC) membrane proteins extrude cyclic nucleotides from various cell types. This study aimed to determine whether MRP proteins regulate cardiac cAMP homeostasis. Here, we demonstrate that MRP4 is the predominant isoform present at the plasma membrane of cardiacmyocytes and that it mediates the efflux of cAMP in these cells. MRP4-deficient mice displayed enhanced cardiac myocyte cAMP formation, contractility, and cardiac hypertrophy at 9 mo of age, an effect that was compensated transiently by increased phosphodiesterase expression at young age. These findings suggest that cAMP extrusion via MRP4 acts together with phosphodiesterases to control cAMP levels in cardiac myocytes.

Critical role for stromal interaction molecule 1 in cardiac hypertrophy.

BACKGROUND: Cardiomyocytes use Ca2+ not only in excitation-contraction coupling but also as a signaling molecule promoting, for example, cardiac hypertrophy. It is largely unclear how Ca2+ triggers signaling in cardiomyocytes in the presence of the rapid and large Ca2+ fluctuations that occur during excitation-contraction coupling. A potential route is store-operated Ca2+ entry, a drug-inducible mechanism for Ca2+ signaling that requires stromal interaction molecule 1 (STIM1). Store-operated Ca2+ entry can also be induced in cardiomyocytes, which prompted us to study STIM1-dependent Ca2+ entry with respect to cardiac hypertrophy in vitro and in vivo. METHODS AND RESULTS: Consistent with earlier reports, we found drug-inducible store-operated Ca2+ entry in neonatal rat cardiomyocytes, which was dependent on STIM1. Although this STIM1-dependent, drug-inducible store-operated Ca2+ entry was only marginal in adult cardiomyocytes isolated from control hearts, it increased significantly in cardiomyocytes isolated from adult rats that had developed compensated cardiac hypertrophy after abdominal aortic banding. Moreover, we detected an inwardly rectifying current in hypertrophic cardiomyocytes that occurs under native conditions (i.e., in the absence of drug-induced store depletion) and is dependent on STIM1. By manipulating its expression, we found STIM1 to be both sufficient and necessary for cardiomyocyte hypertrophy in vitro and in the adult heart in vivo. Stim1 silencing by adeno-associated viruses of serotype 9-mediated gene transfer protected rats from pressure overload-induced cardiac hypertrophy. CONCLUSION: By controlling a previously unrecognized sarcolemmal current, STIM1 promotes cardiac hypertrophy.

Ryanodine receptor leak mediated by caspase-8 activation leads to left ventricular injury after myocardial ischemia-reperfusion.

Myocardial ischemic disease is the major cause of death worldwide. After myocardial infarction, reperfusion of infracted heart has been an important objective of strategies to improve outcomes. However, cardiac ischemia/reperfusion (I/R) is characterized by inflammation, arrhythmias, cardiomyocyte damage, and, at the cellular level, disturbance in Ca(2+) and redox homeostasis. In this study, we sought to determine how acute inflammatory response contributes to reperfusion injury and Ca(2+) homeostasis disturbance after acute ischemia. Using a rat model of I/R, we show that circulating levels of TNF-α and cardiac caspase-8 activity were increased within 6 h of reperfusion, leading to myocardial nitric oxide and mitochondrial ROS production. At 1 and 15 d after reperfusion, caspase-8 activation resulted in S-nitrosylation of the RyR2 and depletion of calstabin2 from the RyR2 complex, resulting in diastolic sarcoplasmic reticulum (SR) Ca(2+) leak. Pharmacological inhibition of caspase-8 before reperfusion with Q-LETD-OPh or prevention of calstabin2 depletion from the RyR2 complex with the Ca(2+) channel stabilizer S107 ("rycal") inhibited the SR Ca(2+) leak, reduced ventricular arrhythmias, infarct size, and left ventricular remodeling after 15 d of reperfusion. TNF-α-induced caspase-8 activation leads to leaky RyR2 channels that contribute to myocardial remodeling after I/R. Thus, early prevention of SR Ca(2+) leak trough normalization of RyR2 function is cardioprotective.

Inhibition of MRP4 prevents and reverses pulmonary hypertension in mice.

