SMC Proliferation Research Articles

Muscle-derived follistatin-like 1 functions to reduce neointimal formation after vascular injury.

It is well-established that exercise diminishes cardiovascular risk, but whether humoral factors secreted by muscle confer these benefits has not been conclusively shown. We have shown that the secreted protein follistatin-like 1 (Fstl1) has beneficial actions on cardiac and endothelial function. However, the role of muscle-derived Fstl1 in proliferative vascular disease remains largely unknown. Here, we investigated whether muscle-derived Fstl1 modulates vascular remodelling in response to injury. The targeted ablation of Fstl1 in muscle led to an increase in neointimal formation following wire-induced arterial injury compared with control mice. Conversely, muscle-specific Fstl1 transgenic (TG) mice displayed a decrease in the neointimal thickening following arterial injury. Muscle-specific Fstl1 ablation and overexpression increased and decreased, respectively, the frequency of BrdU-positive proliferating cells in injured vessels. In cultured human aortic smooth muscle cells (HASMCs), treatment with human FSTL1 protein decreased proliferation and migration induced by stimulation with PDGF-BB. Treatment with FSTL1 enhanced AMPK phosphorylation, and inhibition of AMPK abrogated the inhibitory actions of FSTL1 on HASMC responses to PDGF-BB. The injured arteries of Fstl1-TG mice exhibited an increase in AMPK phosphorylation, and administration of AMPK inhibitor reversed the anti-proliferative actions of Fstl1 on the vessel wall. Our findings indicate that muscle-derived Fstl1 attenuates neointimal formation in response to arterial injury by suppressing SMC proliferation through an AMPK-dependent mechanism. Thus, the release of protein factors from muscle, such as Fstl1, may partly explain why the maintenance of muscle function can have a therapeutic effect on the cardiovascular system.

Thromboxane A(2) receptor stimulation promotes closure of the rat ductus arteriosus through enhancing neointima formation.

Ductus arteriosus (DA) closure follows constriction and remodeling of the entire vessel wall. Patent ductus arteriosus occurs when the DA does not close after birth, and this condition is currently treated using cyclooxygenase inhibitors. However, the efficacy of cyclooxygenase inhibitors is often limited. Our previous study demonstrated that low-dose thromboxane A2 receptor (TP) stimulation constricted the DA with minimal adverse effects in rat neonates. However, its effect on DA remodeling remains unknown. In this study, we focused on the impact of the exogenous TP stimulation on the DA remodeling, especially intimal thickening. Using DA explants from rat fetuses at embryonic day 19 as a ex vivo model and primary cultured rat DA smooth muscle cells from embryonic day 21 as a in vitro model, we evaluated the effect of TP stimulation on the DA remodeling. The selective TP agonists U46619 and I-BOP promoted neointima formation in the ex vivo DA explants, and TP stimulation increased DA SMC migration in a dose-dependent manner. Both effects were inhibited by the selective TP antagonist SQ29548 or the siRNA against TP. TP stimulation also increased DA SMC proliferation in the presence of 10% fetal bovine serum. LC/MS/MS analysis revealed that TP stimulation increased secretion of several extracellular matrix proteins that may contribute to an increase in neointima formation. In conclusion, we uncovered that exogenous administration of TP agonist promotes neointima formation through the induction of migration and proliferation of DA SMC, which could contribute to DA closure and also to its vasoconstrictive action.

Interferon regulatory factor 3 protects against adverse neo-intima formation

Vascular smooth muscle cell (VSMC) proliferation is central to the pathophysiology of neo-intima formation. Interferon regulatory factor 3 (IRF3) inhibits the growth of cancer cells and fibroblasts. However, the role of IRF3 in vascular neo-intima formation is unknown. We evaluated the protective role of IRF3 against neo-intima formation in mice and the underlying mechanisms. IRF3 expression was down-regulated in VSMCs after carotid wire injury in vivo, and in SMCs after platelet-derived growth factor (PDGF)-BB challenge in vitro. Global knockout of IRF3 (IRF3-KO) led to accelerated neo-intima formation and proliferation of VSMCs, whereas the opposite was seen in SMC-specific IRF3 transgenic mice. Mechanistically, we identified IRF3 as a novel regulator of peroxisome proliferator-activated receptor γ (PPARγ), a negative regulator of SMC proliferation after vascular injury. Binding of IRF3 to the AB domain of PPARγ in the nucleus of SMCs facilitated PPARγ transactivation, resulting in decreased proliferation cell nuclear antigen expression and suppressed proliferation. Overexpression of wild-type, but not truncated, IRF3 with a mutated IRF association domain (IAD) retained the ability to exert anti-proliferative effect. IRF3 inhibits VSMC proliferation and neo-intima formation after vascular injury through PPARγ activation.

