Primary Cultures Of Vascular Smooth Muscle Cells Research Articles

Effect of Ang III on Nuclear Factor Kappa Beta in Wistar Rat VSMCs

ObjectiveWe investigated whether angiotensin (Ang) III induces nuclear factor kappa beta (NF‐kb) phosphorylation in isolated rat vascular smooth muscle cells (VSMCs).BackgroundThe molecular mechanisms by which Ang III induces various biological effects have not been fully investigated. Most studies have shown that NF‐kb mediates Ang II inflammatory responses effects in VSMCs. Inflammation of vascular smooth muscle cells (VSMCs) is a critical action associated with cardiovascular diseases including hypertension. The role of Ang III to induce NF‐kb in VSMCs is unknown and is a focus of these studies.MethodsPrimary cultures of VSMCs were isolated from the thoracic aorta of adult Wistar rats by the explant technique. VSMCs were treated with Ang III ranging in concentration from 0.1 nM to 1000 nM for 10 minutes or with 100 nM Ang III for 1 minute to 30 minutes. The western blotting technique was used to determine the effects of the peptide on NF‐kb protein phosphorylation.ResultsConcentration studies showed that Ang III caused a dose‐dependent increase in NF‐kb protein phosphorylation. The effects of the peptide on NF‐kb phosphorylation were maximal between 10 nM and 100 nM. The peptide's effects were rapid and significant; occurring within minutes of treatment and the maximal effects on NF‐kb phosphorylation was observed by 10 minutes.ConclusionThese findings provide insight into the molecular nature of the actions of Ang III and offer possible mechanism by which Ang III physiological actions occur in VSMCs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Porphyromonas gingivalis Lipopolysaccharide Stimulation of Vascular Smooth Muscle Cells Activates Proliferation and Calcification.

Porphyromonas gingivalis (Pg) is a major etiologic agent of periodontitis, whose virulence has been attributed to different factors, including lipopolysaccharide (LPS). Vascular ectopic calcification as a well-known major risk factor for adverse cardiovascular diseases is a highly prevalent vascular pathophenotype, and vascular smooth muscle cells (VSMCs) play an important role in mediating vascular calcification. It was hypothesized that Pg-LPS may stimulate vascular calcification through a direct effect on VSMC function. To test this hypothesis, the effect of Pg-LPS on VSMC calcification was determined. Primary cultures of VSMCs were obtained and identified by immunochemistry in vitro. The proliferation and alkaline phosphatase (ALP) activity of VSMCs were measured using a cell counting kit and an ALP activity test. Mineral deposition was examined using alizarin red staining. Gene (e.g. ALP, core binding factor α1 [Cbfα1], bone sialoprotein [BSP], and osteopontin [OPN]) expression levels altered by Pg-LPS were determined by reverse transcription-polymerase chain reaction array. Pg-LPS could increase the proliferation of VSMCs at different times and enhance ALP activity of VSMCs after 1 day. Alizarin red staining and quantification showed that, with Pg-LPS treatment, VSMCs displayed more obvious calcification nodules. When stimulated with Pg-LPS, the expression of specific osteogenic genes (e.g., ALP, Cbfα1, BSP, and OPN) was significantly promoted in the presence or absence of mineralization-inducing medium, whereas the expression of the OPN gene was inhibited in the mineralization induction group at day 7. Pg-LPS can stimulate VSMC calcification, which results in vascular calcification, further proving the precise relationship between periodontitis and vascular calcification.

Mechanisms involved in extracellular matrix remodeling and arterial stiffness induced by hyaluronan accumulation

