Vascular Smooth Muscle Cell Activation Research Articles

Abstract 11691: Atorvastatin Reverses Impaired Voltage-gated K + Channels-mediated Coronary Vasodilation By Upregulating PPARγ In Diabetes (Best of Basic Science Abstract)

Aims: Voltage-gated K + (K v ) channels in vascular smooth muscle cells (VSMCs) play an important role in the regulation of coronary microcirculation. Atorvastatin (ATV) has shown some beneficial effects on vascular function, but the effect of ATV on K v channels-mediated coronary vasodilation remains unknown. The present study was designed to investigate the role of ATV in improving K v channels-mediated coronary dilator function in diabetes and the underlying mechanisms. Methods: Isolated VSMCs were incubated in normal or high glucose medium plus a different dose of ATV for 24 h at 37 o C. Patch-clamp recording and molecular biological techniques were used to assess the function and expression of K v channels. Control or type 2 diabetic rats were treated with ATV (50 mg/kg daily) by oral gavage for 10 weeks. Vasodilation of isolated rat coronary arteries was measured using a pressurized myograph. GW9662, the peroxisome proliferator-activated receptor gamma (PPARγ) antagonist, was used to determine whether the mechanism of ATV-improved K v channel function can be explained by upregulation of PPARγ pathway. Results: Patch-clamp analysis revealed that high glucose reduced K v current density by 58.7 ± 4.6%, which was accompanied by a downregulation of K v 1.2 and K v 1.5 expression. Treatment with ATV reversed the inhibitory effect of high glucose on K v current density in a dose-dependent manner (1 μmol/L, 15.0 ± 2.6%; 10 μmol/L, 49.1 ± 3.8%; 100 μmol/L, 69.9 ± 4.8%; P < 0.05). In addition, ATV restored high glucose-induced downregulation of K v channel protein expression, and the difference was significant in both 10 μmol/L and 100 μmol/L groups. For in vivo study, K v channels-mediated coronary vasodilation was decreased in diabetic rats, compared with controls (9.1 ± 1.3 vs. 36.7 ± 1.4%, P < 0.05), whereas this decrease was partly corrected by ATV (25.0 ± 2.8 vs. 9.1 ± 1.3%, P < 0.05). Treatment with ATV prominently increased protein expression of PPARγ both in vitro and in vivo . The effect of ATV in regulating K v channels and K v channels-mediated vasodilation was markedly blunted by GW9662. Conclusions: In conclusion, treatment with ATV activates PPARγ pathway and preserves K v channel activity in VSMCs, thus providing improvement of coronary dilator function in diabetic rats.

Abstract 14953: STAT3 in Vascular Smooth Muscle Cells Protects Aorta From Dissection by Reinforcing Aortic Wall

Aortic dissection (AD) is a common and fatal disease for which pathogenesis is largely unknown. Recently we and others reported that JAK/STAT3-activating cytokines are highly expressed in human and mouse models of AD. In this study, we used a mouse model of AD that was induced by continuous infusion of beta-aminopropionitrile, an inhibitor of lysyl oxidase that cross-links collagen and elastin, and angiotensin II (BAPN+AngII). Mouse AD tissue showed STAT3 activation (phosphorylation) both in inflammatory cells and in vascular smooth muscle cells (VSMCs). We used the smooth muscle-specific knockout mice for SOCS3, a negative feedback regulator of STAT3 (smSOCS3-KO), to activate STAT3 specifically in VSMCs. smSOCS3-KO mice developed less severe AD in the aortic arch (WT; 0.093±0.018 vs. smSOCS3-KO; 0.018±0.013 mm/g body weight, P=0.014), as determined by the lesion length. Immunofluorescence staining showed phospho-STAT3 in medial VSMCs by BAPN+AngII, which was more prominent in smSOCS3-KO as expected. Interestingly, smSOCS3-KO aorta showed more collagen fibers, suggesting that STAT3 activation in VSMCs may augment the tensile strength of aorta. Indeed, direct measurement of the tensile strength of the excised aortic rings indicated that smSOCS3-KO aorta had more tensile strength compared to wild type aorta (WT; 0.171±0.018 vs. smSOCS3-KO; 0.234±0.011 N/mm, P=0.004). Consistently, adventitia of smSOCS3-KO aorta showed more fibroblasts, as determined by ER-TR7 staining, compared to WT aorta. We also examined surgical specimens from AD patients for phospho-STAT3 and collagen deposition. Strong signal of phospho-STAT3 was detected at the site of dissection. However, phospho-STAT3 was reduced where strong collagen deposition was observed, suggesting that STAT3 activity is reduced after the completion of collagen deposition. From these results we concluded that activation of STAT3 in VSMCs reinforces aortic wall by enhancing collagen deposition, thus protecting aorta from dissection. Understanding of the protection mechanism of the aortic walls from dissection would lead to better understanding of the molecular pathogenesis, which is essential for developing novel therapeutic strategy for AD.