Integration of proteomic and transcriptomic profiles identifies a novel PDGF-MYC network in human smooth muscle cells

Platelet-derived growth factor-BB (PDGF-BB) has been implicated in the proliferation, migration and synthetic activities of smooth muscle cells that characterize physiologic and pathologic tissue remodeling in hollow organs. However, neither the molecular basis of PDGFR-regulated signaling webs, nor the extent to which specific components within these networks could be exploited for therapeutic benefit has been fully elucidated. Expression profiling and quantitative proteomics analysis of PDGF-treated primary human bladder smooth muscle cells identified 1,695 genes and 241 proteins as differentially expressed versus non-treated cells. Analysis of gene expression data revealed MYC, JUN, EGR1, MYB, RUNX1, as the transcription factors most significantly networked with up-regulated genes. Forty targets were significantly altered at both the mRNA and protein levels. Proliferation, migration and angiogenesis were the biological processes most significantly associated with this signature, and MYC was the most highly networked master regulator. Alterations in master regulators and gene targets were validated in PDGF-stimulated smooth muscle cells in vitro and in a model of bladder injury in vivo. Pharmacologic inhibition of MYC and JUN confirmed their role in SMC proliferation and migration. Network analysis identified the diaphanous-related formin 3 as a novel PDGF target regulated by MYC and JUN, which was necessary for PDGF-stimulated lamellipodium formation. These findings provide the first systems-level analysis of the PDGF-regulated transcriptome and proteome in normal smooth muscle cells. The analyses revealed an extensive cohort of PDGF-dependent biological processes and connected key transcriptional effectors to their regulation, significantly expanding current knowledge of PDGF-stimulated signaling cascades. These observations also implicate MYC as a novel target for pharmacological intervention in fibroproliferative expansion of smooth muscle, and potentially in cancers in which PDGFR-dependent signaling or MYC activation promote tumor progression.

The cyclic AMP response element‐binding protein (CREB) mediates smooth muscle cell proliferation in response to angiotensin II

The cAMP response element-binding protein (CREB) is a transcription factor that mediates the cellular response to metabolic and mitogenic signals. Whether CREB contributes to vascular function has received little attention, especially in relation to the processes associated with atherosclerotic disease progression and restenosis. This study examined the involvement of CREB in the mitogenic actions of angiotensin II (AngII), a growth factor that promotes neointimal hyperplasia in response to vascular injury. Treatments were performed on quiescent vascular smooth muscle cells (VSMCs) obtained from a porcine explant model. Organ culture was performed on porcine hearts subjected to angioplasty ex vivo. Stimulation of VSMCs with AngII resulted in transient CREB phosphorylation. Proliferation of smooth muscle cells in response to AngII was reduced by 90% after infection with adenovirus expressing dominant-negative killer CREB (kCREB) mutant. Likewise, expression of kCREB prevented angioplasty-induced neointimal hyperplasia. AngII-induced CREB phosphorylation was independent of cAMP activation. Examination of putative CREB kinases revealed that MSK was responsible for phosphorylating CREB. In addition, inhibition of PKC revealed that this kinase operates upstream and activates MSK. These results indicate that activation of CREB via PKC and MSK is essential for SMC proliferation in response to AngII.

Decorin mimic inhibits vascular smooth muscle proliferation and migration.

Over the past 10 years, the number of percutaneous coronary intervention procedures performed in the United States increased by 33%; however, restenosis, which inhibits complete functional recovery of the vessel wall, complicates this procedure. A wide range of anti-restenotic therapeutics have been developed, although many elicit non-specific effects that compromise vessel healing. Drawing inspiration from biologically-relevant molecules, our lab developed a mimic of the natural proteoglycan decorin, termed DS-SILY, which can mask exposed collagen and thereby effectively decrease platelet activation, thus contributing to suppression of vascular intimal hyperplasia. Here, we characterize the effects of DS-SILY on both proliferative and quiescent human SMCs to evaluate the potential impact of DS-SILY-SMC interaction on restenosis, and further characterize in vivo platelet interactions. DS-SILY decreased proliferative SMC proliferation and pro-inflammatory cytokine secretion in vitro in a concentration dependent manner as compared to untreated controls. The addition of DS-SILY to in vitro SMC cultures decreased SMC migration and protein synthesis by 95% and 37%, respectively. Furthermore, DS-SILY decreased platelet activation, as well as reduced neointimal hyperplasia by 60%, in vivo using Ossabaw swine. These results indicate that DS-SILY demonstrates multiple biological activities that may all synergistically contribute to an improved treatment paradigm for balloon angioplasty.

