Mechanisms In Vascular Smooth Muscle Cells Research Articles

S-12-2: ENHANCING MAS1:ETBR INTERACTION AS A POTENTIAL VASOPROTECTIVE STRATEGY IN STROKE-PRONE SHR

We previously demonstrated that Ang-(1–7) has anti-inflammatory effects in endothelial cells by inducing interaction between Mas1 and ETBR. Using HTS of > 20,000 druggable compounds, we identified several compounds that enhance Mas1:ETBR interaction as a novel strategy to promote vasoprotection. The objective of this study was to evaluate effects of 2 candidate compounds, termed enhancers 3 and 4 (Enh3 and Enh4), on functional responses in vascular smooth muscle cells (VSMCs) and their therapeutic potential in hypertensive (SHRSP) rats. VSMCs from normotensive (WKY) and SHRSP rats were exposed to Enh3 and Enh4(10–5 M; 5–30 mins). Expression of pro-contractile and mitogenic molecules was assessed by immunoblotting. Ca2+ influx was evaluated using fluorescence microscopy. ROCK2 activity was assessed using a colorimetric immunoassay which detected MYPT1 phosphorylation at Thr696. Male SHRSP rats (18–21weeks) were treated subcutaneously with Enh4 (10 mg/kg/day) for 7 days. Changes in blood pressure and vascular function were assessed via radiotelemetry and wire myography respectively. In WKY VSMCs, Enh3 reduced basal ERK1/2 phosphorylation (26.8 ± 5.9% vs veh,), an effect absent in SHRSP VSMCs, while Enh4 had no effect. Only in SHRSP VSMCs, Enh3, but not Enh4, reduced basal AKT phosphorylation (63.5 ± 8.9% vs veh). Enh4, but not Enh3, reduced basal MLC20 phosphorylation (54.9 ± 7.5% vs veh) in WKY but not in SHRSP VSMCs. ET-1-induced Ca2+ influx in WKY and SHRSP VSMCs was unaffected by Enh3 and Enh4. Assessment of Ca2+ -independent contractile mechanisms in VSMCs showed that ROCK2 activity is increased in SHRSP compared to WKY VSMCs (WKY: 0.27 ± 0.05; SHRSP:4.78 ± 0.34) and Enh4, but not Enh3, reduced ROCK2 activity in SHRSP VSMCs (61.5 ± 0.43% vs veh). Enh4 treatment in SHRSP rats reduced daytime systolic (SBP), diastolic (DBP) and mean arterial pressure (MAP) (SBP:4.26 ± 1.98; DBP:1.48 ± 0.92; MAP:2.07 ± 0.99 vs veh) compared to baseline. Furthermore, Enh4 treatment reduced U46619 induced contraction in SHRSP rat vessels (Emax(Mn): Veh- 13.16 ± 1.81; Enh4- 9.48 ± 1.31). Taken together, enhancing Mas1:ETBR interaction attenuates mitogenic and contractile signaling pathways in WKY and SHRSP VSMCs, and does not increase Ca2+ signaling, important in VSMC contraction. Enhancing Mas1:ETBR interaction reduces blood pressure and improves vascular function in SHRSP rats. Hence, Mas1:ETBR enhancers may be of potential therapeutic use as a new vasoprotective strategy to improve vascular function and to reduce blood pressure in hypertension.

Hsa_circ_0024093 accelerates VSMC proliferation via miR-4677-3p/miR-889-3p/USP9X/YAP1 axis in in vitro model of lower extremity ASO

Arteriosclerosis obliterans (ASO) of the lower extremities is identified as a kind of cardiovascular disease with aberrant proliferation and apoptosis of vascular smooth muscle cells (VSMCs). Accumulating studies have demonstrated the vital role of Yes1-associated transcriptional regulator (YAP1) in VSMCs, while its upstream regulatory mechanism in VSMCs in ASO of the lower extremities needs to be further elucidated. Herein, hsa_circ_0024093, a circular RNA (circRNA) from YAP1, was identified to positively regulate the protein level of YAP1 in VSMCs. Functionally, silencing of hsa_circ_0024093 obviously impeded cell proliferation and migration and promoted apoptosis in VSMCs in the in vitro model of ASO of the lower extremities. Mechanistically, it was found that hsa_circ_0024093 could regulate the expression of USP9X, which further induced YAP1 deubiquitination to stabilize YAP1 protein. In depth, it was revealed from mechanism experiments that hsa_circ_0024093 sequestered miR-889-3p or miR-4677-3p to enhance USP9X expression. Further, rescue assays validated that hsa_circ_0024093 regulated the miR-4677-3p/miR-889-3p/USP9X axis to accelerate the proliferation and migration of VSMCs in the in vitro model of ASO of the lower extremities. These findings may provide a novel perspective for better understanding of ASO of the lower extremities.