Background and aimsHyperglycemia induces hyaluronan (HA) accumulation in the vasculature. Excessive accumulation of HA around the vascular smooth muscle cells (VSMC) results in increased aortic stiffness and strength and accelerated atherosclerosis in ApoE−/− mice. We hypothesized that HA accumulation primes the vasculature for atherosclerosis by crosslinking and reorganizing the extracellular matrix (ECM) and by pushing VSMC differentiation towards a less mature phenotype. MethodsAortas from HAS-2 transgenic (Tg) mice and wild type mice were used for all experiments. Biomechanics and cross-sectional area measurements were performed before and after HA digestion. The vessel and ECM composition was examined by immunoblotting and electron microscopy. Primary VSMC cultures were examined by qPCR and thymidine incorporation. ResultsTg mice aorta cross-sectional area was increased before (14%, p = 0.0148), but not after HA digestion (p = 0.3437). The increase in vessel stiffness (32%, p = 0.0217) and strength (31%, p = 0.0043) in the Tg aorta persisted after HA digestion. Crosslinking of HA by heavy chains from Inter-α-Inhibitor was increased (175%, p = 0.0006). The Tg VSMCs have the appearance of a synthetic phenotype supported by a 40% decrease in α-smooth muscle actin isoform X1 (p = 0.0296) and an increase in proliferation (63%, p = 0.0048) and osteoprotegerin production (133%, p = 0.0010) in cultured Tg VSMCs. ConclusionsOur results show that induced HA accumulation is followed by increased HA crosslinking and create a shift in VSMC phenotype and proliferation. These findings may provide a mechanism for how hyperglycemia through HA accumulation prime the vascular wall for cholesterol and leucocyte accumulation and development of atherosclerosis.

Effect of Angiotensin III on ERK1/2 and p38 mitogen activated protein (MAP) kinases in Wistar rat VSMCs

Objective: We investigated whether angiotensin (Ang) III induces ERK1/2 and p38 mitogen activated protein (MAP) kinases protein phosphorylation in isolated rat vascular smooth muscle cells (VSMCs). Background The molecular mechanisms by which Ang III induces various biological effects have not been fully investigated. Most studies have shown that MAP kinases mediate Ang II apoptosis and growth promoting effects in VSMCs.Moreover, Ang II induces vascular remodeling in these cells leading to increases in blood pressure. MAP kinases regulate or induce two crucial actions in VSMCs, proliferation and migration, which are associated with atherosclerosis and restenosis. Methods: Primary cultures of VSMCs were isolated from the thoracic aorta of adult Wistar rats by the explant technique. VSMCs were treated with Ang III ranging in concentration from 0.1 nM to 1000 nM for 10 minutes or with 100 nM Ang III for 1 minute to 30 minutes. Western blotting technique was used to determine whether Ang III induces ERK1/2 and p38 MAP kinases protein phosphorylation. Results: Concentration studies showed that Ang III caused a dose-dependent increase in ERK1/2 and p38 MAP kinases protein phosphorylation. The effects of Ang III on both MAP kinases phosphorylation were maximal between 10 nM and 100 nM concentrations. The peptide effects were rapid and significant, occurring within minutes of treatment and the maximal effects on MAP kinases phosphorylation was observed by 10 min. Conclusion These findings provide insight into the molecular nature of the actions of Ang III and offer possible mechanism by which Ang III physiological and possibly pathological actions occur in VSMCs.

Abstract 227: Molecular Mechanism of Arhgef11 via the Rho-Rock Pathway in Promoting Kidney Injury in the Dahl S Rat

We have previously implicated genetic variants in Arhgef11 , a Rho guanine nucleotide exchange factor, in kidney injury exhibited in the Dahl salt-sensitive (S) rat, a widely studied model of hypertension and chronic kidney disease. We have demonstrated that genetic knockdown (lentiviral shRNA) of Arhgef11 in HEK293 cells results in reduced RhoA activity (~20%), decreased activation of Rho-ROCK pathway (40-60%), and less stress fiber formation vs. scrambled control. S rat derived primary proximal tubule cells (p-PTC) exhibit increased expression of Arhgef11 , activation of Rho-ROCK, and when stimulated with the profibrotic cytokine, TGFβ1 are also more prone (vs. control) to epithelial-mesenchymal transition (EMT). These data (along with animal studies) suggest that dysregulation of Rho-ROCK in tubular cells likely play an important role in EMT and promote fibrosis; however, chronic stimulation of this pathway in other kidney cell types including vascular smooth muscle cells (VSMC) may also play a role. Accordingly, S rat primary cultures of VSMC isolated from kidney microvessels (k-VSMC) were studied for differences in the RhoA-ROCK, proliferation, migration, apoptosis, and response to cyclical stretch compared to control k-VSMC (minimal congenic). S rat k-VSMC demonstrated higher expression of Arhgef11 and activation of Rho-ROCK pathway, similar to p-PTC studies. Cell migration was significantly enhanced (scratch assay) in k-VSMC from S rats (485.0±26.2 nm) compared to 296.2±37.6 nm in cells from control. Apoptosis, evaluated by flow cytometry, was significantly increased in k-VSMC from S rats stimulated with TGFβ1 (10ng/ml, 24hrs), but not Ang II, at both early (9.9%±0.9%) and late apoptosis (22.0%±3.5%) compared to control (7.71%±0.75% and 15.5%±1.3%, respectively). S rat derived k-VSMC also demonstrated up-regulation and activation of Rho-ROCK pathway after cyclical stretch, which models in vivo stimuli such as shear stress. In summary, increased expression of Arhgef11 (RhoA-ROCK) exhibited by PTC and VSMC cell types in S rat kidney appear to promote epithelial-mesenchymal transition and enhance proliferation, migration, and apoptosis under baseline and profibrotic conditions, which likely contributes to kidney injury.

