Vascular Smooth Muscle Cells In Culture Research Articles

Effect of Lyso-phosphatidylcholine and Schnurri-3 on Osteogenic Transdifferentiation of Vascular Smooth Muscle Cells to Calcifying Vascular Cells in 3D Culture

BackgroundIn vitro cell culture is a widely used technique for investigating a range of processes such as stem cell behavior, regenerative medicine, tissue engineering, and drug discovery. Conventional cell culture is performed in Petri dishes or flasks where cells typically attach to a flat glass or plastic surface as a cell monolayer. However, 2D cell monolayers do not provide a satisfactory representation of in vivo conditions. A 3D culture could be a much better system for representing the conditions that prevail in vivo. Methods and resultsTo simulate 3D conditions, vascular smooth muscle cells (VSMCs) were loaded with gold–polyvmer–iron oxide hydrogel, enabling levitation of the cells by using spatially varying magnetic fields. These magnetically levitated 3D cultures appeared as freely suspended, clustered cells which proliferated 3–4 times faster than cells in conventional 2D cultures. When the levitated cells were treated with 10nM lysophosphatidylcholine (LPC), for 3days, cell clusters exhibited translucent extensions/rods 60–80μm wide and 200–250μm long. When 0.5μg/μl Schnurri-3 was added to the culture containing LPC, these extensions were smaller or absent. When excited with 590–650nm light, these extensions emitted intrinsic fluorescence at >667nm. When the 3D cultures were treated with a fluorescent probe specific for calcium hydroxyapatite (FITC-HABP-19), the cell extensions/rods emitted intensely at 518nm, the λmax for FITC emission. Pellets of cells treated with LPC were more enriched in calcium, phosphate, and glycosaminoglycans than cells treated with LPC and Schnurri-3. ConclusionsIn 3D cultures, VSMCs grow more rapidly and form larger calcification clusters than cells in 2D cultures. Transdifferentiation of VSMC into calcifying vascular cells is enhanced by LPC and attenuated by Schnurri-3. General significanceThe formation of calcified structures in 3D VSMC cultures suggests that similar structures may be formed in vivo.

The effect of telomerase activity on vascular smooth muscle cell proliferation in type 2 diabetes in vivo and in vitro

Serious complications as a result of type2 diabetes mellitus (T2DM) are becoming a major health concern. In the present study, it was hypothesized that telomerase activity is upregulated in vascular smooth muscle cells (VSMCs) during proliferation in T2DM and that the application of telomerase inhibitors impedes the proliferation of VSMCs invitro. Male Wistar rats were randomly allocated into the normal control (NC) or diabetic (DM) group. Diabetes was induced by high‑fat feeding and a low dose of streptozotocin(STZ; 30mg/kg). Primary VSMC cultures were exposed to normal(5.5mM) or high (25mM) glucose and insulin (100nM) in the presence and absence of various concentrations of antisense oligoribonucleotides (ASODNs) for varying lengths of time. Telomerase activity and the proliferation of VSMCs were measured. Results showed that there was a significant increase in the levels of fasting glucose, insulin, triglycerides (TG) and free fatty acids (FFAs) in the diabetic group. Telomerase activity and the proliferation of VSMCs were significantly higher in the diabetic group invivo and in the high glucose and insulin (HGI)-treated group invitro (P<0.01). ASODNs significantly inhibited the proliferation of VSMCs in a concentration- and time‑dependent manner (P<0.01). In conclusion, hyperglycemia and hyperinsulinemia stimulate telomerase activity and the proliferation of VSMCs, while the inhibition of telomerase activity reduces the proliferation of VSMCs, indicating that telomerase may be involved in the pathological process of diabetic vascular disease.