Long Noncoding RNA Hypoxia-Inducible Factor-1 Alpha-Antisense RNA 1 Regulates Vascular Smooth Muscle Cells to Promote the Development of Thoracic Aortic Aneurysm by Modulating Apoptotic Protease-Activating Factor 1 and Targeting let-7g

BackgroundThoracic aortic aneurysm (TAA) is a severe threat that is characterized by the increased aortic diameter. The dysfunction of vascular smooth muscle cells (VSMCs) contributes to the formation of TAA. Previous research indicated that long noncoding RNAs (lncRNAs) were involved in the development of TAA. This article aimed to explore the role of lncRNA hypoxia-inducible factor-1 alpha-antisense RNA 1 (HIF1A-AS1) and potential action mechanisms in VSMCs. MethodsThe expression of HIF1A-AS1, collagen I, collagen III, microRNA let-7g (let-7g) and apoptotic protease-activating factor 1 (APAF1) was detected by quantitative real-time polymerase chain reaction. Cell proliferation and cell apoptosis were assessed by Cell Counting Kit-8 and flow cytometry assays, respectively. The protein levels of proliferating cell nuclear antigen, Cleaved caspase-3 (Cleaved-cas3), B cell lymphoma/leukemia-2 (Bcl-2), Collagen I, Collagen III, and APAF1 were quantified by Western blot. The relationship between let-7g and HIF1A-AS1 or APAF1 was predicted by the online bioinformatics tool and verified by dual-luciferase reporter assay and RNA pull-down assay. ResultsHIF1A-AS1 was upregulated in TAA tissues and was a valuable diagnostic marker of TAA. HIF1A-AS1 overexpression suppressed proliferation, induced apoptosis, and reduced the expression of extracellular matrix proteins in VSMCs. let-7 g was a target of HIF1A-AS1, and its inhibition functioned the same role as HIF1A-AS1 overexpression. APAF1 was a target of let-7g, and its knockdown played the opposite role with HIF1A-AS1 overexpression. The reintroduction of let-7g or APAF1 knockdown reversed the effects of HIF1A-AS1 overexpression in VSMCs. ConclusionsHIF1A-AS1 regulated the proliferation, apoptosis ,and the activity of extracellular matrix proteins in VSMCs through modulating APAF1 by targeting let-7g, leading to the development of TAA.

MiR-145 inhibits the proliferation and migration of vascular smooth muscle cells by regulating autophagy.

miR‐145, the most abundant miRNA in the vascular smooth muscle cells (VSMCs), regulates VSMC function in intimal hyperplasia. It has been reported that autophagy participates in the regulation of proliferation and migration of VSMCs. However, the effect of miR‐145 on autophagy and related mechanism in the proliferation and migration of VSMCs remains unclear. Therefore, we aimed to determine the effect of miR‐145 on autophagy and the mechanism in VSMCs. Cell autophagy was determined by transmission electron microscope, mRFP‐GFP‐LC3 assay and Western blotting. A recombinant lentivirus containing miR‐145 was used to construct VSMCs with miR‐145 overexpression. We found that miR‐145 expression was decreased, and autophagy was increased in the carotid arteries of C57BL/6J mice with intimal hyperplasia and TGF‐β1‐stimulated VSMCs. Furthermore, miR‐145 overexpression inhibited cell autophagy, whereas miR‐145 inhibition promoted autophagy in TGF‐β1‐stimulated VSMCs. Meanwhile, miR‐145 inhibited the proliferation and migration of VSMCs. More importantly, our study showed that autophagy inhibition augmented the inhibitory effect of miR‐145 on the proliferation and migration of VSMCs. In addition, we found that the sirtuins are not direct targets of miR‐145 in the proliferation and migration of VSMCs. These results suggest that miR‐145 inhibits the proliferation and migration of VSMCs by suppressing the activation of autophagy.