Abstract 15762: Gapdh Interaction With Ape1 Endonuclease Protects Vascular Smooth Muscle Cells Against Apoptosis: Potential Role of These Enzymes in Prevention of Atherosclerotic Plaque Destabilization

We have shown that oxidized lipids (OxLDL) co-localized with apoptotic vascular smooth muscle cells (VSMC) in atherosclerotic plaque and that OxLDL downregulated the major glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cultured VSMC via H2O2-dependent mechanism. We hypothesized that maintenance of sufficient GAPDH levels is critical for cell survival under oxidative stress. H2O2 (220 uM, 12h) decreased GAPDH protein (55±5% decrease) and induced apoptosis (TUNEL assay) in human VSMC. Human VSMC transfection with pCMV-GAPDH increased GAPDH protein (3.5-fold) and prevented H2O2-induced apoptosis (74±4% decrease) vs. pCMV-GFP. We generated rat VSMC with constitutive 2-fold overexpression GAPDH (R3-VSMC). R3-VSMC had increased glycolysis (2.5 folds increase, pyruvate levels), ATP levels (38±2% increase) and reduced oxidative DNA damage (40±2% reduction in number of apurinic/apyrimidinic (AP) sites) and these effects correlated with suppressed H2O2-induced cell apoptosis (>90% reduction, TUNEL assay; 45.7±3.6% reduction, Cell Death ELISA). GAPDH-targeted siRNA reduced GAPDH protein (for 55%) and potentiated H2O2-induced apoptosis (9.4±0.3 folds increase, TUNEL; 8.6±0.4 folds increase, ELISA) in WT-VSMC and blocked anti-apoptotic effect seen in R3-VSMC. GAPDH known to bind apurinic/apyrimidinic (AP) endonuclease 1 (APE1), a major DNA repair enzyme. We found that GAPDH was co-immunoprecipitated and co-localized with APE1 in cytosolic/nuclear fraction and H2O2 induced further GAPDH-Ape1 co-localization and migration toward nuclei. APE1 specific activity was increased in H2O2-treated R3-VSMC vs. H2O2-treated WT VSMC, suggesting that GAPDH preserved APE1 activity under oxidative stress. APE1-targeted siRNA abrogated the protection of GAPDH overexpression against H2O2-induced apoptosis in VMSC. In summary, GAPDH downregulation mediates oxidant-induced VSMC apoptosis and forced expression of GAPDH protects VSMC from H2O2-induced cell death potentially via activation of APE1-dependent DNA repair. Our data suggest that preservation of GAPDH and APE1 activity in plaque VSMC could be a potential strategy to stabilize plaque and prevent acute coronary events.

P21WAF1 Is Required for Interleukin-16-Induced Migration and Invasion of Vascular Smooth Muscle Cells via the p38MAPK/Sp-1/MMP-9 Pathway.

Interleukin-16 (IL-16) is a lymphocyte chemoattractant factor well known for its role in immune responses, but its role in vascular disease is unknown. Here, we explored the novel physiological function of IL-16 in vascular smooth muscle cells (VSMCs). The expression of IL-16 and its receptor CD4 was observed in VSMCs. Treatment with IL-16 enhanced the migration and invasion by VSMCs without altering the proliferative potential. IL-16 induced MMP-9 expression via the binding activity of transcription factors NF-κB, AP-1, and Sp-1 motifs in VSMCs. Among the relevant signaling pathways examined, only p38MAPK phosphorylation was significantly stimulated in IL-16-treated VSMCs. Treatment with p38MAPK inhibitor SB203580 prevented the IL-16-induced migration and invasion of VSMCs. SB203580 treatment inhibited the MMP-9 expression and activation of Sp-1 binding in IL-16-treated VSMCs, and siRNA knockdown of CD4 expression blocked the induction of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activation stimulated by IL-16. The IL-16 induced cell-cycle-inhibitor p21WAF1 expression in VSMCs, but had no effect on the expression levels of other cell-cycle negative regulators. Finally, blockage of p21WAF1 function with specific siRNA abolished the IL-16-induced elevation of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activation in VSMCs. Taken together, p21WAF1 was required for the induction of p38MAPK-mediated MMP-9 expression via activation of the Sp-1 binding motif, which led to migration and invasion of VSMCs interacting with IL-16/CD4. These results could provide that IL-16 is a new target in the treatment of vascular diseases such as atherosclerosis and re-stenosis.