Nitric oxide and carbon monoxide antagonize TGF-β through ligand-independent internalization of TβR1/ALK5.

Transforming growth factor (TGF)-β plays a central role in vascular homeostasis and in the pathology of vascular disease. There is a growing appreciation for the role of nitric oxide (NO) and carbon monoxide (CO) as highly diffusible, bioactive signaling molecules in the vasculature. We hypothesized that both NO and CO increase endocytosis of TGF-β receptor type 1 (TβR1) in vascular smooth muscle cells (VSMCs) through activation of dynamin-2, shielding cells from the effects of circulating TGF-β. In this study, primary cultures of VSMCs from Sprague-Dawley rats were treated with NO-releasing molecule 3 (a NO chemical donor), CO-releasing molecule 2 (a CO chemical donor), or control. NO and CO stimulated dynamin-2 activation in VSMCs. NO and CO promoted time- and dose-dependent endocytosis of TβR1. By decreasing TβR1 surface expression through this dynamin-2-dependent process, NO and CO diminished the effects of TGF-β on VSMCs. These findings help explain an important mechanism by which NO and CO signal in the vasculature by decreasing surface expression of TβR1 and the cellular response to TGF-β.

Resveratrol protects vascular smooth muscle cells against high glucose-induced oxidative stress and cell proliferation in vitro.

BackgroundResveratrol exhibits beneficial effects against numerous degenerative diseases at different stages of pathogenesis. This study investigated potential mechanisms and resveratrol effects on high glucose (HG)-induced oxidative stress (30 mM d-glucose, 30 min) and cell proliferation (30 mM d-glucose, 24 h) in vascular smooth muscle cells (VSMCs).Material/MethodsIntracellular reactive oxygen species (ROS) generation was detected by 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Total antioxidant capacity (TAC), malonyldialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) were measured to evaluate oxidative stress. VSMC proliferation was measured by CCK-8 assays and through propidium iodide-based cell cycle analysis. Expression of NAD(P)H oxidase, proliferation proteins, and cell signalling were assessed by immunoblot analysis.ResultsCo-treatment of primary cultures of VSMCs with 1–100 μM resveratrol decreased HG-induced ROS overproduction (P<0.05). Resveratrol also abolished HG-induced phosphorylation of oxidase subunit p47 phox and reduced HG-induced cyclin D1, cyclin E, and PCNA expression in a concentration-dependent manner. Furthermore, resveratrol (10 μM) attenuated HG-induced phosphorylation of Akt, p38 mitogen-activated protein kinase (MAPK), ERK 1/2, and JNK1/2 without affecting total levels. HG stimulation enhanced downstream IκB-α phosphorylation and NF-κB activity, and resveratrol repressed these effects.ConclusionsResveratrol inhibits HG-induced oxidative stress and VSMC proliferation by suppressing ROS generation, NADPH oxidase, Akt phosphorylation, p38 MAPK/JNK/ERK phosphorylation, and IκB-α and NF-κB activities.