Rho-associated protein kinase and cyclophilin a are involved in inorganic phosphate-induced calcification signaling in vascular smooth muscle cells

Arterial calcification, a risk factor of cardiovascular events, develops with differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells. Cyclophilin A (CypA) is a peptidyl-prolyl isomerase involved in cardiovascular diseases such as atherosclerosis and aortic aneurysms, and rho-associated protein kinase (ROCK) is involved in the pathogenesis of vascular calcification. CypA is secreted in a ROCK activity-dependent manner and works as a mitogen via autocrine or paracrine mechanisms in VSMCs. We examined the involvement of the ROCK-CypA axis in VSMC calcification induced by inorganic phosphate (Pi), a potent cell mineralization initiator. We found that Pi stimulated ROCK activity, CypA secretion, extracellular signal-regulated protein kinase (ERK) 1/2 phosphorylation, and runt-related transcription factor 2 expression, resulting in calcium accumulation in rat aortic smooth muscle cells (RASMCs). The ROCK inhibitor Y-27632 significantly suppressed Pi-induced CypA secretion, ERK1/2 phosphorylation, and calcium accumulation. Recombinant CypA was found to be associated with increased calcium accumulation in RASMCs. Based on these results, we suggest that autocrine CypA is mediated by ROCK activity and is involved in Pi-induced ERK1/2 phosphorylation following calcification signaling in RASMCs.

Down-regulation of Suv39h1 attenuates neointima formation after carotid artery injury in diabetic rats.

Patients with diabetes have an increased risk of vascular complications. Suv39h1, a histone methyltransferase, plays a protective role against myocardial injury in diabetes. Herein, we intend to explore whether Suv39h1 could affect neointimal formation after vascular injury in diabetic rats and reveal the underlying mechanism. In this study, we generated adenovirus expressing Suv39h1 as well as lentivirus expressing Suv39h1‐targeting shRNA and evaluated the significance of Suv39h1 in vascular smooth muscle cells (VSMCs) under diabetic conditions. In vitro, we examined proliferative and migratory behaviours as well as the underlying signalling mechanisms in VSMCs in response to high glucose treatment. In vivo, we induced diabetes in SD rats with streptozocin and established the common carotid artery balloon injury model. Suv39h1 was found to be both necessary and sufficient to promote VSMC proliferation and migration under high glucose conditions. We observed corresponding changes in intracellular signalling molecules including complement C3 and phosphor‐ERK1/2. However, either up‐regulating or down‐regulating Suv39h1, phosphor‐p38 level was not significantly affected. Consistently, Suv39h1 overexpression led to accelerated neointima formation, while knocking down Suv39h1 reduced it following carotid artery injury in diabetic rats. Using microarray analyses, we showed that altering the Suv39h1 level in vivo dramatically altered the expression of myriad genes mediating different biological processes and molecular function. This study reveals the novel role of Suv39h1 in VSMCs of diabetes and suggests its potential role as a therapeutic target in diabetic vascular injury.

N‐Cadherin, a novel and rapidly remodelling site involved in vasoregulation of small cerebral arteries

N-cadherin formed punctate adherens junctions (AJ) along the borders between vascular smooth muscle cells (VSMCs) in the pressurized rat superior cerebellar artery. The formation of N-cadherin AJs in the vessel wall depends on the intraluminal pressure and was responsive to treatment with phenylephrine (PE) (10-5 m) and ACh (10-5 m). N-cadherin-coated beads were able to induce clustering of N-cadherin-enhanced green fluorescent protein (EGFP) on the plasma membrane of isolated VSMCs, whereas treatment with PE (10-5 m) or sodium nitroprusside (10-5 m) induced a significant increase or decrease in the N-cadherin-EGFP clustering, respectively. Application of pulling force (∼1nN) to the N-cadherin-coated beads via an atomic force microscope induced a localized mechanical response from the VSMCs that opposed the pulling. N-cadherin is the major cell-cell adhesion molecule in vascular smooth muscle cells (VSMCs). We tested the hypothesis that N-cadherin is part of a novel mechanosensory mechanism in VSMCs and plays an active role in both the arteriolar myogenic response and during changes in vascular tone induced by vasomotor agonists. Intact and pressurized rat superior cerebellar arteries were labelled for confocal immunofluorescence imaging. N-cadherin formed punctate adherens junctions (AJ) along the borders between VSMCs. When the lumen pressure was raised from 50 to 90mmHg, both the density and the average size of N-cadherin AJs increased significantly. Similarly, arteriolar constriction with phenylephrine (PE) (10-5 m) induced a significant increase of N-cadherin AJ density at 50mmHg, whereas vasodilatation induced by ACh (10-5 m) was accompanied by a significant decrease in density and size of N-cadherin AJs. An atomic force microscope (AFM) was employed to further examine the mechano-responsive properties of N-cadherin adhesion sites in isolated VSMCs. AFM probes with an attached N-cadherin-coated microbead (5μm) induced a progressive clustering of N-cadherin-enhanced green fluorescent protein (EGFP) on the VSMC surface. Application of pulling force (∼1nN) to the N-cadherin-coated-beads with the AFM induced a localized mechanical response from the VSMCs that opposed the pulling. Treatment with PE (10-5 m) or sodium nitroprusside (10-5 m) induced a significant increase or decrease of the N-cadherin-EGFP clustering, respectively. These observations provide compelling evidence that N-cadherin AJs are sensitive to pressure and vasomotor agonists in VSMCs and support a functional role of N-cadherin AJs in vasomotor regulation.