Long-term zinc deprivation accelerates rat vascular smooth muscle cell proliferation involving the down-regulation of JNK1/2 expression in MAPK signaling

BackgroundThe accelerated proliferation of vascular smooth muscle cells (VSMCs) is a contributor for atherosclerosis by thickening the vascular wall. Since zinc modulation of VSMC proliferation has not been clarified, this study investigated whether zinc affects VSMC proliferation. Methods and resultsBoth a rat aorta origin vascular smooth muscle cell line (A7r5 VSMCs) and primary VSMCs which were collected from rat aorta (pVSMCs) were cultured with zinc (0–50 μM Zn) for short- (≤12 d) and long-term (28 d) periods under normal non-calcifying (0 or 1 mM P) or calcifying (>2 mM P) P conditions. Mouse vascular endothelial cells (MS I cells) were also cultured (under 0–50 μM Zn and 10 mM P for 20 d) to compare with VSMC cultures. While during short-term culture of VSMCs, zinc deprivation decreased cell proliferation in a zinc-concentration manner both under non-calcifying and calcifying conditions in A7r5 and pVSMCs (P < 0.05), during long-term cultures (28 d), A7r5 VSMC proliferation was inversely related to medium zinc concentration under normal physiological P conditions (regression coefficient r2 = −0.563, P = 0.012). The anti-cell proliferative effect of zinc supplementation (>50 μM) was VSMC-specific. Long-term (35 d), low zinc treatment down-regulated JNK expression and activation, while not affecting ERK1/2 MAPK signaling in A7r5 VSMCs. ConclusionThe results showed that chronic zinc deprivation accelerated VSMC proliferation, perhaps due to down-regulation of MAPK-JNK signaling, and that the anti-cell proliferative role of zinc is VSMC-specific. The findings suggested that zinc may have anti-VSMC proliferative properties in atherosclerosis.

Influence of atorvastatin on angiotensin I metabolism in resting and TNF-α -activated rat vascular smooth muscle cells

Vascular smooth muscle cells (VSMCs) are essential for maintaining vasculature homeostasis and function. By influence on its growth and activation both proinflammatory cytokines and peptides of the renin-angiotensin system (RAS) are potent regulators of VSMCs. Interestingly, angiotensin (Ang) II and Ang-(1-7) elicit opposite effects on VSMC activation, differentiation and proliferation. It has been suggested that statins, besides anti-inflammatory effects, may also modulate VSMC activation by their influence on the RAS. The effect of atorvastatin on Ang I metabolism in a culture of explanted rat VSMCs was examined by liquid chromatography-mass spectrometry (LC-MS); expression of mRNA of the main RAS enzymes in VSMC was assessed by real-time polymerase chain reaction (PCR). In VSMC culture Ang-(1-7) was identified as a major product of Ang I metabolism. In this setting, TNF-α (1 ng/ml) caused a decrease in the conversion of Ang I to Ang-(1-7). This effect was accompanied by a decrease of mRNA expression of neutral endopeptidase (NEP) and angiotensin converting enzyme 2 (ACE2) and increase of mRNA of ACE. Interestingly, atorvastatin (3 μM) attenuated the effects of TNF-α on Ang-(1-7) production as well as reversed the influence of TNF-α on ACE and ACE2 expression. Enhancement by atorvastatin of the ACE2/Ang-(1-7) axis in VSMCs could represent a new and beneficial mechanism on cardiovascular action of this widely used drug.

Protective effect of nectandrin B, a potent AMPK activator on neointima formation: inhibition of Pin1 expression through AMPK activation

Neointima is considered a critical event in the development of vascular occlusive disease. Nectandrin B from nutmeg functions as a potent AMP-activated protein kinase (AMPK) activators. The present study addressed whether nectandrin B inhibits intimal hyperplasia in guide wire-injured arteries and examined its molecular mechanism. Neointima was induced by guide wire injury in mouse femoral arteries. Cell proliferation and mechanism studies were performed in rat vascular smooth muscle cells (VSMC) culture model. Nectandrin B increased AMPK activity in VSMC. Nectandrin B inhibited the cell proliferation induced by PDGF and DNA synthesis. Moreover, treatment of nectandrin B suppressed neointima formation in femoral artery after guide wire injury. We have recently shown that Pin1 plays a critical role in VSMC proliferation and neointima formation. Nectandrin B potently blocked PDGF-induced Pin1 and cyclin D1 expression and nectandrin B's anti-proliferation effect was diminished in Pin1 overexpressed VSMC. PDGF-induced phosphorylation of ERK and Akt was marginally affected by nectandrin B. However, nectandrin B increased the levels of p53 and its downstream target p21 and, also reversibly decreased the expression of E2F1 and phosphorylated Rb in PDGF-treated VSMC. AMPK inhibition by dominant mutant form of adenovirus rescued nectandrin B-mediated down-regulation of Pin1 and E2F1. Nectandrin B inhibited VSMC proliferation and neointima formation via inhibition of E2F1-dependent Pin1 gene transcription, which is mediated through the activation of an AMPK/p53-triggered pathway.

