Muscle Cell Migration Research Articles

Abstract 174: Periadventitial or Intraluminal Delivery of Simvastatin Attenuates Intimal Hyperplasia After Arterial Injury

Introduction: Oral statin administration reduces intimal hyperplasia (IH) after arterial injury by only ~25%. Alternative drug delivery systems have gained attention as carriers for hydrophobic drugs. We studied the effects of simvastatin (free vs hyaluronic acid tagged polysialic acid-polycaprolactone micelles) in vitro on vascular smooth muscle cell (VSMC) migration and in vivo after arterial injury. We hypothesized both free and micelles containing statins would inhibit VSMC chemotaxis and local statin treatment would reduce IH in Sprague-Dawley (SD) rats following carotid balloon injury greater than oral delivery. Methods: VSMCs pretreated with free simvastatin (1 μM, 20 min or 20 hrs) or simvastatin-loaded micelles underwent chemotaxis to thrombospondin-1 (20 μg/mL) or serum-free media using a modified Boyden chamber. Next, SD rats (13-15 wks) who underwent balloon injury of the common carotid artery (5 atm/5 min) received statin therapy – either intraluminal simvastatin loaded micelles (50 μM, pressurized for 5 min) prior to injury or periadventitial pluronic gel (50 μM simvastatin) following injury. Arteries were harvested 14 days postoperatively. Morphometric analysis was performed using the intimal/media area ratio defined as intima/(intima + media). Findings were compared to our historic animals which received oral simvastatin (10 mg/kg/day) or controls without statin therapy. Statistical analysis was by ANOVA and p <0.05 was significant. Results: Simvastatin loaded micelles and free simvastatin inhibited VSMC chemotaxis (54%-60%, p <0.05). IH was induced in all injured vessels. Simvastatin in pluronic gel or micelles reduced IH compared to untreated controls (0.208 ± 0.04 or 0.160 ± 0.03 vs 0.350 ± 0.03, respectively, p <0.05). However, neither gel nor simvastatin-loaded micelles were superior to oral statins (0.261 ± 0.03, p >0.05). Conclusions: Intraluminally and periadventially delivered statins were effective in reducing IH. The efficacy of single dose, locally delivered statins alone may lead to novel treatment modalities for the prevention of IH. The different routes of administration allow for treatment at the time of both open and endovascular procedures, without the need for systemic therapy.

Abstract 374: Inhibition of Mitochondrial CaMKII Reduces Vascular Smooth Muscle Migration and Neointima Formation and Halts Mitochondrial Mobility

Background: Restenosis after angioplasty for coronary vascular disease remains a critical problem in cardiovascular medicine. Vascular smooth muscle cell (VSMC) migration and proliferation cause restenosis through neointima formation. Mitochondrial motility is likely necessary for cell proliferation and migration, and is inhibited in microdomains with increased Ca 2+ . The Ca 2+ /calmodulin-dependent kinase II (CaMKII) in mitochondria (mtCaMKII) is proposed to control mitochondrial matrix Ca 2+ uptake through mitochondrial Ca 2+ uniporter (MCU). Thus, we hypothesized that blocking mtCaMKII decreases VSMC migration and neointima formation by decreasing mitochondrial motility. Methods: mtCaMKII was inhibited by expression of the mitochondria-targeted CaMKII inhibitor peptide (CaMKIIN) in a novel transgenic mouse model in smooth muscle only (SM-mtCaMKIIN) or delivered by adenoviral transduction (Ad-mtCaMKIIN). Results: In our models, mtCaMKIIN was detected selectively in mitochondria of VSMC. mtCaMKIIN significantly reduced mitochondrial Ca 2+ current and Ca 2+ content compared to WT in vivo and in vitro. SM-mtCaMKIIN mice showed significantly reduced neointimal area 28 days after endothelial injury (n=8, p<0.05) and fewer proliferating neointimal cells by PCNA staining. In vitro, Ad-mtCaMKIIN mildly reduced VSMC proliferation and mitochondrial ROS production without altering maximal respiration after PDGF treatment. Ad-mtCaMKIIN abolished VSMC migration, as did mitoTEMPO and MCU inhibitor Ru360. Ad-mtCaMKIIN blocked mitochondrial mobility towards the leading edge, while relocation of mitochondria was seen in WT cells 6 h after PDGF treatment. Mitochondrial redistribution was also inhibited by Ru360, but not by mitoTEMPO or cytoplasmic CaMKII inhibition. Mitochondrial fission promotes cell migration. Accordingly, PDGF increased mitochondrial particles in WT VSMC, while mitochondria in Ad-mtCaMKIIN cells were fragmented and unresponsive to PDGF treatment. Conclusions: mtCaMKIIN prevents mitochondrial distribution to the leading edge and reduces VSMC migration and neointima formation. These data suggest mitochondrial Ca 2+ regulation plays an important role in VSMC migration by altering mitochondrial location.