Functional Cooperation between Vitamin D Receptor and Runx2 in Vitamin D-Induced Vascular Calcification

The transdifferentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells has been implicated in the context of vascular calcification. We investigated the roles of vitamin D receptor (Vdr) and runt-related transcription factor 2 (Runx2) in the osteoblastic differentiation of VSMCs in response to vitamin D3 using in vitro VSMCs cultures and in vivo in Vdr knockout (Vdr -/-) and Runx2 carboxy-terminus truncated heterozygous (Runx2 +/ΔC) mice. Treatment of VSMCs with active vitamin D3 promoted matrix mineral deposition, and increased the expressions of Vdr, Runx2, and of osteoblastic genes but decreased the expression of smooth muscle myosin heavy chain in primary VSMCs cultures. Immunoprecipitation experiments suggested an interaction between Vdr and Runx2. Furthermore, silencing Vdr or Runx2 attenuated the procalcific effects of vitamin D3. Functional cooperation between Vdr and Runx2 in vascular calcification was also confirmed in in vivo mouse models. Vascular calcification induced by high-dose vitamin D3 was completely inhibited in Vdr -/- or Runx2 +/ΔC mice, despite elevated levels of serum calcium or alkaline phosphatase. Collectively, these findings suggest that functional cooperation between Vdr and Runx2 is necessary for vascular calcification in response to vitamin D3.

Urotensin-II promotes vascular smooth muscle cell proliferation through store-operated calcium entry and EGFR transactivation

Urotensin-II (UII) is a vasoactive peptide that promotes vascular smooth muscle cells (VSMCs) proliferation and is involved in the pathogenesis of atherosclerosis, restenosis, and vascular remodelling. This study aimed to determine the role of calcium (Ca(2+))-dependent signalling and alternative signalling pathways in UII-evoked VSMCs proliferation focusing on store-operated Ca(2+) entry (SOCE) and epithelium growth factor receptor (EGFR) transactivation. We used primary cultures of VSMCs isolated from Wistar rat aorta to investigate the effects of UII on intracellular Ca(2+) mobilization, and proliferation determined by the 5-bromo-2-deoxyuridine (BrdU) assay. We found that UII enhanced intracellular Ca(2+) concentration ([Ca(2+)]i) which was significantly reduced by classical SOCE inhibitors and by knockdown of essential components of the SOCE such as stromal interaction molecule 1 (STIM1), Orai1, or TRPC1. Moreover, UII activated a Gd(3+)-sensitive current with similar features of the Ca(2+) release-activated Ca(2+) current (ICRAC). Additionally, UII stimulated VSMCs proliferation and Ca(2+)/cAMP response element-binding protein (CREB) activation through the SOCE pathway that involved STIM1, Orai1, and TRPC1. Co-immunoprecipitation experiments showed that UII promoted the association between Orai1 and STIM1, and between Orai1 and TRPC1. Moreover, we determined that EGFR transactivation, extracellular signal-regulated kinase (ERK) and Ca(2+)/calmodulin-dependent kinase (CaMK) signalling pathways were involved in both UII-mediated Ca(2+) influx, CREB activation and VSMCs proliferation. Our data show for the first time that UII-induced VSMCs proliferation and CREB activation requires a complex signalling pathway that involves on the one hand SOCE mediated by STIM1, Orai1, and TRPC1, and on the other hand EGFR, ERK, and CaMK activation.

Modifications of Chromatin Dynamics Control Smad2 Pathway Activation in Aneurysmal Smooth Muscle Cells

The activation of the Smad2 signaling pathway is thought to play an important role in human aneurysmal diseases as described by an important body of research. We previously showed that constitutive Smad2 activation is associated with Smad2 mRNA overexpression in aneurysmal vascular smooth muscle cells (VSMCs), which is dependent on epigenetic regulation of the SMAD2 promoter involving histone modifications. However, the underlying molecular mechanisms controlling Smad2 overexpression are currently unknown. The aim of the present study is to understand the mechanisms regulating the constitutive Smad2 overexpression in VSMCs by identification of the histone-modifying enzymes, transcription factors, and cofactors responsible for Smad2 promoter activation in aneurysmal disease. This study was performed on medial tissue extracts and primary cultures of VSMCs of human thoracic aneurysms (n=17) and normal thoracic aortas (n=10). Here, we demonstrate that the activation of SMAD2 promoter is driven by the recruitment of a multipartner complex, including the transcription factor p53 and histone acetyltransferases. Remarkably, the transcriptional regulatory network of the SMAD2 promoter is dramatically altered in human aneurysmal VSMCs in vitro and in situ with a switch from Myc-dependent repression of SMAD2 in normal vessel to a p53-dependent activation of SMAD2 in aneurysms. Furthermore, histone acetyltransferases p300 and P300/CBP-associated protein play a major role in SMAD2 promoter activation by acting on histone acetylation, p53 recruitment, and acetylation. These results provide evidence for a major role of p53 and the complex composed of p300 and p300/CBP-associated protein in Smad2 activation in human aneurysmal VSMCs.