Proteomic evidence for the plasticity of cultured vascular smooth muscle cells

Morphological, functional, and gene expression studies have established the phenotypic plasticity of smooth muscle cells (SMCs). These cells have been shown to respond to environmental stimulants such as extracellular matrix and growth factors. Cellular changes can vary between extremes defined as the contractile and synthetic states. Various growth factors have been shown to have profound effects on the phenotype of these cells. In this study, we intended to investigate the effects of growth factor-rich medium on the protein expression of vascular SMCs in culture. Interestingly, transiently changing the type of medium did not result in any apparent morphological differences, yet we hypothesized that some cellular factors might still be altered. In order to understand what kind of intracellular molecular changes should be expected to occur during the medium change, we analyzed global protein expression changes using nano-LC-MS/MS in smooth muscle cell cultures that were isolated and grown in one medium formulation and temporarily switched to the other. Our data indicate that proteins playing a role in energy metabolism (glycolysis), translation, and folding of proteins are affected, along with regulatory molecules and cytoskeletal proteins. The individual proteins and their significance are discussed within the scope of this paper.

Modified explant technique for the culture of human vascular smooth muscle cells

Objective To establish an effective method of human vascular smooth muscle cells in vitro culture.Methods Medial layer of human aorta was separated under sterile conditions,then cut into small pieces (about 1 mm3).After pretreatment with 0.1％ type Ⅰ collagenase,the tissues were transferred into flask and cultured in DMEM containing 20％ fetal bovine serum,penicillin (100 U/ml) and streptomycin (100 g/L).The cell morphology was observed under the inverted microscopy.Smooth muscle cell specific protein (α-SMA) was identified by using immumofluorescence methods.Results Some smooth muscle cells started growing from explants in 4 to 5 days.One to 2 weeks later,the cells grew to confluence by migrated cells.Immumofluorescence staining showed positive expression of α-SMA (fluorescence intensity (++)-(+++)).Conclusion Modified explant technique by pretreating tissues with collagenase was an effective method to get vascular smooth muscle cells in vitro. Key words: Aorta; Vascular smooth muscle cells; Cell culture

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

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

Abstract 384: Nov/ccn3: A Potential Antagonist of Angiotensin Ii Effects in Kidney Disease

Progression of chronic kidney disease (CKD) represents a constant challenge for the identification of new therapeutic targets. The NOV/CCN3 matricellular protein, belonging to the CCN (CYR61/CCN1, CTGF/CCN2, NOV/CCN3) family, could represent a new endogenous inhibitor of the fibrotic process that plays a crucial role in tissue damage and regression of renal function. However, the relation between NOV and renal disease remains unclear. Our objective was to study whether alterations in renal NOV expression are involved in the progression of CKD. NOV mRNA expression was significantly induced by 2.5-3 fold at 12-16 weeks in renin-transgenic mice (RenTg), a well established model of AngII-mediated nephropathy, through the Angiotensin receptor 1 (AT 1 R) compared to normotensive control animals (n=7 mice per group, p&lt;0.05). NOV immunodetection revealed an up-regulation mainly in vascular smooth muscle cells (VSMC) in the renal cortex of RenTg mice. Interestingly, NOV expression was inversely correlated to known fibrotic markers such as Connective tissue growth factor, Transforming growth factor and collagen I, during the progression of renal disease in RenTg mice. NOV up-regulation at mRNA and protein levels (∼ 2.5 fold p&lt;0.05) by AngII through AT 1 R was also observed in VSMC cultures. Furthermore, infection of these cells with an adenovirus recombinant for NOV strongly repressed in a dose-dependent manner the expression of the Angiotensin Receptor 1 reaching 72 and 78% (p&lt;0.05) at mRNA and protein levels respectively. Treatment with exogenous NOV gave similar results. Conversely, knocking down NOV by small interfering RNA increased Angiotensin Receptor 1 by ∼3.5 to 4.5 fold and by ∼7.5 fold (p&lt;0.05) at mRNA and protein levels respectively. These results indicate that NOV is involved in an AngII- AT 1 R regulatory loop. In subsequent studies the NOV-induced downregulation of AT 1 R was abrogated by integrin β1 specific siRNAs. Thus, NOV can attenuate the deleterious profibrotic effects of AngII during the progression of CKD and may provide a potential therapeutic option against this pathology.