The C-Terminal Tail of Aquaporin 1 Is Required for Hypoxia-Induced Migration and Proliferation of Pulmonary Arterial Smooth Muscle Cells

The C-Terminal Tail of Aquaporin 1 Is Required for Hypoxia-Induced Migration and Proliferation of Pulmonary Arterial Smooth Muscle Cells

17β-Estradiol inhibits TNF-α-induced proliferation and migration of vascular smooth muscle cells via suppression of TRAIL

Atherosclerosis is an inflammatory disease and involves migration of vascular smooth muscle cells (VSMCs). Estrogen inhibits VSMCs migration, while the underlying mechanism remains to be revealed. Recent years, there is emerging evidence showing that TNF-related apoptosis-inducing ligand (TRAIL) increases proliferation and migration of VSMCs. In this study, we investigated the regulatory effect of estrogen on TRAIL expression in VSMCs. TNF-α greatly enhanced TRAIL protein expression and stimulated VSMCs proliferation and migration. This effect was partially inhibited by the addition of TRAIL neutralizing antibody, suggesting that TRAIL is important in TNF-α-induced migration. 17β-estradiol (E2) inhibited TRAIL expression under TNF-α stimulation in a time- and concentration-dependent manner. This effect was was mimicked by ERα agonist 4′,4″,4‴-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT), but not ERβ agonist 2,3-bis-(4-hydroxyphenyl)-propionitrile (DPN), indicating that ERα is involved in this action. TNF-α led to nuclear factor kappa B (NF-κB) p65 phosphorylation and the inhibitor pyrrolidine dithiocarbama (PDTC) inhibited TRAIL expression, suggesting that NF-κB signaling is crucial for TARIL production. E2 suppressed p65 phosphorylation in VSMCs and the overexpression of p65 subunit reversed the inhibitory effect of E2 on TRAIL expression and cell proliferation and migration. Taken together, our results indicate that E2 inhibits VSMCs proliferation and migration by downregulation of TRAIL expression via suppression of NF-κB pathway.

The Influence of Caldesmon Suppression on Proliferation and Motilities of Vascular Smooth Muscle Cells

Migration of vascular smooth muscle cells (VSMCs) from the media to intima constitutes a critical step in the development of proliferative vascular diseases. To elucidate the regulatory mechanism of VSMCs motility, the roles of caldesmon (CaD) were investigated previously. CaD is an actin-binding protein dynamically regulating cytoskeleton structure. In this study, the potential role of CaD in mediating proliferation and motility of VSMCs were discussed. First, structural effect of CaD on cytoskeleton integrity was analyzed with CaD knock-down; second, the proliferation of VSMCs was measured in CaD knock-down and control cells; third, the specific role of CaD on VSMCs motilities was evaluated with in vitro migration and invasion assays. We found that CaD is an integral component to maintain cytoskeleton integrity of VSMCs. Our data indicated that CaD suppression does not show significant influence on VSMCs proliferation, but negatively modulates the motilities of VSMCs, and CaD depletion would significantly facilitate migration and invasion of VSMCs.

Abstract 17568: Phospholipase C Gamma-1 Mediated Akt-notch1 Signaling is Essential for Intima Formation