MiR-663 and the miRaculous Vascular Smooth Muscle Phenotypic Switch

Cardiovascular diseases, such as ischemic heart disease and stroke, represent the number one cause of death worldwide.1 In most cases, atherosclerosis and hypertension constitute the underlying causes for cardiovascular disease because it leads to arterial occlusion and impaired cardiac function, respectively. Therapeutic compensation of atherosclerotic artery occlusion by bypass grafting, angioplasty, or stenting bears the risk of intimal hyperplasia and subsequent restenosis.2 In general, a prerequisite for any pathological and physiological vascular remodeling process is the phenotypic switch of vascular smooth muscle cells (VSMCs).3 Even in adult blood vessels, VSMCs retain a remarkable plasticity that is essential for any changes in the vessel wall architecture. Contractile VSMCs representing the majority of VSMCs in healthy vessels guarantee maintenance of vascular tone and thereby the systemic blood pressure and blood flow by controlling the blood vessel diameter. As outlined in the Figure, a plethora of humoral factors, such as platelet-derived growth factor-BB and biomechanical stimuli, especially chronic changes in blood flow and wall stress, is capable to trigger the phenotypic switch of VSMCs from a quiescent, contractile differentiated state to a synthetic, proliferative, and dedifferentiated state.3,4 Figure. The effect of microRNA (miR)-633 on vascular homeostasis and remodeling. Major stimuli, transcription factors, and miRs that are involved in the differentiation and phenotypic switch of vascular smooth muscle cells (VSMCs) are depicted. miR-143/miR-145 promotes the contractile state by inhibiting suppressors of myocardin/serum response factor (SRF) activity (eg, ETS domain-containing protein-1 [Elk-1] and Kruppel-like factor [KLF] 4/5). Interestingly, myocardin activity seems to be regulated by an intrinsic loop because it stabilizes the expression of miR-145, whereas miR-143 seems to inhibit myocardin expression. miR-633 is a novel player with a dual effect on the VSMC phenotype not only by attenuating proliferation, migration, and proteolytic activity, but also by limiting their …

Abstract 306: Transfected Early Growth Response Gene-1 DNA Enzyme Attenuates Autogenous Vein Graft Intimal Hyperplasia in vivo

Introduction Early growth response gene-1 (Erg-1) functions centrally in cell proliferation. We hypothesized that introduction of a novel Egr-1 DNA enzyme (EDRz) via liposomes into the vein graft would attenuate occlusive SMC proliferation and intimal hyperplasia. Methods Rat jugular vein was anastomosed to the infrarenal abdominal aorta end-to-end. EDRz (n=90) was applied to the graft vein adventitia (no therapy control; n=90). Animals were harvested at 1, 2, and 6hr, and 1, 3, 7, 14, 28, and 42d (10 EDRz/10 control per time point). Analyses included light microscopy (morphology), fluorescence (EDRz transfection), RT-PCR and in situ hybridization (Egr-1 mRNA), and Western blot and immunohistochemistry (Egr-1 protein). Results EDRz localized to the vein graft media early; Egr-1 protein was in medial SMCs, monocytes, and ECs; treated grafts had no detectable Egr-1 RNA or protein by 7d. VSMC proliferation (p<0.01 from 3-42d) and intimal thickness were attenuated by EDRz (p<0.01 from 7-42d; Fig ). Conclusions EDRz reduces Egr-1 expression, SMC proliferation, and intimal hyperplasia in autogenous vein grafts. EDRz offers a novel approach to enhance vein graft durability. Figure. The result of thickness of intimal hyperplasia in vein graft and EDRz transfection after 1

Abstract 502: Arterial Calcification Increases Neointimal Proliferation Following Balloon Injury in Rat Carotid Arteries