CTRP3 promotes energy production by inducing mitochondrial ROS and up-expression of PGC-1α in vascular smooth muscle cells

C1q/tumor necrosis factor-related protein-3 (CTRP3) is an adipokine with modulation effects on metabolism and inflammation. Adenosine triphosphate (ATP) exerts multiple biological effects in vascular smooth muscle cells (VSMCs) and energy imbalance is involved in vascular diseases. This study aimed to explore the effect of CTRP3 on energy production and its underlying mechanism in VSMCs. Our results indicated that exogenous CTRP3 increased ATP synthesis and the protein expression of oxidative phosphorylation (OXPHOS)-related molecules, including peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, sirtuin-3 (SIRT3), complex I, II, III, and V in cultured VSMCs. Depletion of endogenous CTRP3 by small interfering RNA (siRNA) reduced ATP synthesis and the expression of those molecules. PGC-1α knockdown abrogated CTRP3-induced ATP production and OXPHOS-related protein expression. Furthermore, CTRP3 increased mitochondrial reactive oxygen species (ROS) production and mitochondrial membrane potential level. Pretreatment with N-acetyl-L-cysteine, a reactive oxygen species scavenger, and cyanidem-chlorophenylhydrazone, an uncoupler of OXPHOS, suppressed CTRP3-induced ROS production, PGC-1α expression and ATP synthesis. In conclusion, CTRP3 modulates mitochondrial energy production through targets of ROS and PGC-1α in VSMCs.

Homocysteine induces the expression of C-reactive protein via NMDAr-ROS-MAPK-NF-κB signal pathway in rat vascular smooth muscle cells

ObjectiveHomocysteine (Hcy) is known as an independent risk factor for atherosclerosis. C-reactive protein (CRP) directly participates in initiation and progression of atherosclerosis. However, there is no direct evidence to demonstrate pro-inflammatory effect of Hcy on vascular smooth muscle cells (VSMCs) through CRP. In the present study, we examined the effect of Hcy on CRP expression and investigated the related mechanism in VSMCs. Methods and resultsProtein expression and secretion were detected by Western blot and ELISA, respectively. mRNA expression was detected by RT-PCR. Superoxide anion was detected by lucigenin chemiluminometry and the immunofluorescence staining was observed by a fluorescence microscope. The results revealed that Hcy significantly induced mRNA and protein expressions of CRP in VSMCs both in vitro and in vivo, and anti-IL-1β or anti-IL-6 neutralizing antibody alone or in combination partially reduced Hcy-induced CRP expression. Hcy increased the expression of NR1 subunit of N-methyl-d-aspartate receptor (NMDAr), and MK-801 alleviated Hcy-induced CRP expression in VSMCs. Further studies showed that Hcy-stimulated superoxide anion generation in VSMCs. Nevertheless, pretreatment of the cells with MK-801, TTFA and DPI significantly reduced Hcy-stimulated superoxide anion generation, and antioxidant NAC decreased Hcy-induced CRP expression in VSMCs. Additionally, PD98059, SB205380 or PDTC antagonized Hcy-induced CRP expression, and MK-801, NAC, PD98059 or SB205380 inhibited Hcy-activated phosphorylations of ERK1/2 and p38. ConclusionThe present study demonstrates that Hcy is able to initiate an inflammatory response in VSMCs by stimulating CRP production, which is mediated through NMDAr-ROS-ERK1/2/p38-NF-κB signal pathway. These findings provide new evidence for a role of Hcy in pathogenesis of atherosclerosis.