Introduction: Vascular smooth muscle cell (VSMC) proliferation, migration and dedifferentiation are critical for vascular diseases, such as atherosclerosis and restenosis after angioplasty. Recently, it is demonstrated that after vascular injury Notch 1and Notch 3 knockout (KO) mice have decreased intima formation whereas Notch 2 KO mice have increased one. Therefore, it is important to investigate the specific regulatory pathways that activate the different Notch receptors. Methods and Results: There was a time and dose dependent activation of Notch1, as determined by western blot of N1-ICD but not Notch 2 and 3 in VSMC. As Akt activates Notch signaling in endothelial cells and phospholipase C gamma-1 (PLCγ1) binds to and activates Akt in tumor cells, we therefore propose that PLCγ1 mediated Akt -Notch1 pathway is critical for intima formation by increasing VSMC proliferation, migration and dedifferentiation. To test our hypothesis, we measured PLCγ1 and Akt interaction in VSMC. We observed an association between PLCγ1 and Akt, which was increased upon AngII or PDGF stimulation. Depletion of PLCγ1 by siRNA significantly inhibited Akt and Notch1 activation. As the major target gene of Notch1, Hey2 protein expression was increased 1.8 fold and 2.3 fold respectively after AngII and PDGF stimulation. PLCγ1 siRNA treatment inhibited it by 68.3% and 64.7%. Depletion of PLCγ1 significantly inhibited PDGF induced VSMC proliferation, migration and dedifferentiation but increased apoptosis. In vivo, PLCγ1 and control siRNA were delivered periadventitially in pluronic gel after carotid ligation. Morphometric analysis 21days after ligation demonstrated that PLCγ1 depletion attenuated intima area (2.2 ± 0.7 vs 4.3 ± 1.0, *104 μm2) and intima/media ratio (0.21 ± 0.09 vs 0.43 ± 0.12) compared to the control group. Conclusions: In conclusion, PLCγ1 mediated Akt-Notch1 signaling is crucial for intima formation. The PLCγ1 and Akt interaction will be a promising target for therapeutic stratergies that inhibit vascular remodeling.

Abstract 19838: The Atypical Cadherin Fat1 Suppresses Vascular Smooth Muscle Mesenchymal Markers and Limits Atherosclerosis

Introduction: Fat1 controls vascular smooth muscle cell (SMC) growth and migration. In recent studies, we found increased Fat1 expression in atherosclerotic lesions from apoE -/- (KO) mice, and identified vimentin, an intermediate filament protein and mesenchymal marker, as a novel Fat1 interactor also expressed in SMCs. Hypothesis: Fat1 regulation of SMCs limits atherosclerosis, at least in part via Fat1 effects on vimentin. Methods: We generated fat1 SMC-KO ; apoE double knockout (DKO) mice, fed them a high fat diet for 24 weeks, and assessed atherosclerosis analyzing aortic roots and brachiocephalic arteries (BCA) for lesion area, immunohistochemical features, and gene expression. Results: DKO mice (N=10) exhibited larger lesions at the aortic root (80837.0±7942.4) and BCA (36017.2±10496.4) compared to KO controls (64190.2±6539.1 and 19462.9±5246.3, respectively, p<0.05). DKO lesions had larger fibrous caps (40.6±5.5%, N=7) and necrotic cores (30.3±3.2%, N=8) compared to KO samples (26.3±2.9 and 21.6±2.2%, respectively, p<0.05), plus a trend towards increased cellularity (+24%, N=7, p=0.0573). Macrophage number was not different, but SMC area (staining for SM22α) was larger in DKO lesions (1.15±0.25, vs. KO, 0.37±0.08, N=9, p<0.01). Proliferation (phospho-histone H3 staining) was also higher (DKO, 52.6±13.3 vs. KO, 31.4±10.1, N=7 p<0.05). Fat1 and vimentin expression colocalized within fibrous caps, and Fat1-deficient SMCs and lesions expressed more vimentin (>3-fold, p<0.05 by immunoblotting). By co-immunoprecipitation, Fat1 interacted with vimentin and limited its abundance in SMCs; knockdown studies suggested that increased proliferation of Fat1-deficient SMCs depends on vimentin. In addition, Fat1-deficient SMCs showed evidence of E- to N-cadherin switching and upregulation of the Snail1 transcription factor. Conclusions: Our in vivo data support a role for Fat1 in control of SMC proliferation during atherogenesis, and suggest that Fat1-vimentin interaction is important for this regulation. These findings are reminiscent of the epithelial-mesenchymal transition (EMT) program described in proper epithelial cells, suggest that components of this regulatory network are operative in SMCs, and are controlled by Fat1.

A fragmented form of annexin A1 is secreted from C2C12 myotubes by electric pulse-induced contraction.