Introduction Arterial calcification predicts accelerated restenosis after angioplasty and stenting. We sought to determine how calcification affected neointimal accumulation after balloon injury in the rat carotid. Hypothesis We propose that transformed SMCs in calcified arteries respond to balloon injury in an accelerated manner through a mechanism involving bone-related factors. Methods Arterial calcification was induced in male Sprague-Dawley rats weighing 350-400 grams by subcutaneous administration of vitamin D3. Control rats received vehicle. Both groups underwent left carotid balloon injury one week following injection, a time at which significant medial calcification has developed. Arteries were harvested twenty-one days following balloon injury. The degree of neointimal cell proliferation was next assessed by immunohistochemical staining for PCNA. Neointimal hyperplasia was measured by morphometric analysis using standard imaging analysis software. Results At 3 weeks after carotid balloon injury, mean cellular proliferation as measured by PCNA staining was higher in the intima of calcified than control arteries (63.8 ± 6.0 versus 40.5 ± 9.5 cells/hpf, P=ns). Calcified arteries from Vitamin D3-treated rats, however, had significantly more intimal hyperplasia than control uncalcified arteries (intima-to-media ratio = 1.374 ± 0.071 versus 0.758 ± 0.080, P=0.0021). Intimal and medial areas were also individually larger in calcified than control arteries. Conclusions Neointimal hyperplasia is accelerated in Vitamin D3-treated rats through a mechanism that partially involves increased SMC proliferation. Calcified vessels may respond differently to balloon angioplasty and this may explain the poorer results of endovascular interventions in patients with arterial calcification. Other factors including accelerated migration and increased matrix synthesis may also be involved. Treatments to inhibit restenosis in calcified arteries may differ from those that work in uncalcified vessels.

Abstract 2426: In vitro and in vivo evaluation of dasatinib and imatinib on physiological parameters of pulmonary arterial hypertension .

Abstract Pulmonary arterial hypertension (PAH), defined by increase in mean pulmonary arterial pressure (PAP), results from occlusion or vasoconstriction of the pulmonary veins and can lead to progressive right ventricular failure. The tyrosine kinase inhibitor (TKI) imatinib, indicated for chronic myelogenous leukemia (CML) is being investigated as a potential treatment for PAH, due to its anti-vasoproliferative properties. However, the TKI dasatinib also used for CML is associated with PAH in a small percentage of heavily pre-treated patients although partial or complete reversibility is seen after discontinuation of treatment. Despite the different kinase profiles, studies in rat models of PAH have shown that both TKIs are equally efficacious in reversing functional and structural PAH-related changes. Therefore, nonclinical in vivo (SD rat) and in vitro (human pulmonary artery endothelial cells/smooth muscle cells [hPAEC/hPASM) studies evaluating direct effects of the two TKIs were conducted to understand the potential mechanistic differences for the apparently different clinical effect. The in vivo study explored direct effects of vehicle (80 mM citric acid; control), clinically relevant doses of imatinib and dasatinib (30 or 8 mg/kg/day, oral) and monocrotaline (single i.p. dose, 70 mg/kg; positive control) on PAH-related pulmonary changes in rats (1-month treatment). Monocrotaline reduced nitric oxide (NO; vasodilation) and increased endothelin-1 (ET-1; vasoconstriction) levels in plasma, induced structural changes (perivascular inflammation, EC injury and SMC proliferation) in PA and lungs, and increased (2-5× control) systolic and diastolic PAP and right ventricular pressure. In contrast, both imatinib and dasatinib increased NO in plasma (2.5×), did not any induce PAH-related structural changes (PA or lungs) and did not alter hemodynamic function compared to controls. The in vitro hPAEC/PASM co-culture model demonstrated that imatinib and dasatinib at clinically relevant (Cmax) concentrations increased NO and decreased ET-1 protein and mRNA. Our results demonstrate that dasatinib, as imatinib, does not have the potential to directly induce PAH-related changes in vivo or in vitro. In addition, both molecules induce biochemical changes in vivo and in vitro consistent with a protective effect on PAP. Citation Format: Mausumee Guha, James Hennan, Julia Li, Damir Simic, Jochen Woiche, Bethany Baumgart, Thomas Sanderson, Michael Graziano, Roderick Bunch. In vitro and in vivo evaluation of dasatinib and imatinib on physiological parameters of pulmonary arterial hypertension . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2426. doi:10.1158/1538-7445.AM2013-2426 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

P58: Secondary amines containing one aromatic nitro group: Preparation, nitrosation, sustained nitric oxide release, and the synergistic effects of released nitric oxide and an arginase inhibitor on vascular smooth muscle cell proliferation