Abstract 664: Vascular Smooth Muscle Toll-like Receptor-2 (tlr2) Is Upregulated During Hyperglycemia

Hyperglycemia, the major initiator of diabetic vascular dysfunction, is related with heightened vascular contractility and inflammation. Toll-like receptor-2 (TLR2), a key effector of innate immunity, has recently emerged as a crucial mediator of proinflammatory signaling during diabetes; however, whether or not TLR2 is altered during diabetic vascular complications remains to be elucidated. We hypothesize that upregulation of TLR2 contributes to vascular dysfunction and inflammation in the aorta of diabetic rats. This study was performed in streptozotocin (STZ)-induced diabetic rats (65mg/kg; 5 weeks) and primary aortic vascular smooth muscle cell (VSMC) cultures stimulated with high glucose (HG; 25mM), in vivo and in vitro models of hyperglycemia, respectively. To confirm the diabetic vascular dysfunction condition, myograph studies were performed. The results showed that thoracic aortic rings from STZ-induced diabetic rats have increased contractile response to phenylephrine (PE, 1ηM to 0.1mM) compared to control (E max 206.20%±8.99% vs 98.41%±3.65%, respectively). TLR2 was increased significantly in the aorta from STZ-induced diabetic rats (1.8 fold vs. control) and in aortic VSMCs stimulated with HG for 6 hours (1.5 fold vs control). Elevated tumor necrosis factor α (TNF-α) and decreased interleukin-10 (IL-10) protein levels confirmed the proinflammatory state of aortas from STZ-diabetic rats (2.5 and 0.4 fold vs. control, respectively). Furthermore, expression of MyD88, a downstream adaptor protein for TLR2 signaling, is augmented significantly in diabetic aorta and aortic VSMCs under HG conditions (2.6 and 1.5 fold vs. control, respectively). Also, considering that protein kinase C (PKC) is a potential candidate of upstream activation of TLR2, we evaluated the expression the PKC isoforms. Our results show that PKCε is the only isoform increased significantly (1.7 fold vs. control). Our findings suggest that activating TLR2/MyD88 signaling pathway may play a role in mediating augmented proimflammatory response, which in turn contributes to vascular dysfunction during hyperglycemic conditions.

Abstract 172: Toll-like Receptor-4 Mediates Augmented Contractility In Diabetic Resistance Vessels