Multidrug resistance-associated protein 4 (MRP4, also known as Abcc4) regulates intracellular levels of cAMP and cGMP in arterial SMCs. Here, we report our studies of the role of MRP4 in the development and progression of pulmonary arterial hypertension (PAH), a severe vascular disease characterized by chronically elevated pulmonary artery pressure and accompanied by remodeling of the small pulmonary arteries as a prelude to right heart failure and premature death. MRP4 expression was increased in pulmonary arteries from patients with idiopathic PAH as well as in WT mice exposed to hypoxic conditions. Consistent with a pathogenic role for MRP4 in PAH, WT mice exposed to hypoxia for 3 weeks showed reversal of hypoxic pulmonary hypertension (PH) following oral administration of the MRP4 inhibitor MK571, and Mrp4-/- mice were protected from hypoxic PH. Inhibition of MRP4 in vitro was accompanied by increased intracellular cAMP and cGMP levels and PKA and PKG activities, implicating cyclic nucleotide-related signaling pathways in the mechanism underlying the protective effects of MRP4 inhibition. Our data suggest that MRP4 could represent a potential target for therapeutic intervention in PAH.

SERCA2a controls the mode of agonist-induced intracellular Ca2+ signal, transcription factor NFAT and proliferation in human vascular smooth muscle cells.

In blood vessels, tone is maintained by agonist-induced cytosolic Ca(2+) oscillations of quiescent/contractile vascular smooth muscle cells (VSMCs). However, in synthetic/proliferative VSMCs, Gq/phosphoinositide receptor-coupled agonists trigger a steady-state increase in cytosolic Ca(2+) followed by a Store Operated Calcium Entry (SOCE) which translates into activation of the proliferation-associated transcription factor NFAT. Here, we report that in human coronary artery smooth muscle cells (hCASMCs), the sarco/endoplasmic reticulum calcium ATPase type 2a (SERCA2a) expressed in the contractile form of the hCASMCs, controls the nature of the agonist-induced Ca(2+) transient and the resulting down-stream signaling pathway. Indeed, restoring SERCA2a expression by gene transfer in synthetic hCASMCs 1) increased Ca(2+) storage capacity; 2) modified agonist-induced IP(3)R Ca(2+) release from steady-state to oscillatory mode (the frequency of agonist-induced IP(3)R Ca(2+) signal was 11.66 ± 1.40/100 s in SERCA2a-expressing cells (n=39) vs 1.37 ± 0.20/100 s in control cells (n=45), p<0.01); 3) suppressed SOCE by preventing interactions between SR calcium sensor STIM1 and pore forming unit ORAI1; 4) inhibited calcium regulated transcription factor NFAT and its down-stream physiological function such as proliferation and migration. This study provides evidence for the first time that oscillatory and steady-state patterns of Ca(2+) transients have different effects on calcium-dependent physiological functions in smooth muscle cells.

Protease-activated receptor-1 antagonist F 16618 reduces arterial restenosis by down-regulation of tumor necrosis factor α and matrix metalloproteinase 7 expression, migration, and proliferation of vascular smooth muscle cells.

Wound healing after angioplasty or stenting is associated with increased production of thrombin and the activation of protease-activated receptor 1 (PAR1). The aim of the present study was to examine the effects of a new selective PAR1 antagonist, 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)-penta-1,3-dienyl]-benzonitrile (F 16618), in restenosis and vascular smooth muscle cell (SMC) proliferation and migration using both in vivo and in vitro approaches. Daily oral administration of F 16618 inhibited the restenosis induced by balloon angioplasty on rat carotid artery in a dose-dependent manner. Furthermore, single intravenous administration of F 16618 during the angioplasty procedure was sufficient to protect the carotid artery against restenosis. In vitro, F 16618 inhibited the growth of human aortic SMCs in a concentration-dependent manner with maximal effects at 10 µM. At that concentration, F 16618 also prevented thrombin-mediated SMC migration. In vivo, oral and intravenous F 16618 treatments reduced by 30 and 50% the expression of the inflammatory cytokine tumor necrosis factor α (TNFα) 24 h after angioplasty. However, only acute intravenous administration prevented the induction of matrix metalloproteinase 7 expression. In contrast, F 16618 treatments had no effect on early SMC de-differentiation and transcription of monocyte chemoattractant protein-1 and interleukin-6 and late re-endothelialization of injured arteries. Furthermore, F 16618 compensated for the carotid endothelium loss by inhibiting PAR1-mediated contraction. Altogether, these data demonstrate that PAR1 antagonists such as F 16618 are a highly effective treatment of restenosis after vascular injury, by inhibition of TNFα, matrix metalloproteinase 7, and SMC migration and proliferation in addition to an antithrombotic effect.