Background Nitric oxide (NO) is synthesized by vascular endothelial cells in vivo from l -arginine by nitric oxide synthases (NOS). Initial events in the onset of atherosclerosis lead to endothelial dysfunction and subsequent decrease of endogenous NO. Within the vasculature NO inhibits smooth muscle cell proliferation (SMC), therefore decreased concentrations lead to rapid SMC division, a key event in the formation of atherosclerotic plaques. Reestablishing physiological levels exogenously with NO-donors, aims to stop uncontrolled SMC proliferation and the progression of atherosclerosis. However current NO donors rapidly release NO in highly concentrated, single bursts, leading to cellular oxidative stress. In contrast, N-nitroso (N–NO) NO donors synthesized in our lab through the nitrosation of secondary amines, release NO in a slow, sustained manner, establishing and maintaining low NO concentrations that effectively reduce SMC proliferation, without onset of oxidative stress.Additionally, NOS competes for l -arginine with arginase, which catalyzes its conversion to l -ornithine, the precursor to various polyamines-known stimulators of SMC proliferation. Arginase inhibition would reduce SMC proliferation by decreasing l -ornithine concentrations and providing increased l -arginine for endogenous NO production. Combining arginase inhibition with NO-donor application may prevent SMC proliferation more effectively than either method individually, and may have a synergistic effect. Methods Two isomeric families of NO-donors, prepared in our laboratory, were applied to SMC cultures at various concentrations. Each secondary amine precursor was also tested for any toxic or anti-proliferative effects. Likewise the potent arginase inhibitor, ABH was tested to determine the most effective concentration for proliferation reduction. Finally, optimized concentrations of NO-donors and ABH were added in tandem. Proliferation and toxicity were monitored using a cell viability and LIVE/DEAD assay. A third assay was implemented to monitor changes in intracellular NO concentrations due to the addition of each compound. Results Cellular viability assays showed eight of ten amines exerted no toxic or anti-proliferative effects in the range tested (0.1–20 μM), nor did they increase intracellular NO concentrations. These compounds were nitrosated and tested in same concentration range. All donors exhibited proliferation inhibition without inducing cell death between 5% and 20 μM in a concentration dependent manner. At 20 μM, the decrease in viable cells ranged from 5% to 22%. Likewise, ABH decreased the number of viable cells in a concentration dependent manner from 0.001 to 5.0 mM by 9.9–30.0%, respectively. Both the NO-donors and ABH increased intracellular NO with increasing concentrations. Finally, the most effective NO-donor from each family (20 μM) in combination with ABH (5 mM) inhibited SMC proliferation in a synergistic manner, reducing the number of viable cells by an additional 2% and 16% compared to the sum of individual components alone. These results were supported by the synergistic increase of intracellular NO. Conclusion Inhibition of SMC proliferation after endothelial dysfunction could slow down the establishment of arterial plaques, reducing the occurrence of cardiovascular diseases including atherosclerosis. Reestablishing NO concentrations both exogenously with NO-donors and endogenously by arginase inhibition with ABH simultaneously has shown to more effectively reduce SMC proliferation than either method individually, while avoiding onset of oxidative stress. Disclosure NIH, Award # R15HL 106600.

TRPV4‐mediated Ca2+ influx promotes airway smooth muscle cell proliferation via NFATc3

Bronchial asthma is a highly prevalent chronic respiratory disease with significant impact on human health. Airway remodeling due to proliferation of airway smooth muscle cells (ASMC) causes irreversible airflow obstruction during asthma, but the underlying molecular mechanisms are unclear. We hypothesized that Ca2+ influx via TRPV4 contributes to ASMC proliferation. We find that the selective TRPV4 agonist GSK1016790A elicited concentration‐dependent Ca2+ influx (EC50 = 18.1±2.4 nM) in primary rat ASMC that was blocked by the TRPV4 antagonist HC067047 (500 nM). GSK1016790A enhanced ASMC proliferation (EC50=31.0±7.5 nM) whereas HC067047 (IC50=220 ±46 nM) had the opposite effect. Involvement of the Nuclear Factor of Activated T cells (NFAT), a Ca2+‐activated transcription factor important for SMC proliferation, was examined. The calcineurin blocker cyclosporine (10 μM), and expression of the NFAT‐inhibiting peptide VIVIT attenuated GSK1016790A‐induced ASMC proliferation. In addition, GSK1016790A induced translocation of a cytoplasmic NFATc3‐GFP fusion protein to the nucleus of ASMC. Our findings show that Ca2+ influx via TRPV4 induces proliferation of ASMC by activating NFATc3. Supported by HL091905.