Hyperglycemia, the hallmark of diabetes, plays a central role in the pathogenesis of diabetic vascular dysfunction; however the underlying mechanisms are not entirely understood. We previously reported that inhibition of Toll-Like Receptor- 4 (TLR4), a key receptor of the innate immune system, improves vascular function in spontaneously hypertensive rats (SHR) revealing a novel role of TLR4 in the vascular system. In our current study, we hypothesized that upregulation of TLR4 in the vasculature following hyperglycemia leads to augmented vascular contractility. Third order mesenteric arteries from streptozotocin (STZ)-induced diabetic Sprague-Dawley rats (glucose 279 ± 11.6mg/dL) and their respective controls (98 ± 8.03mg/dL ), were incubated with either neutralizing antibody to TLR4 (1ug/mL) or IgG (1ug/mL) for 30 min and contractile responses to phenylephrine (PE) were evaluated ex vivo. Vessels from STZ rats exhibited greater maximal contractile force to PE compared to control (Emax 38.99 ± 1.45 vs. 23.98 ± 0.73 (mN), p&lt;0.05, respectively). Anti-TLR4 incubation attenuated this response to PE in vessels from STZ rats (29.71 ± 1.69mN) but had no effect in control vessels (21.03 ± 1.02mN). To test whether hyperglycemia modulates vascular TLR4, mesenteric vascular smooth muscle cell (MVSM) cultures were stimulated with high glucose (HG 25mM) or normal glucose (NG 5mM) for 48 hours. HG increased protein expression of TLR4 in MVSM compared to MVSM stimulated with NG (2.74±0.34 vs 1.38±0.20, p&lt;0.05). Concurrently, HG caused activation of mitogen-activated protein/extracellular signal-regulated kinase (ERK1/2) (5.04±0.83 vs 3.80±0.90, p&lt;0.05), increased levels of PCNA, a marker of proliferation, (1.34±0.11 vs 0.35±0.04, p&lt;0.05), and decreased levels of calponin, a marker of contractile phenotype, (1.45±0.14 vs 3.62±0.40, p&lt;0.05). Levels of total intracellular reactive oxygen species (ROS) were robustly augmented under HG condition (2.2 fold of the normal glucose). Our results suggest that hyperglycemia upregulates vascular TLR4 leading to augmented vascular contractility, potentially due to activation of ERK1/2, increased ROS and MSMCs proliferation.

Abstract 603: Silencing Of PKG1 Gene Mimics Effect Of Aging And Sensitizes Vascular Smooth Muscle Cells To Pro-fibrotic Effect Of Cardiotonic Steroids

Marinobufagenin (MBG), an endogenous Na/K-ATPase inhibitor and a vasoconstrictor, induces vascular fibrosis via PKC-dependent inhibition of Fli-1, a nuclear transcription factor and a negative regulator of collagen synthesis. In vascular smooth muscle cells (VSMC), ANP, via cGMP/PKG1 dependent mechanism, reduces sensitivity of Na/K-ATPase to MBG. Because vascular fibrosis is a hallmark of cardiovascular aging, we hypothesized that VSMC from aged rats, in which cGMP/PKG-dependent signaling is down-regulated, would exhibit heightened sensitivity to the pro-fibrotic effect of MBG. In primary culture of VSMC from thoracic aorta from young (3 month old) rats, 1 nmol/L MBG inhibited Na/K-ATPase by 15%, and induced a 60% down-regulation of Fli-1 and a concomitant 50% increase in the levels of collagen-1. Alpha-ANP (1 nmol/L) blocked these effects. In VSMC from aged (24 months old) rats levels of PKG1 and Fli-1 in VSMC were markedly reduced, while PDE-5 was markedly upregulated. MBG (1 nmol/L) inhibited Na/K-ATPase by 26%, suppressed Fli-1 two-fold, and increased level of collagen-1 more than two-fold (P&lt;0.01). Unlike that in young rats, alpha-ANP failed to oppose these effects of MBG in aged animals. Silencing of the PKG1 gene in VSMC from young rats produced a marked increase in the sensitivity of VSMC to the pro-fibrotic effect of MBG; 1 nmol/L MBG increased levels of collagen-1 2.5-fold (P&lt;0.01). These results demonstrate that age-associated reduction in vascular PKG1 levels and resultant decline in cGMP signaling sensitize VSMC to pro-fibrotic effect of MBG. Silencing of PKG1 in VSMC from young rats mimics these effects of aging.

Regulatory effects of 1,25-dihydroxyvitamin D3 on vascular smooth muscle cells.

Inflammatory response has been recognized as a central feature in the development and progression of atherosclerosis, and VSMCs (Vascular Smooth Muscle Cells) - the main cellular component of media, play an important role in this process. Many reports indicate that the biologically active vitamin D metabolite - 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3) = calcitriol), besides its well established role in calcium homeostasis, plays an essential role in the regulation of the inflammation process. The aim of this study was to determine the regulatory effects of calcitriol, applied at two supra-physiological doses (10 nM and 100 nM), in VSMC culture. Secretion of the pro-inflammatory cytokines, IL-6 and TNF-α, was significantly attenuated in calcitriol-treated VSMC culture, but the level of anti-inflammatory TGF-β was generally unchanged. Since in advanced atherosclerosis lesions several cell types, including VSMCs, overproduce the HSP70 chaperone protein, we also checked the effects of calcitriol on its synthesis. The presence of 1,25(OH)(2)D(3) did not affect HSP70 synthesis under physiological conditions but the synthesis of HSP70 in VSMCs exposed to heat shock was significantly inhibited by calcitriol (=100 nM). We observed that 1,25(OH)(2)D(3) induced SOD 1 activity, stimulated the expression of IκB-α, and did not influence the level of NF-κB-p65 in VSMCs. The results of our study suggest that 1,25(OH)(2)D(3) may serve as a natural anti-inflammatory agent and may therefore play a beneficial role in the physiology of VSMC in some contexts of atherosclerosis.