The main function of annexin A1 (ANXA1), a member of the annexin superfamily, is to bind to cellular membranes in a Ca(2+)-dependent manner. In skeletal muscle, ANXA1 is thought to be involved in the repair of damaged membrane tissue and in the migration of muscle cells. We hypothesized that ANXA1 is one of the myokines secreted during muscle contractions to accelerate the repair of cell damage after contraction. Here we performed cell contractions by electric pulse stimulation; the results revealed that a fragmented form of ANXA1 was cleaved by calpain and selectively secreted from skeletal muscle cells by contraction. We therefore realized that muscle-contraction-induced calpain-dependent ANXA1 fragmentation has a wound-healing effect on damaged cells. This suggested that not the intact form but rather fragmented ANXA1 is a contraction-induced myokine.

GW26-e1796 Effect of Ruanmailing containing serum on twinfilin-1 in PDGF-activated vascular smooth muscle cells

It was confirmed previously by our group that Ruanmailing component, one of traditional Chinese Medicine, may inhibit migration of vascular smooth muscle cells (VSMCs) induced by PDGF. In this study, we tested the effect of Ruanmailing containing serum on twinfilin-1 in PDGF-induced VSMCs to further

MicroRNA-199a Regulates Hypoxia-Mediated Pulmonary Artery Smooth Muscle Cell Proliferation and Migration by Targeting ROCK1

MicroRNA-199a Regulates Hypoxia-Mediated Pulmonary Artery Smooth Muscle Cell Proliferation and Migration by Targeting ROCK1

Dyslipidemia regulates thrombospondin-1-induced vascular smooth muscle cell chemotaxis.

Dyslipidemia is a risk factor for intimal hyperplasia (IH). Key to IH is vascular smooth muscle cell (VSMC) migration. Thrombospondin-1 (TSP-1) is a matricellular protein that stimulates VSMC migration. HDL will inhibit and LDL will augment TSP-1-induced VSMC chemotaxis. VSMC chemotaxis will be inhibited by the HDL moiety, S1P, through the S1PR1 receptor, and augmented by the LDL component, LPA, through the LPAR1 receptor. The goal of this study was to determine the effect of HDL and LDL and their receptors on TSP-1-induced VSMC chemotaxis. For VSMC chemotaxis to TSP-1 cells received the following pretreatments: low (25 µg/ml) or optimal (75 µg/ml) concentration of HDL, S1P, optimal (75 µg/ml) or high (175 µg/ml) concentration of LDL, or LPA. For the receptor studies, VSMCs were transfected with siRNA to S1PR1, S1PR3, LPAR1, LPAR2, LPAR3, or a S1PR2 receptor antagonist. The TSP-1-induced chemotaxis results were (1) HDL (25 µg/ml) or LDL (75 µg/ml) exhibited no effect on chemotaxis; (2) HDL (75 µg/ml) inhibited chemotaxis by 50.9 ± 8 % and S1P by 43.4 ± 11.6 %; (3) LDL (175 µg/ml) augmented chemotaxis by 30 ± 10.4 % and LPA by 25.6 ± 12.3 %; (4) S1PR1 and S1PR3 knockdown and S1PR2 antagonist-treated cells augmented chemotaxis; and (5) LPAR1 and LPAR2 knockdown inhibited and LPAR3 knockdown had no effect on chemotaxis. In conclusion, HDL/S1P inhibits, while LDL/LPA stimulates TSP-1-induced VSMC chemotaxis. The HDL/S1P effect is mediated by the S1PR1-3 receptors. The LDL/LPA effects are mediated by the LPAR1 and LPAR2 receptors, but not LPAR3. Therefore, lipids have significant effects on TSP-1-induced VSMC chemotaxis.

Atherosclerosis at your fingertips?

The endothelium separates blood and the vascular wall. Endothelial stimulation by agonists or by shear stress has effects on the smooth musculature in the vessel wall by paracrine communication. After the finding that intact endothelium is essential for acetylcholine to produce arterial vasodilation, various agents were discovered that stimulated the endothelium to produce endothelium-derived relaxing or contracting factors (EDRF/EDCF), the major EDRF being nitric oxide (NO). Additionally to vasodilation, NO inhibits platelet and monocyte adhesion and smooth muscle cell proliferation and migration, all key factors in the genesis and progression of atherosclerosis. Thus, dysfunctional endothelium, characterised by limited NO production, has lost part of its vasodilatory function and promotes atherosclerosis. In 1986, Ludmer and colleagues infused acetylcholine and the NO donor nitro-glycerine in the left anterior descending artery (LAD). Nitro-glycerine invariably caused LAD vasodilation but acetylcholine caused only vasodilation in the normal arteries, while arteries with stenosis showed vasoconstriction or even temporal occlusion. This study paved the road for invasive assessment of coronary endothelial integrity and its association with atherosclerosis in the coronary circulation. In 1989, Cox and colleagues measured flow-mediated LAD diameter changes by quantitative angiography, using adenosine infusions to increase blood flow. Dilation was markedly impaired in atherosclerotic vessels. However, all vessels dilated with nitro-glycerine. Hence, absence of flow-mediated dilation in atherosclerosis reflects impaired endothelial vasodilator function. Later, in 2009, Lavi and colleagues demonstrated that coronary segments with a higher degree of endothelial dysfunction were associated with necrotic plaque.