Smad2-Dependent Protease Nexin-1 Overexpression Differentiates Chronic Aneurysms From Acute Dissections of Human Ascending Aorta

Tissue activation of proteolysis is involved in acute intramural rupture (dissections, acute ascending aortic dissection) and in progressive dilation (aneurysms, thoracic aneurysm of the ascending aorta) of human ascending aorta. The translational aim of this study was to characterize the regulation of antiproteolytic serpin expression in normal, aneurysmal, and dissecting aorta. We explored expression of protease nexin-1 (PN-1) and plasminogen activator inhibitor-1 and their regulation by the Smad2 signaling pathway in human tissue and cultured vascular smooth muscle cells (VSMCs) of aneurysms (thoracic aneurysm of the ascending aorta; n=46) and acute dissections (acute ascending aortic dissection; n=10) of the ascending aorta compared with healthy aortas (n=10). Both PN-1 and plasminogen activator inhibitor-1 mRNA and proteins were overexpressed in medial tissue extracts and primary VSMC cultures from thoracic aneurysm of the ascending aorta compared with acute ascending aortic dissection and controls. Transforming growth factor-β induced increased PN-1 expression in control but not in aneurysmal VSMCs. PN-1 and plasminogen activator inhibitor-1 overexpression by aneurysmal VSMCs was associated with increased Smad2 binding on their promoters and, functionally, resulted in VSMC self-protection from plasmin-induced detachment and death. This phenomenon was restricted to aneurysms and not observed in acute dissections. These results demonstrate that epigenetically regulated PN-1 overexpression promotes development of an antiproteolytic VSMC phenotype and might favor progressive aneurysmal dilation, whereas absence of this counter-regulation in dissections would lead to acute wall rupture.

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.

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.

Peroxisome proliferator-activated receptor-γ stimulates 11β-hydroxysteroid dehydrogenase type 1 in rat vascular smooth muscle cells

Glucocorticoids are metabolized in vascular tissue by two types of 11β-hydroxysteroid dehydrogenases (11HSD1, 11HSD2) and thus these enzymes are considered to be important factors that modulate the diverse and complex effects of glucocorticoids on cardiovascular function. The present study evaluated the effect of peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone on 11HSD1 vascular smooth muscle cells (VSMC) and compared the effect with that of corticosterone. Using primary cultures of VSMC derived from rat aorta, we showed that pioglitazone significantly increases 11HSD1 activity and mRNA expression in a dose-dependent manner with EC 50 243 nM and that this effect is not blocked by RU 486, an antagonist of the glucocorticoid receptor. In contrast, corticosterone had no effect on 11HSD1. Pioglitazone positively regulated transcription of two CCAAT/enhancer-binding proteins (C/EBPs), specifically C/EBPα a potent activator of 11HSD1 gene transcription in some cells types, and C/EBPζ, whereas C/EBPβ and C/EBPδ were not changed. In contrast, corticosterone stimulated the expression of C/EBPβ and C/EBPδ, but the levels of C/EBPα and C/EBPζ were not changed. In conclusion, activation of PPARγ in VSMC up-regulates vascular 11HSD1 and thus reactivates 11-oxo metabolites to biologically active glucocorticoids through a mechanism that seems to involve C/EBPα and C/EBPζ. Our data provide one of the possible explanations for PPARγ agonists’ effects on the cardiovascular system.