Resveratrol and Quercetin Interact to Inhibit Neointimal Hyperplasia in Mice with a Carotid Injury

Restenosis is a critical complication of angioplasty and stenting. Restenosis is multifactorial, involving endothelial injury, inflammation, platelet activation, and vascular smooth muscle cell (VSMC) proliferation. Thus, dietary strategies to prevent restenosis likely require the use of more than one agent. Resveratrol (R) and quercetin (Q) are polyphenols that are known to exhibit vascular protective effects. We tested whether R and Q administered in the diet interact to inhibit vessel stenosis in mice with a carotid injury. B6.129 mice were administered a high-fat diet containing 21% fat and 0.2% cholesterol along with R (25 mg/kg), Q (10 mg/kg), or R + Q for 2 wk. A carotid injury was induced and the mice were again administered the enriched diet for 2 wk. Compared with the controls, R significantly decreased stenosis, assessed as an intima:media ratio, by 76%. Although Q treatment alone exhibited no effect, it potentiated the effect of R in that treatment with R + Q significantly decreased the intima:media ratio by 94%. Moreover, this effect was greater than that of R treatment alone (P < 0.05). Although treatments with R, Q, and R + Q significantly affected platelet activation and endothelial function, the responses observed for R + Q were less than additive. Specifically, the effects of R + Q were less than the sum of effects for treatments with R and Q alone. In contrast, treatment with R + Q exhibited more-than-additive effects on inflammatory markers and significant interactions between R and Q were observed. The presence of synergy between R and Q was thus tested in cultures of VSMC and macrophages. Isobolographic analysis revealed that 2:1 molar ratios of R:Q exhibited synergistic inhibition of VSMC proliferation and macrophage chemotaxis. In conclusion, in combination, R and Q can interact to reduce the extent of restenosis, perhaps due to their synergistic inhibition of VSMC proliferation and inflammation.

Inhibition of vascular smooth muscle cell proliferation and migration in vitro and neointimal hyperplasia in vivo by adenoviral‐mediated atrial natriuretic peptide delivery

Vascular smooth muscle cell (VSMC) proliferation and migration are important components of the remodeling process in atherosclerosis or following angioplasty. Atrial natriuretic peptide (ANP) inhibits the growth of VSMCs in vitro but this effect has not been proven in vivo. In the present study, we examined the effects of local overexpression of ANP following gene transfer on in vitro VSMC proliferation and migration and in vivo neointimal formation in a rat carotid artery model of vascular injury. ANP gene transfer was performed using a recombinant adenovirus containing the ANP cDNA controlled by the Rous sarcoma virus (RSV) long terminal repeat (Ad-RSV-ANP). A recombinant adenovirus expressing the RSV-controlled β-galactosidase gene (Ad-RSV-β-gal) was used as the control. Rat VSMC culture was used for in vitro studies. In the in vivo experiments, carotid arteries were analyzed after balloon injury and local infusion of the viral solution. VSMCs transfected by Ad-RSV-ANP produced a significant amount of ANP detected by immunoreactive assay and accumulated about 6.5 times more cGMP than the viral control. VSMC proliferation stimulated with 10% fetal calf serum was reduced by 31% and migration by 25%. Fourteen days after injury, neointimal formation and the intima/media ratio were reduced by 25% and 28%, respectively, in the Ad-RSV-ANP-treated group compared to the control group. The present study demonstrates the efficacy of recombinant adenovirus Ad-RSV-ANP with respect to inhibiting rat VSMC proliferation and migration. Our findings also provide evidence that ANP is implicated in the modulation of vascular remodeling following endothelial injury.