Microsomal Prostaglandin E Synthase-1–Derived PGE 2 Inhibits Vascular Smooth Muscle Cell Calcification

Chronic administration of selective cyclooxygenase-2 (COX-2) inhibitors leads to an increased risk of adverse cardiovascular events, including myocardial infarction and stroke. Vascular smooth muscle cell (VSMC) calcification, a common complication of chronic kidney disease, is directly related to cardiovascular morbidity and mortality. Here, we tested whether specific COX-2 inhibition affects vascular calcification during chronic renal failure. The COX-2-specific inhibitors NS398 and SC236 significantly increased high-phosphate (Pi)-induced VSMC calcification. Similarly, COX-2(-/-) VSMCs, COX-2(-/-) aortas rings treated with high Pi and adenine diet-induced COX-2(-/-) chronic renal failure mice displayed enhanced calcium deposition. Metabolomic analysis revealed the differential suppression of PGE2 production by COX-1- and COX-2-specific inhibitors in high-Pi-stimulated VSMCs, indicating the involvement of PGE2 during COX-2 inhibition-aggravated vascular calcification. Indeed, exogenous PGE2 reduced alkaline phosphatase activity, osteogenic transdifferentiation, apoptosis, and calcification of VSMCs. In accordance, downregulation of microsomal prostaglandin E synthase (mPGES)-1 in VSMCs, mPGES-1(-/-) aorta with high-Pi stimulation and mPGES-1(-/-) chronic renal failure mice resulted in enhanced vascular mineralization. Further applications of RNAi and specific antagonists for PGE2 receptors indicated EP4 may mediate PGE2-inhibited vascular calcification. Our data revealed the pivotal role of COX-2-mPGES-1-PGE2 axis in vascular calcification. The selective inhibition of COX-2 or mPGES-1 may increase the risk of calcification and subsequent adverse cardiovascular events during chronic renal failure.

MiRNA-34a reduces neointima formation through inhibiting smooth muscle cell proliferation and migration

AimsWe have recently reported that microRNA-34a (miR-34a) regulates vascular smooth muscle cell (VSMC) differentiation from stem cells in vitro and in vivo. However, little is known about the functional involvements of miR-34a in VSMC functions and vessel injury-induced neointima formation. In the current study, we aimed to establish the causal role of miR-34a and its target genes in VSMC proliferation, migration and neointima lesion formation. Methods and resultsVarious pathological stimuli regulate miR-34a expression in VSMCs through a transcriptional mechanism, and the P53 binding site is required for miR-34a gene regulation by these stimuli. miR-34a over-expression in serum-starved VSMCs significantly inhibited VSMC proliferation and migration, while knockdown of miR-34a dramatically promoted VSMC proliferation and migration, respectively. Notch homolog 1 (Notch1), a well-reported regulator in VSMC functions and arterial remodeling, was predicted as one of the top targets of miR-34a by using several computational miRNA target prediction tools, and was negatively regulated by miR-34a in VSMCs. Luciferase assay showed miR-34a substantially repressed wild type Notch1–3′-UTR-luciferase activity in VSMCs, but not mutant Notch1–3′-UTR-luciferease reporter, confirming the Notch1 is the functional target of miR-34a in VSMCs. Data from co-transfection experiments also revealed that miR-34a inhibited VSMC proliferation and migration through modulating Notch gene expression levels. Importantly, the expression level of miR-34a was significantly down-regulated in injured arteries, and miR-34a perivascular over-expression significantly reduced Notch1 expression levels, decreased VSMC proliferation, and inhibited neointima formation in wire-injured femoral arteries. ConclusionOur data have demonstrated that miR-34a is an important regulator in VSMC functions and neointima hyperplasia, suggesting its potential therapeutic application for vascular diseases.

Angiotensin II Type 2 Receptor Inhibits Vascular Intimal Proliferation With Activation of PPARγ.

Angiotensin II type 2 (AT2) receptor stimulation could exert beneficial effects on vascular remodeling. Previously, we reported that AT2 receptor stimulation ameliorated insulin resistance in diabetic mice accompanied by PPARγ activation which also plays a variety of crucial roles in the vasculature. Therefore, this study aimed to investigate the vascular protective effect of the AT2 receptor with activation of PPARγ involving AT2 receptor-interacting protein (ATIP). Vascular injury was induced by polyethylene-cuff placement around the femoral artery in C57BL/6J mice. Treatment with compound 21 (C21), an AT2 receptor agonist, decreased neointimal formation, cell proliferation, and the mRNA levels of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF)-α, and interleukin-1β, and phosphorylation of nuclear factor-kappa B, and increased PPARγ DNA-binding activity in the injured artery, whereas these inhibitory effects of C21 were attenuated by co-treatment with a PPARγ antagonist, GW9662. Treatment of vascular smooth muscle cells (VSMC) with C21 prepared from smAT2 transgenic mice, which highly express the AT2 receptor in VSMC, increased both PPARγ activity and its DNA-binding activity determined by dual-luciferase assay and electrophoresis mobility shift assay (EMSA), respectively. We observed that ATIP was involved in PPARγ complex formation, and that transfection of siRNA of ATIP1 attenuated the AT2 receptor-mediated increase in PPARγ activity in VSMC. In response to AT2 receptor stimulation, ATIP was translocated from the plasma membrane to the nucleus. Our results suggest a new mechanism by which AT2 receptor stimulation activates PPARγ, thereby resulting in amelioration of vascular intimal proliferation, and that ATIP plays an important role in AT2 receptor-mediated PPARγ activation.