Mitochondrial Activity - a Novel Strategy for Prevention and Reversion of Transplant Vasculopathy

Background: This study aimed at investigating the efficacy of dichloracetate (DCA) to prevent the development and progression of chronic allograft vasculopathy (CAV) by regulating the mitochondrial metabolism of aberrant proliferating smooth muscle cells. Methods: Orthotopic aortic transplantations were performed in the PVG-to-ACI rat model and followed over 120 days. Immunosuppression with DCA (0.75mg/L) or everolimus+clopidogrel (4 mg/kg and 1mg/kg per os, respectively) was either started early or delayed when CAV was already present. Through levels were monitored. Aortic luminal obliteration was quantified using computer morphometry and confocal microscopy. Donor-reactive antibodies were quantified by flow cytometry. Results: Untreated animals developed CAV with luminal obliteration of 40.1±24.2% after 120 days. Continuous immunosuppression with DCA or everolimus/clopidogrel effectively prevented the development of vasculopathy (7.9±4 and 8.1±12.9, respectively; p< 0.001 vs. no treatment). When the start of the immunosuppressive regimen was delayed until postoperative day 80, DCA and everolimus/clopidogrel inhibited a progression of established CAV (6.6±8 and 5.3±4.6; p< 0.001 vs. no treatment). Even more importantly, both treatment regimens did also reverse the luminal obliteration (p< 0.001). Interestingly, delayed treatment of DCA induced apoptosis resulting in shrinking hyperplasia area and inhibition of aberrant SMC proliferation. A stable elevation of donor-reactive IgG-antibody levels was found over 120 days in the absence of treatment. DCA was very well tolerated. Conclusions: DCA prevents the development of CAV and not only inhibits a progression of established disease, but also reverses the established disease by inducing apoptosis. It shows a similar efficacy but a safer drug profile when compared to everolimus/clopidogrel, since only aberrant SMC are affected.

Abstract 185: The Impact of Cyclic Strain on Focal Adhesion Signaling and miRNA Profile in Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSCM) are major contributors to the regulation of vascular tone through their contractile flexibility. Along with other stimulus, they are exposed to mechanical cyclic strain. This is induced by changes in arterial blood pressure waveforms, generated from the left ventricle. It is thought such stimulus can be a major regulator of phenotypic modulation in smooth muscle cells. These altered states of VSMC fate can have a direct influence on cell function, including proliferation, apoptosis, migration and adhesion. This is considered to play a critical role in the pathogenesis of cardiovascular disease. We have shown that cyclic strain, an important physiological stimulus, has a compounding influence on smooth muscle cell function via mechanotransduction. Increased strain has shown to increase SMC adhesion, proliferation and migration. We hypothesise that the effect of mechanical cyclic strain is transduced through the cell via integrin rich focal adhesions, which can alter the miRNA signature of vascular smooth muscle cells and ultimately play a pivotal role in cell function. The Focal Adhesion Kinase (FAK) protein and its endogenous negative regulator FAK Related Non Kinase (FRNK) are known to play a commanding role in integrin signaling. We propose that down regulation of FAK by either siRNA technology or over expression of FRNK will negate the effect of pathological mechanical strain on cell function. We also predict regulation of expression on individual miRNA, through use of Pre-Mir and Anti-Mir technology could yield a similar effect. In the current study we exposed primary derived human aortic smooth muscle cells, in an in vitro model, to cyclic strain to assess its influence on focal adhesions, integrin and miRNA expression. We have concluded thus far that increased cyclic strain shows increased cell migration, adhesion and proliferation. It also alters the miRNA signature as we have shown with a panel of mechano-sensitive miRNA. Coinciding with this we have also demonstrated the impact on cellular function from knockdown of miRNA biogenesis molecules such as Drosha, Dicer, Exportin 5 and Argonaute2 through use of siRNA.

Physical association of Angiotensin‐II type 1 Receptor (AT1R) and NOX‐1 mediates NF‐κB and AP‐1‐dependent Interleukin‐ 18 induction and Aortic SMC Migration and Proliferation

Persistent elevation of angiotensin (Ang)‐II promotes atherosclerosis via oxidative stress and induction of inflammatory cytokines. Since NF‐κB and AP‐1 are important mediators in Ang‐ II signaling pathways, and since the pro‐mitogenic and pro‐migratory factor interleukin (IL)‐18 is an NF‐κB and AP‐1 responsive gene, we investigated whether Ang‐II‐mediated SMC migration and proliferation are IL‐18 dependent. Ang‐II stimulated the migration and proliferation of SMC isolated from rat thoracic aorta. Ang‐II induced O2− generation, IKKβ and JNK activation, p65 and c‐Jun phosphorylation, NF‐κB and AP‐1 activation, and IL‐18 transcription, effects that were attenuated by losartan, DPI, and NOX‐1 knockdown. Both αIL‐18 Ab and IL‐18BP attenuated Ang‐II induced SMC migration and proliferation. Importantly, AT1R binds NOX‐1 in vitro and in vivo. Ang‐II also induced Nox1 transcription via AP‐1. Ang‐II infusion (7 days) increased NOX‐1 mRNA expression, DPI‐inhibitable O2− generation, p65 and c‐Jun phosphorylation, and IL‐18 induction in endothelium‐denuded thoracic aorta in an AT1R‐dependent manner.ConclusionsAng‐II stimulates SMC migration and proliferation via AT1R/NOX‐1‐ mediated IKK/NF‐κB and JNK/AP‐1‐dependent IL‐18 induction, and this process may involve AT1R/NOX‐1 physical association. The AT1R/NOX‐1/IL‐18 pathway may be a critical mechanism in atherogenesis and restenosis.