Abstract 417: Adenosine Diphosphate Ribosyl Cyclase via PI3K-Akt and FOXO3a Up-regulating MMP-9 to Enhance Vascular Smooth Muscle Cell Proliferation and Migration in Mice

The current study was designed to explore the mechanisms of vascular smooth muscle cell (VSMC) proliferation and migration induced by adenosine diphosphate ribosyl cyclase(ADPRC). In this study, 32 Male ApoE-/- mice(6 weeks old, 18-22g)on a C57BL/6J background were divided into four groups, which received normal chow (n=8, NC group), high-fat Western-type diet (n=8, 0.25% cholesterol, 21% fat,HFD group), high-fat Western-type diet,infusion of 2,2′-dihydroxyazobenzene(DHAB, a ADPRC inhibitor, 2mg/kg/day, n=8, HFD-DHAB group) intraperitoneally or high-fat Western-type diet,infusion of LY294002(a Inhibitor of Akt, 5mg/kg/d, n=8, HFD-LY group) intraperitoneally, for 10 weeks. 8 male C57BL/6J mice served as control. After 10 weeks, mice were anesthetized with chloral hydrate, aorta was removed and immediately frozen in liquid nitrogen. Aortic atherosclerotic lesions, VSMC proliferation and migration were assessed by histomorphological observation, smooth muscle actin-α(α-SMA)and proliferating cell nuclear antigen (PCNA) examination. ADPRC expression and alterations of Akt, FOXO3a, phospho-FOXO3a and MMP-9 were determined by RT-PCR, Western Blot, Immunohistochemistry or Immunofluorescence. The results showed that, in aortic atherosclerotic lesions derived from atherosclerotic mice of HFD group, an increased VSMC proliferation and migration, reflected by the up-regulation of α-SMA and PCNA expression, were observed followed by increased expression of ADPRC, Akt, FOXO3a, phospho-FOXO3a and MMP-9. The enhanced expression of ADPRC and followed alterations of FOXO3a, phospho-FOXO3a, MMP-9 as well as α-SMA, PCNA, VSMC proliferation and migration were absent in NC group and C57BL/6J control mice. Treatment with DHAB or LY294002 reversed VSMC proliferation, migration and expression of Akt, FOXO3a, phospho-FOXO3a and MMP-9 in HFD-DHAB and HFD-LY group. These data shows that high-fat Western-type diet induced ADPRC may via PI3K-Akt to phosphorylate FOXO3a up-regulating MMP-9 to enhance vascular smooth muscle cell proliferation and migration in mice.

Identification of the origin of circulating endothelial progenitor cells

Identification of the origin of circulating endothelial progenitor cells

Angiotensin-(1-7) and angiotensin-(1-9) inhibit vascular smooth muscle cell growth and migration in vitro and vascular remodelling in vivo