PGC-1α limits angiotensin II-induced rat vascular smooth muscle cells proliferation via attenuating NOX1-mediated generation of reactive oxygen species.

AngII (angiotensin II)-induced excessive ROS (reactive oxygen species) generation and proliferation of VSMCs (vascular smooth muscle cells) is a critical contributor to the pathogenesis of atherosclerosis. PGC-1α [PPARγ (peroxisome-proliferator-activated receptor γ) co-activator-1α] is involved in the regulation of ROS generation, VSMC proliferation and energy metabolism. The aim of the present study was to investigate whether PGC-1α mediates AngII-induced ROS generation and VSMC hyperplasia. Our results showed that the protein content of PGC-1α was negatively correlated with an increase in cell proliferation and migration induced by AngII. Overexpression of PGC-1α inhibited AngII-induced proliferation and migration, ROS generation and NADPH oxidase activity in VSMCs. Conversely, Ad-shPGC-1α (adenovirus-mediated PGC-1α-specific shRNA) led to the opposite effects. Furthermore, the stimulatory effect of Ad-shPGC-1α on VSMC proliferation was significantly attenuated by antioxidant and NADPH oxidase inhibitors. Analysis of several key subunits of NADPH oxidase (Rac1, p22(phox), p40(phox), p47(phox) and p67(phox)) and mitochondrial ROS revealed that these mechanisms were not responsible for the observed effects of PGC-1α. However, we found that overexpression of PGC-1α promoted NOX1 degradation through the proteasome degradation pathway under AngII stimulation and consequently attenuated NOX1 (NADPH oxidase 1) expression. These alterations underlie the inhibitory effect of PGC-1α on NADPH oxidase activity. Our data support a critical role for PGC-1α in the regulation of proliferation and migration of VSMCs, and provide a useful strategy to protect vessels against atherosclerosis.

Intermittent hypoxia in rats reduces activation of Ca2+ sparks in mesenteric arteries.

Ca(+) sparks are vascular smooth muscle cell (VSMC) Ca(2+)-release events that are mediated by ryanodine receptors (RyR) and promote vasodilation by activating large-conductance Ca(2+)-activated potassium channels and inhibiting myogenic tone. We have previously reported that exposing rats to intermittent hypoxia (IH) to simulate sleep apnea augments myogenic tone in mesenteric arteries through loss of hydrogen sulfide (H2S)-induced dilation. Because we also observed that H2S can increase Ca(2+) spark activity, we hypothesized that loss of H2S after IH exposure reduces Ca(2+) spark activity and that blocking Ca(2+) spark generation reduces H2S-induced dilation. Ca(2+) spark activity was lower in VSMC of arteries from IH compared with sham-exposed rats. Furthermore, depolarizing VSMC by increasing luminal pressure (from 20 to 100 mmHg) or by elevating extracellular [K(+)] increased spark activity in VSMC of arteries from sham rats but had no effect in arteries from IH rats. Inhibiting endogenous H2S production in sham arteries prevented these increases. NaHS or phosphodiesterase inhibition increased spark activity to the same extent in sham and IH arteries. Depolarization-induced increases in Ca(2+) spark activity were due to increased sparks per site, whereas H2S increases in spark activity were due to increased spark sites per cell. Finally, inhibiting Ca(2+) spark activity with ryanodine (10 μM) enhanced myogenic tone in arteries from sham but not IH rats and blocked dilation to exogenous H2S in arteries from both sham and IH rats. Our results suggest that H2S regulates RyR activation and that H2S-induced dilation requires Ca(2+) spark activation. IH exposure decreases endogenous H2S-dependent Ca(2+) spark activation to cause membrane depolarization and enhance myogenic tone in mesenteric arteries.

Abstract MP05: Knockdown of Gamma-adducin Expression Impairs the Myogenic Response of the Cerebral and Renal Arterioles