357 Comparative Effects of the Drugs Used for Pulmonary Hypertension Reversibility Testing: A Meta-Analysis

pyruvate dehydrogenase kinase (PDK) 2, catalyzing the phosphorylation of pyruvate dehydrogenase (PDH) and thus regulating the flux of pyruvate into the mitochondria, were investigated during SMC proliferation and the development of human IH. Methods and Materials: A novel human intimal hyperplasia (IH) model was established in which balloon-injured human internal mammary arteries (hMA) were transplanted into the abdominal aortic position of nude rats. Results: IH rapidly developed over 21 days and was morphologically and in immunohistochemistry identical to the neointima of human coronary arteries. Over 21 days, we found increased proliferative and decreased apoptotic activity within the IH lesion and luminal obliteration progressed to approx. 60%. Tissue PDK2 expression was significantly elevated and PDH was inhibited.PDGF was markedly elevated in the animal serum during neointima formation. Activation of SMC in vitro with PDGF caused increased PDK2 activity via both PI3K/Akt and Ras/Raf/Erkpathways. We observed an increase of the mitochondrial membrane potential ( m), a decreased release of mitochondrial reactive oxygen species (mROS), as well as enhanced cell proliferation. PDK2 inhibition using dichloroacetate(DCA) or PDK2 shRNA prevented PDGF-induced mitochondrial hyperpolarization and SMC proliferation.In vivo, oral DCAtreatment of rats or pre-implant PDK2 shRNA-transduction of the hMAs prevented the injuryinduced increase of SMC proliferation and significantly decreased IH development. Conclusions: PDK2 is the link between PDGF downstream pathways and mitochondrial metabolic modulation and its activation facilitates SMC proliferation. Pharmacological or genetic inhibition of PDK2 in SMC attenuates PDGF-induced increase in m, cell proliferation and the development of human IH. PDK2 may be a novel target for the prevention of restenosis.

T6 TRAIL is a potential novel therapeutic target in pulmonary arterial hypertension

Introduction and ObjectivesPulmonary Arterial Hypertension (PAH) is a life threatening disease characterised by the progressive narrowing and occlusion of small pulmonary arteries, driven by the dysregulated growth of vascular cells....

Xanthine Oxidase-Derived ROS Upregulate Egr-1 via ERK1/2 in PA Smooth Muscle Cells; Model to Test Impact of Extracellular ROS in Chronic Hypoxia

Exposure of newborn calves to chronic hypoxia causes pulmonary artery (PA) hypertension and remodeling. Previous studies showed that the redox-sensitive transcription factor, early growth response-1 (Egr-1), is upregulated in the PA of chronically hypoxic calves and regulates cell proliferation. Furthermore, we established in mice a correlation between hypoxic induction of Egr-1 and reduced activity of extracellular superoxide dismutase (EC-SOD), an antioxidant that scavenges extracellular superoxide. We now hypothesize that loss of EC-SOD in chronically hypoxic calves leads to extracellular superoxide-mediated upregulation of Egr-1. To validate our hypothesis and identify the signaling pathways involved, we utilized PA tissue from normoxic and chronically hypoxic calves and cultured calf and human PA smooth muscle cells (PASMC). Total SOD activity was low in the PA tissue, and only the extracellular SOD component decreased with hypoxia. PA tissue of hypoxic calves showed increased oxidative stress and increased Egr-1 mRNA. To mimic the in vivo hypoxia-induced extracellular oxidant imbalance, cultured calf PASMC were treated with xanthine oxidase (XO), which generates extracellular superoxide and hydrogen peroxide. We found that 1) XO increased Egr-1 mRNA and protein, 2) XO induced the phosphorylation of ERK1/2 and, 3) pretreatment with an ERK1/2 inhibitor prevented induction of Egr-1 by XO. siRNA knock-down of EC-SOD in human PASMC also upregulated Egr-1 mRNA and protein, activated ERK1/2, and enhanced SMC proliferation and reduced apoptosis. We conclude that an oxidant/antioxidant imbalance arising from loss of EC-SOD in the PA with chronic hypoxia induces Egr-1 via activation of ERK1/2 and contributes to pulmonary vascular remodeling.