Background: Vascular smooth muscle cell(VSMC) proliferation and migration underlies the pathogenesis of atherosclerosis, vein graft failure and in-stent restenosis. Angiotensin II(AngII), acting via the AT1R, is integral in these processes. AngII is inhibited by the counter-regulatory axis of the renin angiotensin system, which is centered around the actions of angiotensin-converting-enzyme-2 and the production of Ang-(1-7) and Ang-(1-9), which act via the Mas receptor and AT2R, respectively. Here we investigated the role of Ang-(1-9) and Ang-(1-7) in primary human VSMC migration and proliferation, and in a mouse model of vascular injury. Methods: Migration was assessed via scratch assay and proliferation using the MTS-assay. Vascular injury was induced in vivo via wire injury to the left carotid artery. Ang-(1-7) and Ang-(1-9) were delivered via osmotic minipump and vascular remodeling quantified at 28 days post-injury. Results: VSMC migration and proliferation was inhibited by Ang-(1-9) and Ang-(1-7); these effects were selectively blocked by the pharmacological antagonists PD123,319 and A779, respectively, suggesting Ang-(1-9) acts via the AT2R and Ang-(1-7) via Mas. In vivo wire injury of the mouse carotid artery induced significant neointimal formation(NI) at 28 days post-injury; this was attenuated by Ang-(1-7) and Ang-(1-9) via Mas and the AT2R, respectively. Conclusion: These data demonstrate that Ang-(1-7) and Ang-(1-9) inhibit VSMC proliferation and migration in vitro and neointimal formation in vivo via the AT2R and Mas receptor, respectively. Here we demonstrate for the first time a direct biological effect of Ang-(1-9) within the vasculature. These findings highlight the potential of Ang-(1-9) and Ang-(1-7) as therapeutic agents in vascular injury.

Impact and related mechanism of exogenous receptor activity modifying protein 1 on calcitonin gene-related peptide modified bone marrow mesenchymal stem cells on the migration of vascular smooth muscle cells in vitro

To investigate the impact of calcitonin gene-related peptide (CGRP) modified bone marrow mesenchymal stem cell (MSC) on the migration of vascular smooth muscle cell (VSMC) and related mechanisms. The MSC and VSMC were isolated from rats and cultured, CGRP was transfected to MSC with the high expression lentivirus vector, VSMC was transfected with high expression lentivirus vector of receptor activity modifying protein 1 (RAMP1) and the silence expression lentivirus vector of RAMP1. Then MSC was co-cultured with VSMC. Experimental groups were as follows: (1) Ang II group (MSC + VSMC + Ang II); (2) MSC(CGRP+) group (MSC(CGRP+) + VSMC + Ang II); (3) MSC(CGRP+) RAMP1(-) group (MSC(CGRP+) + VSMC(RAMP1-) + Ang II); (4) MSC(CGRP+) RAMP1(+) group (MSC(CGRP+) + VSMC(RAMP1+) + Ang II); (5) RAMP1(+) group (MSC + VSMC(RAMP1+) + Ang II). Transwell assay was applied to detect the migration of smooth muscle cells, Western blot was applied to detect the protein expression of cells in various groups. VSMC migration number was significantly lower in MSC(CGRP+) group compared with Ang II group (50.8 ± 2.6 vs. 71.4 ± 2.3, P < 0.05), but higher than in MSC(CGRP+) RAMP1(+) group (50.8 ± 2.6 vs. 30.4 ± 3.0, P < 0.05). When RAMP1 expression reduced in VSMC, compared with MSC(CGRP+) RAMP1(+) group, VSMC migration increased in the MSC(CGRP+) RAMP1(-) group compared to MSC(CGRP+)RAMP1(+) (69.0 ± 5.6 vs. 30.4 ± 3.0, P < 0.05) and was similar to Ang II group (69.0 ± 5.6 vs. 71.4 ± 2.3, P > 0.05) and RAMP1(+) group (71.6 ± 3.4). According to the result of Western blot, P-P65 protein expression in MSC(CGRP+) group was lower than that in Ang II group (0.475 ± 0.022 vs.0.642 ± 0.035, P < 0.05). P-P65 protein expression in MSC(CGRP+)RAMP1(-) group was higher than that in MSC(CGRP+) RAMP1(+) group (0.670 ± 0.030 vs. 0.373 ± 0.041, P < 0.05), and there was no difference between MSC(CGRP+)RAMP1(-) group and Ang II group (P > 0.05). P-P65 protein expression was similar between RAMP1(+) group (0.643 ± 0.039) and Ang II group (P > 0.05). CGRP inhibits VSMC migration through RAMP1. NF-κB and RAMP1 play crucial role in the inhibiting effects of CGRP on VSMC migration. Thus, RAMP1-CGRP signaling inhibits VSMC migration through NF-κB signal pathways.