We recently identified a region on chromosome 1 containing 15 genes that rescues the impaired myogenic response in the afferent arteriole (Af-art) and the development of renal injury in Fawn Hood Hypertensive (FHH) rats. We also found an inactivating K572Q mutation in the gamma-Adducin (Add3) gene in FHH rats in this region that may play a causal role. To test this hypothesis, the present study examed the effect of knockdown of the expression of Add3 with DsiRNA on the myogenic response of renal and cerebral arterioles. A newly designed 27-mer Add3 DsiRNA blocked the expression of the Add3 protein in 293 cells and reduced Add3 mRNA expression in a dose-dependent manner up to 75% in cultured middle cerebral arteries (MCA). The inner diameter of MCA and renal Af-art 36 hours after co-transfection of Add3 DsiRNA dilated by 9 ± 2% and 4 ± 3%, respectively, in response to an elevation in transmural pressure from 60 to 140 mmHg. In contrast, these vessels still constricted normally by 11± 2% and 10 ± 1% in vessels transfected with scrambled siRNA. Add3 DsiRNA had no effect on the vasoconstrictor response of the Af-art to NE (10-7 M). The large conductance calcium sensitive potassium (BK) current was 5-fold higher in vascular smooth muscle cells (VSMC) freshly isolated from the MCA that were transfected with Add3 DsiRNA as indicated by the appearance of SiGLO red fluorescence compared with that seen in non-transfected cells. Administration of IBTX normalized the elevated BK channel current recorded from VSMC transfected with Add3 DsiRNA, but it had little effect in non-transfected cells. These results indicate that knockdown of the expression of Add3 impairs the myogenic response of both MCA and Af-art and it is associated with an elevation in BK channel activity. It also supports the hypothesis that the K572Q mutation of Add3 found in FHH rats may play a causal role in the impaired myogenic response and autoregulation of renal and cerebral blood flow by elevating BK channel activity in VSMC.

Abstract P601: Matrix Metalloproteinase 2 Plays An Important Role In Angiotensin Ii-induced Vascular Injury Mediated In Part Through Epidermal Growth Factor Receptor Activation In Vascular Smooth Muscle Cells

Background: Matrix metalloproteinase 2 (MMP2) is involved in cardiovascular disease. Whether MMP2 plays a role in hypertension and vascular damage is unknown. We hypothesized that Mmp2 knockout will prevent angiotensin (Ang) II-induced blood pressure (BP) rise and vascular injury. Methods: Ten to 12-week-old male Mmp2 knockout (Mmp2-/-) and wild-type (WT) mice were infused with Ang II (1000 ng/kg/min, SC) for 14 days. Systolic BP was measured by telemetry, mesenteric arteries (MA) endothelial function and vascular remodeling by pressurized myography. In aortic wall or perivascular fat (PVAT), reactive oxygen species (ROS) generation was determined using dihydroethidium staining, and vascular cell adhesion protein 1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1) expression and monocyte/macrophage infiltration by immunofluorescence. Spleen T cells and monocyte profile were assessed by flow cytometry. Vascular smooth muscle cells (VSMCs) were isolated from MA of WT and Mmp2 knockout mice, stimulated 5 min with 100 nM Ang II and epidermal growth factor receptor (EGFR) phosphorylation measured by Western-Blotting. Results: Ang II increased Systolic BP (172±7 vs 122±3, P<0.01), decreased MA vasodilatory responses to acetylcholine (33±5% vs 83±3%, P<0.01) and increases MA media-to-lumen ratio (5±0% vs 3±0%, P<0.01), media cross-sectional area (7224±467 vs 5345±336 μm2, P<0.05), and stiffness (P<0.01), as shown by a leftward shift of the stress/strain relationship, in WT. Furthermore, Ang II enhanced aortic ROS generation (73±11 vs 6±1 RFU/μm2, P<0.01), aortic VCAM-1 (17±3 vs 5±3 RFU/μm2, P<0.01) and MCP-1 expression (71±14 vs 11±3 RFU/μm2, P<0.01) and PVAT monocyte/macrophage infiltration (32±5 vs 4±0 RFU/μm2, P<0.05), and spleen activated CD4+CD69+ and CD8+CD69+ T cells and pro-inflammatory Ly-6Chi monocytes (17±2 vs 10±1%, 11±1 vs 5±1% and 53±6 vs 25±2%, respectively, P<0.05) in WT. Ang II increased EGFR phosphorylation in VSMCs in vitro (1.91±0.19 vs 1.0±0.0%, P<0.05). Mmp2 knockout prevented or reduced all of the above except BP elevation (P<0.05). Conclusion: MMP2 plays an important role in Ang II-induced vascular injury, which could be mediated at least in part through EFGR activation in VSMCs.

Receptor for Advanced Glycation End-Products Signaling Interferes with the Vascular Smooth Muscle Cell Contractile Phenotype and Function.

Increased blood glucose concentrations promote reactions between glucose and proteins to form advanced glycation end-products (AGE). Circulating AGE in the blood plasma can activate the receptor for advanced end-products (RAGE), which is present on both endothelial and vascular smooth muscle cells (VSMC). RAGE exhibits a complex signaling that involves small G-proteins and mitogen activated protein kinases (MAPK), which lead to increased nuclear factor kappa B (NF-κB) activity. While RAGE signaling has been previously addressed in endothelial cells, little is known regarding its impact on the function of VSMC. Therefore, we hypothesized that RAGE signaling leads to alterations in the mechanical and functional properties of VSMC, which could contribute to complications associated with diabetes. We demonstrated that RAGE is expressed and functional in the A7r5 VSMC model, and its activation by AGE significantly increased NF-κB activity, which is known to interfere with the contractile phenotype of VSMC. The protein levels of the contraction-related transcription factor myocardin were also decreased by RAGE activation with a concomitant decrease in the mRNA and protein levels of transgelin (SM-22α), a regulator of VSMC contraction. Interestingly, we demonstrated that RAGE activation increased the overall cell rigidity, an effect that can be related to an increase in myosin activity. Finally, although RAGE stimulation amplified calcium signaling and slightly myosin activity in VSMC challenged with vasopressin, their contractile capacity was negatively affected. Overall, RAGE activation in VSMC could represent a keystone in the development of vascular diseases associated with diabetes by interfering with the contractile phenotype of VSMC through the modification of their mechanical and functional properties.