Abstract 16803: Endothelial Cells Communicate with Vascular Smooth Muscle Cells Via Transfer of microRNA Contained in Secreted Exosomes

Background: Endothelial cell (EC) mediated inhibition of vascular smooth muscle cell (VSMC) proliferation plays an important role in maintaining vascular wall homeostasis and preventing atherosclerosis. The mechanism that mediates this effect has not been clarified. MicroRNAs (miR) are ∼23nt RNAs that regulate many important cellular processes by post-transcriptional regulation of gene expression. Exosomes are small (30-100nm) vesicles secreted from cells, and previously shown to contain miR. We hypothesized that intercellular transfer of miRNA from EC to VSMC via exosomes contributes to the inhibition of VSMC proliferation. Results: We show that EC secreted exosomes contain abundant miRs and protect their contents from degradation by circulating RNases. The miR population in secreted exosomes differs significantly from the cytoplasmic miR population, indicating regulation of miR packaging in ECs. Conditioned media of ECs inhibited Human Aortic VSMC (HAVSMC) proliferation by 37.4% (n=4, p=0.0006) at 120 hrs, while exosome-depleted conditioned media loses this inhibitory effect (n=4, p=0.0001). Purified exosomes applied directly to VSMCs inhibited HAVSMC proliferation by 28.8% at 72hrs (n= 8, p= 0.0107) and by 26.9% at 96 hrs (n=8, p=0.0596). Similar results were found with Coronary Artery SMC proliferation (data not shown). EC-derived exosomes are taken up into recipient SMCs, as seen by ICC to an exosome marker, GFP-CD63, and by qPCR to transferred miRNAs. Transfection into VSMC of RNA purified from EC secreted exosomes is sufficient to inhibit SMC proliferation (34.8%, n= 9, p=0.0001), and provides a similar level of inhibition compared to whole exosome treatment (33.5%, n=9, p=0.001). The miRNA population of exosomes can be engineered through transfection of the progenitor EC; transfection with pre-mir-203 leads to a 317-fold increase in exosomal mir-203 levels, and a 126-fold increase in SMCs treated with EC-derived conditioned media. Conclusions: This is the first evidence that ECs can communicate with VSMCs via transfer of exosomal miRs and further, identify this process as a mechanism for EC-mediated inhibition of VSMC proliferation. These findings also identify exosomal miRs as a potential therapeutic agent in vascular disease.

Abstract 8089: A Novel Method to Inhibit Diabetes Accelerated Atherosclerosis in a Pig Model

Smooth muscle cells grown in high glucose are more responsive to IGF-I than those grown in normal glucose. This requires ligand binding to a unique region of the β3 subunit (C-loop) of αVβ3 integrin. This study was undertaken to determine whether systemic administration of an anti-C-loop β3 (Cloop) antibody to hyperglycemic pigs would inhibit the IGF-I mediated signaling events and the development of atherosclerosis. Pigs were placed on a high fat diet containing between 0.5 and 2% cholesterol to maintain circulating cholesterol levels &gt; 500mg/dL. Hyperglycemia (glucose levels &gt; 350mg/dL) was induced with a single injection of streptozotocin (50mg/kg). At the onset of hyperglycemia subcutaneous injections of either control or Cloop antibody were given every 72 hours for 16 weeks (0.3mg/kg). The mean neointimal area (defined as the area inside the internal elastic lamina minus the luminal area) of the aorta was reduced by 70% in the pigs treated with the anti-Cloop antibody compared with the diabetic pigs treated with control antibody. There was a &gt; 90% reduction in neointimal area in the left main artery in the treated compared with the untreated control pigs. Similarly, there was a greater than 75% reduction in neointimal area in the right coronary artery and circumflex artery. Treatment with the antibody reduced the neointimal formation in the left anterior descending artery by 30%. Biochemical analysis of femoral artery homogenates demonstrated that treatment with the anti-Cloop antibody reduced MAPK and AKT signaling by 36 and 40 % respectively compared with control treated animals. We have previously shown that IGF-I stimulation of these pathways is increased in hyperglycemia in a β3 ligand occupancy dependent manner and results in SMC migration and proliferation. To confirm inhibition of β3 activation we determined that there was a 43% reduction in β3 phosphorylation, a marker of its activation state. Our results demonstrate that systemic administration of the C-loop antibody to hyperglycemic hyperlipidemic pigs reduces IGF-I signaling and this is associated with a significant reduction in the development of atherosclerotic lesions in the coronary arteries and aorta.