Phenotypic modulation of cultured vascular smooth muscle cells: a functional analysis focusing on MLC and ERK1/2 phosphorylation

Primary cultures of vascular smooth muscle cells (VSMCs) from rats offer a good model system to examine the molecular basis of mechanism of vascular contraction-relaxation. However, during pathological conditions such as atherosclerosis and hypertension, VSMCs characteristically exhibit phenotypic modulation, change from a quiescent contractile to a proliferative synthetic phenotype, which impairs this mechanism of vascular contraction-relaxation. Taking in account that Myosin light chain (MLC) and ERK1/2 directly participate in the process of vascular contraction, the aim of the current study was to analyze the involvement of MLC and ERK1/2 signaling during the process of VSMCs phenotypic modulation. Primary cultures of VSMCs from rat thoracic aortas were isolated and submitted to different number of passages or to freezing condition. Semi-quantitative RT-PCR was used to evaluate the mRNA levels of VSMCs differentiation markers, and western blot assays were used to determine the MLC and ERK1/2 phosphorylation levels during VSMCs phenotypic modulation. Also, immunocytochemical experiments were performed to evaluate morphological alterations occurred during the phenotypic modulation. Elevated number of passages (up to 4) as well as the freezing/thawing process induced a significant phenotypic modulation in VSMCs, which was accompanied by diminished MLC and ERK1/2 phosphorylation levels. Phosphorylation of MLC was suppressed completely by the treatment with a synthetic inhibitor of MEK-1, a direct upstream of ERK1/2, PD98059. These findings provide that ERK1/2-promoted MLC phosphorylation is impaired during VSMCs phenotypic modulation, suggesting that ERK1/2 signaling pathway may represent a potential target for understanding the pathogenesis of several vascular disease processes frequently associated to this condition.

Akt Regulates the Survival of Vascular Smooth Muscle Cells via Inhibition of FoxO3a and GSK3

Apoptosis of vascular smooth muscle cells (VSMCs) may lead to atherosclerotic plaque instability and rupture, resulting in myocardial infarction, stroke, and sudden death. However, the molecular mechanisms mediating survival of VSMCs in atherosclerotic plaques remain unknown. Although plaque VSMCs exhibit increased susceptibility to apoptosis and reduced expression of the IGF1 receptor (IGF1R) when compared with normal VSMCs, a causative effect has not been established. Here we show that increased expression of the IGF1R can rescue plaque VSMCs from oxidative stress-induced apoptosis, demonstrating that IGF-1 signaling is a critical regulator of VSMC survival. Akt mediates the majority of the IGF1R survival signaling, and ectopic activation of Akt was sufficient to protect VSMCs in vitro. Both IGF1R and phospho-Akt expression were reduced in human plaque (intimal) VSMCs when compared with medial VSMCs, suggesting that Akt mediates survival signaling in atherosclerosis. Importantly, downstream targets of Akt were identified that mediate its protective effect as inhibition of FoxO3a or GSK3 by Akt-dependent phosphorylation protected VSMCs in vitro. We conclude that Akt and its downstream targets FoxO3a and GSK3 regulate a survival pathway in VSMCs and that their deregulation due to a reduction of IGF1R signaling may promote apoptosis in atherosclerosis.

Characterization of Phosphate Transport in Rat Vascular Smooth Muscle Cells

Hyperphosphatemia and inorganic phosphate (Pi) transport by vascular smooth muscle cells (VSMCs) have been implicated in the pathogenesis of vascular calcification. The aim of this work has been to characterize Pi transport in VSMCs. Primary cultures of VSMCs express both high affinity Na-dependent and Na-independent components of Pi transport. Under physiological conditions both transport systems are saturated, show similar activity, and are inhibited by increasing pH. The Na-dependent transport is also weakly inhibited by phosphonoformic acid (PFA) (3.9 mmol/L IC50 at 0.05 mmol/L Pi). Real-time polymerase chain reaction shows that Pit1 and Pit2 are expressed to the same degree, and no other Pi transporters are significantly expressed. When expressed in Xenopus oocytes they are strictly Na-dependent, with high affinities for Pi, and are inhibited by increasing pH, but only weakly inhibited by PFA. We have used RNA interference to demonstrate that Pit1 and Pit2 are the transporters responsible for Na-dependent Pi transport in VSMCs. Taken together these novel findings suggest new roles of Pi transport in the pathogenesis of VC and have implications as potential future clinical targets.

Artesunate reduces proliferation, interferes DNA replication and cell cycle and enhances apoptosis in vascular smooth muscle cells.