Inhibition of TLR4 attenuates vascular dysfunction and oxidative stress in diabetic rats.

Hyperglycemia-induced reactive oxygen species (ROS) production plays a major role in the pathogenesis of diabetic vascular dysfunction. However, the underlying mechanisms remain unclear. Toll-like receptor 4 (TLR4), a key component of innate immunity, is known to be activated during diabetes. Therefore, we hypothesize that hyperglycemia activates TLR4 signaling in vascular smooth muscle cells (VSMCs) that triggers ROS production and causes vascular dysfunction. Rat mesenteric VSMCs exposed to high glucose (25 mmol/l) increased TLR4 expression and activated TLR4 signaling via upregulation of myeloid differentiation factor 88 (MyD88). TLR4 inhibitor CLI-095 significantly attenuated elevated levels of ROS and nuclear factor-kappa B (NF-κB) activity in VSMCs exposed to high glucose. Mesenteric arteries from streptozotocin-induced diabetic rats treated with CLI-095 (2 mg/kg/day) intraperitoneally for 2 weeks exhibited reduced ROS generation and attenuated noradrenaline-induced contraction. These results suggest that hyperglycemia-induced ROS generation and NF-κB activation in VSMCs are at least, in part, mediated by TLR4 signaling. Therefore, strategies to block TLR4 signaling pathways pose a promising avenue to alleviate diabetic-induced vascular complications. High glucose-induced TLR4 activation in vascular smooth muscle cells. Inhibition of TLR4 attenuated high glucose-induced ROS production and NF-κB activity in VSMC. Suppression of TLR4 signaling attenuated mesenteric contraction in diabetic rat.

PDGFRβ signalling regulates local inflammation and synergizes with hypercholesterolaemia to promote atherosclerosis.

Platelet-derived growth factor (PDGF) is a mitogen and chemoattractant for vascular smooth muscle cells (VSMCs). However, the direct effects of PDGF receptor β (PDGFRβ) activation on VSMCs have not been studied in the context of atherosclerosis. Here, we present a new mouse model of atherosclerosis with an activating mutation in PDGFRβ. Increased PDGFRβ signaling induces chemokine secretion and leads to leukocyte accumulation in the adventitia and media of the aorta. Furthermore, PDGFRβD849V amplifies and accelerates atherosclerosis in hypercholesterolemic ApoE−/− or Ldlr−/− mice. Intriguingly, increased PDGFRβ signaling promotes advanced plaque formation at novel sites in the thoracic aorta and coronary arteries. However, deletion of the PDGFRβ-activated transcription factor STAT1 in VSMCs alleviates inflammation of the arterial wall and reduces plaque burden. These results demonstrate that PDGFRβ pathway activation has a profound effect on vascular disease and support the conclusion that inflammation in the outer arterial layers is a driving process for atherosclerosis.

Dipeptidyl peptidase-4 inhibition by gemigliptin prevents abnormal vascular remodeling via NF-E2-related factor 2 activation

Dipeptidyl peptidase-4 (DPP-4) inhibitors exert a potent anti-hyperglycemic effect and reduce cardiovascular risk in type 2 diabetic patients. Several studies have shown that DPP-4 inhibitors including sitagliptin have beneficial effects in atherosclerosis and cardiac infarction involving reactive oxygen species. Here, we show that gemigliptin can directly attenuate the abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) via enhanced NF-E2-related factor 2 (Nrf2) activity. Gemigliptin dramatically prevented ligation injury-induced neointimal hyperplasia in mouse carotid arteries. Likewise, the proliferation of primary VSMCs was significantly attenuated by gemigliptin in a dose-dependent manner consistent with a decrease in phospho-Rb, resulting in G1 cell cycle arrest. We found that gemigliptin enhanced Nrf2 activity not only by mRNA expression, but also by increasing Keap1 proteosomal degradation by p62, leading to the induction of Nrf2 target genes such as HO-1 and NQO1. The anti-proliferative role of gemigliptin disappeared with DPP-4 siRNA knockdown, indicating that the endogenous DPP-4 in VSMCs contributed to the effect of gemigliptin. In addition, gemigliptin diminished TNF-α-mediated cell adhesion molecules such as MCP-1 and VCAM-1 and reduced MMP2 activity in VSMCs. Taken together, our data indicate that gemigliptin exerts a preventative effect on the proliferation and migration of VSMCs via Nrf2.