Functional analyses of coronary artery disease associated variation on chromosome 9p21 in vascular smooth muscle cells

Variation on chromosome 9p21 is associated with risk of coronary artery disease (CAD). This genomic region contains the CDKN2A and CDKN2B genes which encode the cell cycle regulators p16(INK4a), p14(ARF) and p15(INK4b) and the ANRIL gene which encodes a non-coding RNA. Vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of atherosclerosis which causes CAD. We ascertained whether 9p21 genotype had an influence on CDKN2A/CDKN2B/ANRIL expression levels in VSMCs, VSMC proliferation and VSMC content in atherosclerotic plaques. Immunohistochemical examination showed that VSMCs in atherosclerotic lesions expressed p16(INK4a), p14(ARF) and p15(INK4b). Analyses of primary cultures of VSMCs showed that the 9p21 risk genotype was associated with reduced expression of p16(INK4a), p15(INK4b) and ANRIL (P = 1.2 × 10(-5), 1.4 × 10(-2) and 3.1 × 10(-9)) and with increased VSMC proliferation (P = 1.6 × 10(-2)). Immunohistochemical analyses of atherosclerotic plaques revealed an association of the risk genotype with reduced p15(INK4b) levels in VSMCs (P = 3.7 × 10(-2)) and higher VSMC content (P = 5.6 × 10(-4)) in plaques. The results of this study indicate that the 9p21 variation has an impact on CDKN2A and CDKN2B expression in VSMCs and influences VMSC proliferation, which likely represents an important mechanism for the association between this genetic locus and susceptibility to CAD.

Implication for transglutaminase 2-mediated activation of β-catenin signaling in neointimal vascular smooth muscle cells in chronic cardiac allograft rejection

Cardiac allograft vasculopathy (CAV) remains the main cause of long-term transplant rejection. CAV is characterized by hyperproliferation of vascular smooth muscle cells (VSMCs). Canonical β-catenin signaling is a critical regulator of VSMC proliferation in development; however, the role of this pathway and its regulation in CAV progression are obscure. We investigated the activity of β-catenin signaling and the role for a putative activating ligand, transglutaminase 2 (TG2), in chronic cardiac rejection. Hearts from Bm12 mice were transplanted into C57BL/6 mice (class II mismatch), and allografts were harvested 8 weeks after transplantation. Accumulation and sub-cellular distribution of β-catenin protein and expression of several components of β-catenin signaling were analyzed as hallmarks of pathway activation. In vitro, platelet-derived growth factor treatment was used to mimic the inflammatory milieu in VSMC and organotypic heart slice cultures. Activation of β-catenin in allografts compared with isografts or naïve hearts was evidenced by the augmented expression of β-catenin target genes, as well as the accumulation and nuclear localization of the β-catenin protein in VSMCs of the occluded allograft vessels. Expression of TG2, an activator of β-catenin signaling in VSMCs, was dramatically increased in allografts. Further, our ex vivo data demonstrate that TG2 is required for VSMC proliferation and for β-catenin activation by platelet-derived growth factor in cardiac tissue. β-Catenin signaling is activated in occluded vessels in murine cardiac allografts. TG2 is implicated as an endogenous activator of this signaling pathway and may therefore have a role in the pathogenesis of CAV during chronic allograft rejection.

Abstract 197: MARCKS Signaling Modulates Vascular Smooth Muscle Cell Proliferation