Optogenetic Control of Vascular Tone with High Temporal Resolution

Blood pressure is directly controlled by the contractile state of vascular smooth muscle cells (VSMCs) which is regulated by membrane potential-dependent Ca2+-entry through Ca2+-channels and Ca2+-release from intracellular stores. Modulation of membrane potential in VSMCs in intact vessels can be performed by electrical field stimulation or elevation of extracellular potassium concentration, but both methods are not cell-specific and have low spatio-temporal resolution. To overcome these limitations, we have used the light-gated cation channel channelrhodopsin2 for optogenetical control of VSMCs membrane potential. ChR2 was expressed in a7r5 VSMCs and patch clamp experiments showed light-induced inward currents. Brief light pulses with increasing light intensities led to graded depolarization and eventually initiation of action potentials. Prolonged illumination resulted in constant depolarization and elevated intracellular Ca2+-concentrations. Optogenetic stimulation of VSMCs in intact vessels was performed using transgenic mice that express ChR2 (Nat Methods. 2010;7:897-900) in VSMCs but not endothelial cells. Isometric force was analyzed in aortic rings with a wire-myograph. Illumination with blue light (475nm, 2.7mW/mm2) reliably induced contractions with very fast on- (< 800ms) and off-kinetics (< 2.5s) and contraction could be maintained up to 10 min. The amplitude of contraction could be graded by variation of light intensity and reached a maximum comparable to stimulation with noradrenalin (10μM). Control vessels from wild-type mice or transgenic mice expressing EGFP did not show light-induced contractions. Taken together ChR2 enables light-induced contraction of VSMCs with high temporal resolution and represents a powerful technique to analyze membrane-potential dependent mechanisms in VSMCs. Optogenetic stimulation allows cell-specific and localized stimulation down to single cell level and therefore can be used in the future to investigate the interplay of endothelial cells and VSMCs as well as the electrical coupling between cells within intact vessels.

Key Fatty Acid Combinations Define Vascular Smooth Muscle Cell Proliferation and Viability

High plasma concentrations of free fatty acids (FFA) and insulin are common features in atherosclerotic patients with type 2 diabetes. FFA, according to their nature, can have various effects on vascular smooth muscle cells (VSMC). These cells play important roles throughout atherosclerosis pathogenesis, from plaque development to plaque instability. Thus, this study aims to assess the impact of two FFA combinations and insulin on murine VSMC viability. The two combinations contain the same FFA but at different ratios, one being richer in saturated fatty acids (SFA) and the other having a higher proportion of monounsaturated fatty acids (MUFA). Both combinations inhibited VSMC proliferation due to their pro-apoptotic potential, with SFA being the major inducers of apoptosis. However, the presence of oleic acid (OLA) attenuated this impact in a dose-dependent manner. OLA had also the capacity to reduce apoptosis rates more strongly when combined with a SFA than when used alone in VSMC treatments. This effect was significant only for specific proportions of these FFA and was even more effective in presence of insulin. These results highlight the presence of a competition between pro-apoptotic and anti-apoptotic mechanisms in VSMC that is dependent on FFA ratios (saturated vs. monounsaturated) and on insulinemia. They also underline the importance of the presence of MUFA such as OLA in diets containing high proportions of SFA.

Drugs Targeting Epigenetic Histone Acetylation in Vascular Smooth Muscle Cells for Restenosis and Atherosclerosis

It is well known that atherosclerosis and restenosis are common cardiovascular diseases and major health care problems. Vascular remodeling and migration and proliferation of vascular smooth muscle cells (VSMCs) are key features of these pathologies. There has been enormous progress in drug development and clinical management of these disorders, with angioplasty and drug eluting stents being standard procedures to treat vascular obstruction. However, despite their benefits, these current treatment modalities are not always efficacious. Furthermore, they can also be associated with postoperative complications and graft failures, some of which can be life threatening. Evaluation of newer mechanisms involved in VSMC proliferation aimed at uncovering additional therapeutic approaches to curb VSMC dysfunction in cardiovascular diseases is clearly warranted. See accompanying article on page 851 Accumulating evidence suggests that several common diseases, including cardiovascular disorders, diabetes, and the vascular complications of diabetes, are governed by a combination of genetic and environmental factors and that epigenetic mechanisms, such as DNA methylation and histone modifications in chromatin, form a key link between them.1–3 Epigenetics is the added layer of gene regulation that occurs in chromatin without changes in the actual underlying DNA sequence and plays a major role in dictating cell-specific gene expression patterns and transcriptional outcomes.4,5 Along with DNA methylation, key posttranslational modifications of histone N-terminal tails can alter chromatin structure to form an added layer of gene regulation and modulate gene transcription.5 Therefore, gene transcription depends on chromatin structure, which is very dynamic, depending on a multitude of histone posttranslational modifications that allow for the conversion of inaccessible, compact, or …

IL‐19 decreases ox‐LDL induced VSMC activation by suppression of scavenger receptor expression and reduction of ox‐LDL uptake