Sphingosine-1-phosphate (S1P) inhibits TNFA-induced MMP-9 expression (TIME) through S1P2-mediated activation of PTEN

S1P regulates numerous functions in vascular smooth muscle cells (VSMC). We have shown that high S1P levels promoted neointima formation after carotid injury, a process that depends on matrix metalloproteinases (MMP). Here, we tested whether S1P affects basal and cytokine-induced MMP-9 activity in rat and mouse VSMC. S1P potently inhibited TIME as seen on gelatin zymography of the secreted enzyme and gene expression by real time PCR, respectively, without any effect on MMP-2. Using S1P receptor antagonists and VSMC isolated from S1P receptor knockout mice we have identified S1P2 as the S1P receptor responsible for TIME inhibition. S1P had no effect on IkBa phosphorylation and NF-kB activation by TNFa but potently inhibited Akt phosphorylation after TNFa that we found to be required for TIME. S1P inhibited PI3K/Akt by activating the Phosphatase and Tensin homolog (PTEN) pathway by engaging the S1P2 receptor as we could show using PI3K inhibitors, siRNA to PTEN and pharmacological PTEN blockade (bpVHO(pic)). The functional consequence of TIME inhibition by S1P was the abrogation of MMP-dependent TNFa-stimulated VSMC migration in Boyden chamber experiments. In vivo, downregulation of all S1P receptors by maintaining high S1P levels through pharmacological S1P lyase inhibition promoted neointima formation after carotid ligation, while the S1P analogue FTY720 had no effect. FTY720 acts on all S1P receptors except S1P2, and S1P2-deficient mice exhibit the same phenotype as S1P lyase-inhibited. Thus lack of MMP-9 inhibition due to downregulated or missing S1P2 may be responsible for increased neointima formation, a process shown to causally depend on MMP-9.

211 Mirna-34a Reduces Neointima fommation through Inhibiting Smooth Muscle Cell Proliferation and Migration

<h3>Background/objective</h3> We have recently reported that microRNA-34a (miR-34a) regulates vascular smooth muscle cell (VSMC) differentiation from stem cells <i>in vitro</i> and <i>in vivo</i>. However, little is known about the functional involvements of miR-34a in VSMC functions and vessel injury-induced neointima formation. In the current study, we aimed to establish the causal role of miR-34a and its target genes in VSMC proliferation, migration and neointima lesion formation. <h3>Methods and results</h3> miR-34a was significantly down-regulated in VSMCs upon various pathological stimuli, and in injured arteries. miR-34a over-expression in serum-starved VSMCs significantly inhibited VSMC proliferation and migration, while knockdown of miR-34a dramatically promoted VSMC proliferation and migration, respectively. Notch homolog 1 (Notch1), a well-reported regulator in VSMC functions and arterial remodelling, was predicted as one of the top targets of miR-34a by using several computational miRNA target prediction tools, and was negatively regulated by miR-34a in VSMCs. Luciferase assay showed miR-34a substantially repressed wild type Notch1-3’-UTR-luciferase activity in VSMCs, but not mutant Notch1-3’-UTR-luciferease reporter, confirming the Notch1 is the functional target of miR-34a in VSMCs. Data from co-transfection experiments also revealed that miR-34a inhibited VSMC proliferation and migration through modulating Notch gene expression levels. Importantly, miR-34a perivascular overexpression significantly reduced Notch1 expression levels, decreased VSMC proliferation, and inhibited neointima formation in wire-injured femoral arteries. <h3>Conclusions/implications</h3> Our data have demonstrated that miR-34a is an important regulator in VSMC functions and neointima hyperplasia, suggesting its potential therapeutic application for vascular diseases.

NADPH oxidase activation contributes to native low-density lipoprotein-induced proliferation of human aortic smooth muscle cells.

Elevated plasma concentration of native low-density lipoprotein (nLDL) is associated with vascular smooth muscle cell (VSMC) activation and cardiovascular disease. We investigated the mechanisms of superoxide generation and its contribution to pathophysiological cell proliferation in response to nLDL stimulation. Lucigenin-induced chemiluminescence was used to measure nLDL-induced superoxide production in human aortic smooth muscle cells (hAoSMCs). Superoxide production was increased by nicotinamide adenine dinucleotide phosphate (NADPH) and decreased by NADPH oxidase inhibitors in nLDL-stimulated hAoSMC and hAoSMC homogenates, as well as in prepared membrane fractions. Extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase C-θ (PKCθ) and protein kinase C-β (PKCβ) were phosphorylated and maximally activated within 3 min of nLDL stimulation. Phosphorylated Erk1/2 mitogen-activated protein kinase, PKCθ and PKCβ stimulated interactions between p47phox and p22phox; these interactions were prevented by MEK and PKC inhibitors (PD98059 and calphostin C, respectively). These inhibitors decreased nLDL-dependent superoxide production and blocked translocation of p47phox to the membrane, as shown by epifluorescence imaging and cellular fractionation experiments. Proliferation assays showed that a small interfering RNA against p47phox, as well as superoxide scavenger and NADPH oxidase inhibitors, blocked nLDL-induced hAoSMC proliferation. The nLDL stimulation in deendothelialized aortic rings from C57BL/6J mice increased dihydroethidine fluorescence and induced p47phox translocation that was blocked by PD98059 or calphostin C. Isolated aortic SMCs from p47phox−/− mice (mAoSMCs) did not respond to nLDL stimulation. Furthermore, NADPH oxidase 1 (Nox1) was responsible for superoxide generation and cell proliferation in nLDL-stimulated hAoSMCs. These data demonstrated that NADPH oxidase activation contributed to cell proliferation in nLDL-stimulated hAoSMCs.