178 Angiotensin-(1–9) inhibits vascular smooth muscle cell proliferation and migrationin vitroand neointimal formationin vivo

Vascular smooth muscle cell (SMC) proliferation and migration underlie the pathogenesis of vein graft failure and in-stent restenosis. Current interventions inhibit both SMC and endothelial cell (EC) growth, leading to thrombosis and re-occlusion. Angiotensin II (AngII) is a key regulator of VSMC proliferation and migration. A counter-regulatory axis of the renin angiotensin system (RAS) has been identified which inhibits AngII and is centred around angiotensin converting enzyme2 and Ang-(1–7) acting at Mas. We recently reported Ang-(1–9) as a novel member of this axis, acting at the angiotensin type 2 receptor (AT 2 R) to inhibit cardiac remodelling. Here we investigated the role of Ang-(1–9) in human SMC and EC migration and proliferation and vascular injury in vivo , and compared it to Ang-(1–7). SMC and EC were isolated from human saphenous veins. To assess migration, EC and SMC were stimulated with Ang II and Ang-(1–9) or Ang-(1–7) +/- the AT 1 R, AT 2 R or Mas antagonists losartan, PD123,319 (PD) or A779, respectively, and a wound healing assay performed. To assess proliferation, EC and SMC were stimulated with fetal calf serum (FCS) and Ang-(1–9) or Ang-(1–7) ± losartan, PD or A779. Proliferation was assessed using MTS and Edu assays. An in vivo mouse model was established via wire injury to the carotid artery. Ang-(1–7) or Ang-(1–9) ± PD or A779 were delivered subcutaneously via minipumps and neointima (NI) formation quantified 28 days post injury. Ang-(1–9) and Ang-(1–7) inhibited Ang II induced VSMC migration (Ang II 98.9 ± 1.1%, Ang-(1–9) 43.3 ± 3.3% and Ang-(1–7) 41.8 ± 4.6% wound closure; P 2 R and Ang-(1–7) via Mas. Neither Ang-(1–9) or Ang-(1–7) prevented EC migration or proliferation. In vivo wire injury of the mouse carotid artery induced significant NI formation at 28days (NI/media area (NI/MA) 0.80 ± 0.07 injured control vs 0.01 ± 0.01 sham; P in vivo Ang-(1–9) acts via the AT 2 R and Ang-(1–7) acts via Mas. We demonstrate for a novel, direct effect of Ang-(1–9) in inhibiting VSMC proliferation and migration in vitro , and reducing neointimal formation in vivo via the AT 2 R. These data provide further insight into the role of the Ang-(1–9)/AT 2 R interaction in the vasculature and highlights the potential of Ang-(1–9) as a therapeutic agent for vascular remodelling.

Catheter-based Renal Denervation as a Treatment for Pulmonary Hypertension: Hope or Hype?

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance (PVR) that causes right ventricular (RV) failure and premature death. This hypertensive condition leads to precapillary pulmonary hypertension, defined as a mean pulmonary artery pressure (mPAP) 25 mmHg, pulmonary capillary wedge pressure 15 mmHg, and PVR > 3 Wood units at rest. The prevalence of PAH is estimated to range from 15 to 50 patients/million, with a female predominance (men:women, 1:2) and 1-year and 5-year survival rates of 85% and 57%, respectively. During the last 15 years, treatments for PAH have been studied in multiple clinical trials, with approval subsequently granted for 9 distinct drugs belonging to 4 pharmacological families: prostanoids (epoprostenol, iloprost, treprostinil), endothelin receptor antagonists (bosentan, ambrisentan, macitentan), phosphodiesterase-5 inhibitors (sildenafil, tadalafil), and adenylate cyclase stimulators (riociguat). Despite these therapeutic advances, many patients with PAH show disabling symptoms and an ominous prognosis, underscoring the need for new therapeutic strategies to combat PAH. The pathophysiology of PAH involves excessive vasoconstriction and vascular remodeling. The increased PVR is initially reversible and is due to an imbalance between vasodilatory and vasoconstrictive agents, but can become permanent in later stages. The vascular remodeling is characterized by intimal thickening and fibrosis and the proliferation and migration of vascular smooth muscle cells, with hypertrophy and fibrosis of the tunica media, inflammation, and in situ thrombosis, which trigger the formation of plexiform and obstructive lesions (the most characteristic finding of PAH), all of which lead to RV remodeling. Given our poor understanding of the pathophysiology of PAH, the development of new preclinical models of PAH and their evaluation using appropriate imaging techniques are essential to further our understanding of PAH.