Background: The myrisolated alanine rich C-kinase substrate (MARCKS) is over expressed in animal models of intimal hyperplasia (IH) and in several human neoplasms. Further it has been demonstrated that MARCKS knock-down in vivo arrests vascular smooth muscle cell (VSMC) proliferation. We hypothesize that MARCKS arrests VSMC proliferation through a p27 dependent mechanism. Materials and Methods: Human coronary artery VSMC (Lonza) were grown in culture. Cell proliferation was determined by the AlamarBlue assay. Protein expression was determined by Western Blot analysis. MARCKS signaling was stimulated by treatment with the phorbol ester PMA, and MARCKS signaling was inhibited by either siRNA mediated knock-down or with one of two peptides that act as selective competitive antagonists. Results: Stimulation of VSMC in culture with PMA for 30 minutes resulted in and increased fraction of phosphor-MARCKS and complete knock-down of p27 protein. Treatment of VSMC in culture with siRNA resulted in arrest of cell proliferation, an increase in p27 expression with no change in p21 expression. Similarly, the peptide designed to specifically inhibit the functions of the N-terminal domain also resulted in similar effects in proliferation, p27, and p21 expression. The same results were noted when a separate peptide designed to inhibit the functions of the C-terminus domain was used to treat VSMC in culture. Conclusion: MARCKS signaling results in decreased p27 expression, which possibly explains proliferative VSMC phenotype observed in animal models of IH. Blockade of MARCKS signaling by siRNA mediated knock-down results in decreased cell proliferation and increased p27 protein expression. Antagonists to either the N- and C-terminus domains yield similar results in proliferation and p27 expression. VSMC proliferation requires both functional domains of MARCKS.

Observing better transfection efficiency in primary cultured VSMCs: Comparison and development of two different protocols

Aim: In this study, we have evaluated the procedure of transient transfection efficiency of vascular smooth muscle cells (VSMCs) with Lipofectamine (Life Technologies) and FuGENE (ROCHE, FuGENE HD Reagent) transfection reagents using the pCH110 eukaryotic assay vector containing the lacZ reporter gene. We also did try these attainments to transfect VSMCs with Ras N17 DNA and affirmed our findings. Material Methods: Plasmid pcH110, which has been purified by cesium chloride gradient centrifugation, was used in all transfections as the assay vector. And c-H-Ras was observed via Western-blot technique. Results: Briefly, the transfection with FuGENE has been given the best results, comparing with Lipofectamin. Under our culture conditions for VSMCs, FuGENE transfection efficiency could be augmented by simply increasing the amount of plasmid DNA 1.5–3 times above the recommended concentration without any visible cytotoxicity. With the FuGENE reagent, optimal transfection efficiency was obtained for primary culture of VSMCs within the recommended concentrations, but at the top of the range. The results indicate that optimization of the transfection process should include plasmid DNA concentrations above the levels suggested by the manufacturers, in order to accomplish the highest transfection efficiency. And, these finding was also supported with our Western-blot results when VSMCs have been transiently transfected with Ras N17 DNA.

Potential role of insulin signaling on vascular smooth muscle cell migration, proliferation, and inflammation pathways

To investigate the role of insulin signaling pathways in migration, proliferation, and inflammation of vascular smooth muscle cells (VSMCs), we examined the expression of active components of the phosphatidyl inositol 3 (PI-3) kinase (p-Akt) and mitogen-activated protein kinase (MAPK) (p-Erk) in primary cultures of VSMCs from human coronary arteries. VSMCs were treated in a dose-response manner with insulin (0, 1, 10, and 100 nM) for 20 min, and Akt and Erk phosphorylation were measured by Western blot analysis. In separate experiments, we evaluated the effect of 200 μM palmitate, in the presence and absence of 8 μM pioglitazone, on insulin-stimulated (100 nM for 20 min) Akt and Erk phosphorylation. The phosphorylation of Akt and Erk in VSMCs exhibited a dose dependency with a three- to fourfold increase, respectively, at the highest dose (100 nM). In the presence of palmitate, insulin-induced Akt phosphorylation was completely abolished, and there was a threefold increase in p-Erk. With addition of pioglitazone, the phosphorylation of Akt by insulin remained unchanged, whereas insulin-stimulated Erk phosphorylation was reduced by pioglitazone. These data in VSMCs indicate that high palmitate decreases insulin-stimulated Akt phosphorylation and stimulates MAPK, whereas preexposure peroxisome proliferator-activated receptor-γ agonist pioglitazone preserves Akt phosphorylation and simultaneously attenuates MAPK signaling. Our results suggest that metabolic and mitogenic insulin signals have different sensitivity, are independently regulated, and may play a role in arterial smooth muscle cells migration, proliferation, and inflammation in conditions of acute hyperinsulinemia.