PP.41.11

Objective: Hypoxia is a pathological process, emerging due to oxygen starvation of cells and leading to destructive changes in tissues. It is shown that hypoxia triggers of metabolic disorders, which can lead to damage of contractile ability of the vascular smooth muscle cells (VSMC). It is assumed that one of the possible mechanisms of action of hypoxia on the contractile function of smooth muscle cells (SMCs) is activated K+ channels of plasma membrane. Design and method: Contractile activity of endothelium-denuded SMCs from rat aorta in hypoxia was studied by the method of mechanography (Myobath II/Lab-Trax-4/16). Hypoxic conditions were created by bubbling nitrogen gas (95%N2/5%O2) through the solutions for 15 minutes immediately before the experiment. Contractions induced by membrane depolarization of VSMC in a high-potassium solution (30 mM KCl) or phenylephrine (PE, 1 μM). Solutions of tetraethylammonium (TEA,10 mM), 4-aminopyridine (4-AP, 1 mM) and glibenclamide (10 μM) were tested. Results: Under hypoxic conditions decreased the amplitude of the contractile response SMCs to high-potassium solution and PE (83.1 ± 2.9%, 73.4 ± 2.5% (n = 8, p < 0.05), respectively) from the control in normoxic conditions. Addition of TEA on the background of a high-potassium contraction in hypoxia resulted in increased mechanical tension of segments to 109.7 ± 2.1% (n = 8, p < 0.05), and phenylephrine to 125.3 ± 3.4% (n = 8, p < 0.05) from control during hypoxia. 4-AP in hypoxia increases the amplitude of the contractile response SMCs to high-potassium solution (110.9 ± 2.8% (n = 8, p < 0.05)) and PE-induced (111.3 ± 1.9% (n = 8, p < 0.05)) from control in hypoxia. Glibenclamide increases amplitude of PE-induced contraction of SMC to 107.8 ± 2.6% (n = 8, p < 0.05) and had no significant effect on high-potassium contractions of SMCs in hypoxia. Conclusions: Thus, hypoxia has an inhibitory effect on the contractile activity of VSMC. Hypoxia cause decreased amplitude of high-potassium solution and PE-induced contractions, in a greater extent in PE-induced contractions. This effect was associated with opening of voltage-dependent potassium channels, and by the action of phenylephrine - ATP-sensitive potassium channels of SMCs.

Cytochrome P450 1B1 Is Essential For Angiotensin II‐ And Arachidonic Acid‐Induced Activation Of NADPH Oxidase In Vascular Smooth Muscle Cells Of Mice

Previously, we have shown that angiotensin II (Ang II) and arachidonic acid (AA) via its metabolism by cytochrome P450 (CYP1B1) activates NADPH oxidase and generates superoxide in rat vascular smooth muscle cells (VSMCs). This study was conducted to determine the effect of Ang II and AA on NADPH oxidase activity in VSMCs from Cyp1b1-/- mice with and without transduction of adenovirus cDNA Cyp1b1. VSMCs were isolated from thoracic aorta of wild type (Cyp1b1+/+) and CYP1B1 knockout (Cyp1b1-/-) mice. And treated with Ang II (200 nM) or AA (30 mM) for 15 min. In another series of experiments, VSMCs isolated and cultured from Cyp1b1-/- mice were transduced with 120 MOI and after 24 hrs were treated with Ang II or AA for 15 min. The cells were sonicated for 10 second and ultracentrifuged at 100,000g to separate cell membranes. NADPH oxidase activity in the membranes was measured by using 5 nM lucigenin enhanced-chemiluminescence based assay. Ang II and AA increased NADPH oxidase activity in VSMC from Cyp1b1+/+ but not Cyp1b1-/- mice. However, in Cyp1b1-/- VSMCs transduced with adenovirus cDNA Cyp1b1, Ang II and AA increased NADPH oxidase activity. These data suggest that CYP1B1 is essential for Ang II-induced activation of NADPH oxidase and is most likely mediated by AA metabolites and/or lipid peroxides in VSMCs.