This study examined the effect of artesunate (Art) on the proliferation, DNA replication, cell cycles and apoptosis of vascular smooth muscle cells (VSMCs). Primary cultures of VSMCs were established from aortas of mice and artesunate of different concentrations was added into the medium. The number of VSMCs was counted and the curve of cell growth was recorded. The activity of VSMCs was assessed by using MTT method and inhibitory rate was calculated. DNA replication was evaluated by [3H]-TdR method and apoptosis by DNA laddering and HE staining. Flowmetry was used for simultaneous analysis of cell apoptosis and cell cycles. Compared with the control group, VSMCs proliferation in Art interfering groups were inhibited and [3H]-TdR incorprating rate were decreased as well as cell apoptosis was induced. The progress of cell cycle was blocked in G0/G1 by Art in a dose-dependent manner. It is concluded that Art inhibits VSMCs proliferation by disturbing DNA replication, inducing cell apoptosis and blocking cell cycle in G0/G1 phase.

Endogenous estrogen deficiency reduces proliferation and enhances apoptosis-related death in vascular smooth muscle cells: insights from the aromatase-knockout mouse.

Altered proliferation and death of vascular smooth muscle cells (VSMCs) are important mechanisms in vascular growth and remodeling. This study examined the effect of endogenous estrogens on VSMC proliferation. An estrogen-deficient animal model, the aromatase-knockout (ArKO) mouse, was used. Primary cultures of VSMCs were established from aortas of 11-week-old male and female ArKO and wild-type (WT) mice. In ArKO cells, the absence of aromatase cytochrome P450 mRNA expression was demonstrated by reverse transcription-polymerase chain reaction; Western blotting showed normal expression of estrogen receptor protein. Proliferative responses to serum or platelet-derived growth factor-BB were lower in ArKO than WT cells; 17beta-estradiol (E2, 10 nmol/L) enhanced the response to platelet-derived growth factor-BB in ArKO cells but inhibited the response in WT cells. E2 inhibited activity of mitogen-activated protein kinase ERK1/2 in WT but not ArKO cells. Apoptosis-related death caused by tumor necrosis factor-alpha was greater in ArKO than in WT cells; this effect in ArKO was attenuated by E2. Differences in VSMC proliferation between ArKO and WT occurred in both sexes. Morphological studies in aortas derived from male mice at 1 year of age demonstrated that medial smooth muscle area was approximately 10% less in ArKO than WT mice at this age. Deficiency of endogenous estrogens reduces proliferation and enhances apoptosis-related death in VSMCs; exogenous E2 corrects these abnormalities.

Estrogen activates phosphatases and antagonizes growth-promoting effect of angiotensin II.

Accumulating evidence suggests that estrogen exerts cardioprotective effects and protects against neointima formation in response to vascular injury in vivo, whereas angiotensin (Ang) II stimulation via the Ang II type 1 (AT(1)) receptor exaggerates vascular injury. We postulate that estrogen treatment antagonizes the AT(1) receptor-mediated growth-promoting effects in vascular smooth muscle cells (VSMCs). The present in vitro study was designed to explore this possibility and to establish the cellular mechanism whereby estrogen attenuates the growth of VSMCs. Primary cultures of VSMCs derived from male adult Sprague-Dawley rats express exclusively AT(1) receptors. Treatment with Ang II enhanced proliferation of VSMC and c-fos expression, whereas 17beta-estradiol (E2) attenuated these vasotrophic effects of Ang II. We also demonstrated that E2 attenuated AT(1) receptor-mediated extracellular signal-regulated kinase activation and that this effect of E2 was restored by pretreatment with vanadate or okadaic acid. Moreover, we demonstrated that E2 enhanced SHP-1 activity, rapidly reaching a peak after 3 minutes of E2 stimulation, whereas E2 transactivated mitogen-activated protein kinase phosphatase-1 expression, showing a peak after 60 minutes of E2 treatment. SHP-1 activation was not influenced by actinomycin D treatment, whereas E2-mediated mitogen-activated protein kinase phosphatase-1 expression was attenuated. Taken together, our results suggest a novel mechanism of vasoprotection by which estrogen antagonizes the effect of the AT(1) receptor via the activation and induction of phosphatases through nongenomic as well as genomic signaling.