Atherosclerosis is inflammatory in nature and oxidized low density lipoprotein (ox‐LDL) has activating effects on vascular smooth muscle cells (VSMC). Interleukin‐19 (IL‐19) is a recently described Th2 anti‐inflammatory cytokine. We have previously reported that IL‐19 expression is induced in VSMC by inflammatory cytokines and in arteries by injury. Our hypothesis is that IL‐19 participates in a negative autocrine regulatory feedback mechanism to ameliorate inflammation. The purpose of this study is to determine the molecular mechanism(s) of IL‐19's anti‐inflammatory role in atherosclerosis. IL‐19 decreases the abundance of ox‐LDL induced COX‐2 protein expression. Fluorescently labeled ox‐LDL uptake is inhibited by IL‐19, as determined by flow cytometry and immunocytochemistry. IL‐19 reduces the abundance of LOX‐1 and CX‐CL16 mRNA, VSMC receptors which internalize ox‐LDL. Each of these receptors have AU‐rich elements (ARE) in their 3' UTR which act as binding sites for the RNA‐binding protein HuR, an inflammatory mRNA stabilizing protein. The mRNA stabilizing function of HuR is linked to its cytoplasmic translocation. IL‐19 inhibits HuR protein abundance and ox‐LDL induced HuR cytoplasmic translocation. Together, these data indicate that at least one of IL‐19's anti‐atherosclerotic mechanisms in VSMC involves reduction of ox‐LDL uptake, primarily through reduced scavenger receptor abundance.Work supported by grant HL063810 and HL090885 from NHLBI, and grant 0455562U from AHA, to M.V.A.. A.A.C. received NHLBI Ruth L. Kirchstein Predoctoral fellowship F30HL095329.

Requirement of c‐Src in Angiotensin II and Endothelin 1‐induced Activation of MAPKinases in Vascular Smooth Muscle Cells (VSMC)

Endothelin‐1 (ET‐1) and Angiotensin II (Ang II), two important vasoactive peptides, have been implicated in the pathogenesis of vascular abnormalities such as hypertension and atherosclerosis, through the activation of growth promoting signal transduction pathways, which include mitogen‐activated protein kinases (MAPKs) and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (PKB). We have previously shown that c‐Src, a non‐receptor protein tyrosine kinase (NR‐PTK), is an upstream regulator of ET‐1 and Ang II‐induced activation of PKB in VSMC. However, the role of c‐Src in ET‐1‐induced MAPK signaling remains controversial in VSMC. Therefore, in the present studies, we have investigated the involvement of c‐Src in this process. Treatment of VSMC with ET‐1 and Ang II induced phosphorylation of ERK1/2, JNK and p38MAPK in A10 VSMC and in aortic VSMC isolated from 12 week old Wistar Kyoto rats (WK‐VSMC). PP2, an inhibitor of the Src family of NR‐PTK, decreased phosphorylation of ERK1/2, JNK and p38MAPK induced by both ET‐1 and Ang II in a dose‐dependent fashion. Furthermore, knockdown of c‐Src by siRNA also exhibited a similar response, and inhibited ET‐1 and Ang II‐induced ERK1/2, JNK and p38MAPK phosphorylation. In summary, these data suggest that both ET‐1 and Ang II signal the activation of MAPKs through a c‐Src dependent mechanism in VSMC. (Supported by grants from Canadian Institutes of Health Research).

Impact of Insulin-like Growth Factor-I on Migration, Proliferation and Akt-ERK Signaling in Early and Late-passages of Vascular Smooth Muscle Cells

Migration and proliferation of vascular smooth muscle cells (VSMCs) are important events in the progression of atherosclerosis. Insulin-like growth factor I (IGF-1) possesses both antiapoptotic and mitogenic/motogenic effects in VSMCs although the influence of life cycle on IGF-1-induced effects is unclear. This study was designed to evaluate the effect of IGF-1 on migration, proliferation, and signaling mechanisms in VSMCs from early (3-5) to late (20-22) passages. Migration, proliferation, and cell survival were measured using monolayer wounding, 3[H]-thymidine incorporation and MTT assay, respectively. Akt and ERK, which are critical to proliferation, differentiation and migration, were examined using Western blot analysis. DCF-DA fluorescence was used to quantify Reactive Oxygen Species (ROS) production. Late-passage VSMCs exhibited significantly higher basal cell proliferation and enhanced sensitivity to IGF-1-stimulated migration compared to cells from early-passages. Phosphorylated Akt and ERK levels were significantly higher in late-passage cells compared to early-passage, which was further enhanced by IGF-1 treatment. Late-passage cells exhibited higher levels of ROS production compared to early-passage, cells. IGF-1 did not significantly alter ROS levels in either passage. Expression of the cell cycle regulator p53, p21, and p16 was not affected by repeated passaging of cells. These results indicated that repeated passaging of VSMCs exhibits a phenotype which has higher proliferative capacity. Activation of trophic signaling molecules such as ERK1/2 and Akt and generation of ROS may represent the mechanisms by which repeated passages of VSMCs acquire a motogenic and mitogenic phenotype.