Abstract 440: Increased Migration and Proliferation of Human Venous Valve Wall Smooth Muscle Cells are Mediated by FGF2

Introduction: Lesions causing coronary and peripheral vein graft failure often occur at valves. To test the hypothesis that intrinsic differences in cells of the valve wall contribute to graft failure we measured cell migration from tissue explants and migration and proliferation of cultured cells from valve and non-valve sections of human saphenous vein (HSV). Methods: After removal of endothelium, HSVs were dissected into an outer wall with adventitia and some media and an inner wall with intima and some media. Cell migration from explants and migration (microchemotaxis chambers) and proliferation of cultured cells at passage 6 were measured. Results: Cells migrated faster from valve compared to non-valve tissue of the inner wall (Figure A; P&lt;0.01, N=24 veins), but not the outer wall (not shown). PDGF-BB- or serum-mediated migration of valve inner wall smooth muscle cells (VSMC) was greater than with non-valve SMCs (NVSMC; P&lt;0.05, N=6 pairs, 6.5 ± 1.2 vs. 4.2 ± 0.8 fold of control for PDGF-BB and 4.7 ± 0.6 vs. 3.4 ± 0.4 for serum). VSMC also proliferated faster than NVSMC after stimulation with PDGF-BB plus 2% serum (P&lt;0.01, N=6 pairs, 3.3 ± 0.4 vs. 2.2 ± 0.2 fold of control). Finally, a blocking antibody to FGF2 had no effect on PDGF-mediated migration or proliferation of NVSMC (migration: IgG 2.2 ± 0.3 vs anti-FGF2 2.0 ± 0.4, N=6; proliferation: IgG 2.6 ± 0.1 vs anti-FGF2 2.5 ± 0.2, N=7), but largely blocked the enhanced migration and proliferation of VSMC (migration: Figure B; P&lt;0.01, N=6; proliferation: control IgG 3.4 ± 0.3 vs. Anti-FGF2 Ab 2.7 ± 0.3 fold; P&lt;0.01, N=7). Conclusion: The increased migration and proliferation of VSMC compared to NVSMC is largely mediated by FGF2.

Abstract 451: Oxygen Regulation of Human Coronary Artery Smooth Muscle Cell Proliferation and Migration

Introduction: Intimal thickening of blood vessels, a hallmark of several vascular diseases including atherosclerosis and a potential point of therapeutic intervention, is caused by vascular smooth muscle cell proliferation and migration. It has been suggested that oxygen availability in vessels not only regulates behavior of smooth muscle cells but also serves as a trigger that may lead to pathological responses. In this study we determined whether hypoxia elicits proliferative and migratory responses in Human Coronary Artery SMCs (HCASMCs). Methods: Proliferation of HCASMCs was assessed using a 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. SMCs were plated in 96-well plates (n=5), serum starved, and then placed under hypoxic or normoxic conditions for 2, 4 and 6 days (2D/4D/6D) before MTT was added to each well. Absorbance at the wavelength 570 nm was read on an ELISA plate reader, and percent change in cell viability was determined and normalized to control (cell viability under normoxia). Cell migration was characterized by scratch-wound assay. SMCs were seeded in 6 well plates overnight (n=3), then a ‘scratch’ on the cell monolayer was created for each well before putting into different oxygen levels for 4 hours, 12 hours and 24 hours. Images were captured at the beginning and at intervals during cell migration to close the scratch, and the degree of migration was determined by comparing the images. Results: Compared to normoxic condition, cell number changed to 118.1%±1.3% in 5% O 2 (p&lt;0.05) and 98.2%%±1.9% in 1% O 2 after 2D; to 151.9% ±8.5% in 5% O 2 (p&lt;0.001) and 119.4%±5.0% in 1% O 2 (p&lt;0.05) after 4D; and to 163.0%±4.3% in 5% O 2 (p&lt;0.001) and 120.3%±2.2% in 1% O 2 (p&lt;0.05) after 6D. In the cell migration assay, the difference in migration rate between different groups after 4 hours was not obvious, but there was a significant difference after 12 hours (29.3%±1.3% closure in normoxia vs 39.8%±1.9% in 5% O 2 vs 40.9%±3.5% in 1% O 2 , p&lt;0.05) and 24 hours (71.5%±4.4% in normoxia vs 87.2%±2.2% in 5% O 2 vs 87.5%±3.1% in 1% O 2 , p&lt;0.05). Conclusion: Our studies reveal that hypoxia induces both proliferation and migration of HCASMCs.