Homocysteine-Mediated Expression of SAHH, DNMTs, MBD2, and DNA Hypomethylation Potential Pathogenic Mechanism in VSMCs

Homocysteine (Hcy) is a well-established risk factor for atherosclerosis and may cause dysregulation of gene expression, but the characteristics and the key links involved in its pathogenic mechanisms are still poorly understood. The aim of this study was to explore (i) the effects of Hcy on DNA methylation in vascular smooth muscle cells (VSMCs) and (ii) the underlying mechanism of Hcy-induced changes in DNA methylation patterns in relation to atherosclerosis. We examined the levels of gDNA methylation, namely, the Alu and line-1 element sequences, which can serve as a surrogate marker for gDNA methylation, and also investigated the effects of Hcy on the intracellular S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) concentrations as well as the expressions of SAH hydrolase (SAHH), DNA methyltransferase3a (DNMT3a), DNMT3b, and methyl-CpG-binding domain 2 (MBD2). We found that clinically relevant levels of Hcy (0-500 microM) induced elevation of SAH, declination of SAM and SAM/SAH ratio, and reduction in expression of SAHH and MBD2, but increased the activity of DNMT3a and DNMT3b compared to the control group (p < 0.05). We found also that the genome-wide hypomethylation is a common feature of gDNA in the VSMCs cultured with Hcy. In conclusion, these results suggest that Hcy-induced DNA methylation may be an important potential pathogenic mechanism in the development of atherosclerosis, and may become a therapeutic target for preventing Hcy-induced atherosclerosis.

Preconditioning with low concentration NO attenuates subsequent NO-induced apoptosis in vascular smooth muscle cells via HO-1-dependent mitochondrial death pathway

Nitric oxide (NO) signaling pathways are important in both the maintenance of vascular homeostasis and disease progression. Overproduction of NO has been associated with ischemia/reperfusion (I/R) injury. Growing evidences suggest that NO preconditioning has cytoprotective effects against I/R injury. However, the mechanism with which NO mediates these effects remains to be elucidated. The purpose of this study was to examine the mechanism of how NO preconditioning inhibits subsequent NO-induced apoptosis in vascular smooth muscle cells (VSMC), specifically focusing on heme oxygenase-1 (HO-1). According to our data, sodium nitroprusside (SNP) increased HO-1 expression in a concentration dependent manner. Preconditioning with low concentration SNP (0.3mM) inhibited subsequent high concentration SNP (1.5mM)-induced apoptosis, and this effect was reversed by the HO-1 inhibitor SnPP. Low concentration SNP-mediated protection involved p38 kinase inactivation and increased Bcl-2 expression. Furthermore, mitochondrial membrane potential was concomitantly increased with decreased expressions of Bax, Apaf-1, and activity of caspase-3, which was reversed by SnPP treatment. Our results show that low concentration SNP preconditioning suppresses subsequent high concentration SNP-induced apoptosis by inhibiting p38 kinase and mitochondrial death pathway via HO-1-dependent mechanisms in VSMC.

Store-Operated Ca2+ Channel in Renal Microcirculation and Glomeruli

Store-operated Ca2+ channel (SOC) is defined as a channel that opens in response to depletion of the internal Ca2+ stores. During the last decade, many investigators have made a great effort to identify and characterize SOC, and to evaluate its physiologic function and pathophysiologic relevance in a variety of cell lines, primary cultures, and native tissues. To date, accumulating evidence has demonstrated that SOC is an essential Ca2+ entry mechanism in vascular smooth-muscle cells of renal microvasculature and glomerular mesangial cells, both of which tightly control glomerular hemodynamics and filtration. Store-operated Ca2+, combined with other types of Ca2+ entry channels, constitutes a profile of Ca2+ changes in response to physiologic vasoconstrictors and, thereby, regulates renal microcirculation and mesangial function. In addition, SOC is associated with altered Ca2+ signaling occurring in diseased kidneys, such as diabetic nephropathy. Although the gating mechanism and molecular identity of SOC are still enigmatic and may be cell-type and tissue specific, data from several independent groups suggest that protein kinase C plays an important role in SOC activation and that certain isoforms of canonical transient receptor potential (TRPC) proteins are candidates of SOC in renal microvessels and mesangial cells.

Regulation of K-Cl Cotransport: from Function to Genes

This review intends to summarize the vast literature on K-Cl cotransport (COT) regulation from a functional and genetic viewpoint. Special attention has been given to the signaling pathways involved in the transporter's regulation found in several tissues and cell types, and more specifically, in vascular smooth muscle cells (VSMCs). The number of publications on K-Cl COT has been steadily increasing since its discovery at the beginning of the 1980s, with red blood cells (RBCs) from different species (human, sheep, dog, rabbit, guinea pig, turkey, duck, frog, rat, mouse, fish, and lamprey) being the most studied model. Other tissues/cell types under study are brain, kidney, epithelia, muscle/smooth muscle, tumor cells, heart, liver, insect cells, endothelial cells, bone, platelets, thymocytes and Leishmania donovani. One of the salient properties of K-Cl-COT is its activation by cell swelling and its participation in the recovery of cell volume, a process known as regulatory volume decrease (RVD). Activation by thiol modification with N-ethylmaleimide (NEM) has spawned investigations on the redox dependence of K-Cl COT, and is used as a positive control for the operation of the system in many tissues and cells. The most accepted model of K-Cl COT regulation proposes protein kinases and phosphatases linked in a chain of phosphorylation/dephosphorylation events. More recent studies include regulatory pathways involving the phosphatidyl inositol/protein kinase C (PKC)-mediated pathway for regulation by lithium (Li) in low-K sheep red blood cells (LK SRBCs), and the nitric oxide (NO)/cGMP/protein kinase G (PKG) pathway as well as the platelet-derived growth factor (PDGF)-mediated mechanism in VSMCs. Studies on VSM transfected cells containing the PKG catalytic domain demonstrated the participation of this enzyme in K-Cl COT regulation. Commonly used vasodilators activate K-Cl COT in a dose-dependent manner through the NO/cGMP/PKG pathway. Interaction between the cotransporter and the cytoskeleton appears to depend on the cellular origin and experimental conditions. Pathophysiologically, K-Cl COT is altered in sickle cell anemia and neuropathies, and it has also been proposed to play a role in blood pressure control. Four closely related human genes code for KCCs (KCC1-4). Although considerable information is accumulating on tissue distribution, function and pathologies associated with the different isoforms, little is known about the genetic regulation of the KCC genes in terms of transcriptional and post-transcriptional regulation. A few reports indicate that the NO/cGMP/PKG signaling pathway regulates KCC1 and KCC3 mRNA expression in VSMCs at the post-transcriptional level. However, the detailed mechanisms of post-transcriptional regulation of KCC genes and of regulation of KCC2 and KCC4 mRNA expression are unknown. The K-Cl COT field is expected to expand further over the next decades, as new isoforms and/or regulatory pathways are discovered and its implication in health and disease is revealed.

Antioxidant effect of adrenomedullin on angiotensin II-induced reactive oxygen species generation in vascular smooth muscle cells.

Recent adrenomedullin (AM) gene-targeting studies have proposed a novel concept that AM plays a protective role against oxidative stress in vivo. The present study was undertaken to explore the underlying molecular mechanism of the putative antioxidant action of AM against angiotensin II (Ang II)induced reactive oxygen species (ROS) generation in rat vascular smooth muscle cells (VSMCs). Intracellular ROS levels were measured by dichlorofluoroscein fluorescence. Redox-sensitive c-Jun amino-terminal kinase (JNK) and ERK1/2 activation and gene expression induced by Ang II in VSMCs were also studied. AM dose-relatedly (10(-8)-10(-7) m) inhibited intracellular ROS generation stimulated by Ang II (10(-7) m), as mimicked by dibutyl-cAMP, the effect of which was inhibited by the pretreatment with N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride, a protein kinase A inhibitor, and calcitonin gene-related peptide(8-37), an AM/calcitonin gene-related peptide receptor antagonist. Ang II induced JNK and ERK1/2 activation via a redox-sensitive manner, whereas AM inhibited JNK, but not ERK1/2, activation by Ang II. Furthermore, AM inhibited Ang II-induced redox-sensitive gene expression (plasminogen activator inhibitor-1 and monocyte chemoattractant protein-1) in the same manner as N-acetyl-l-cysteine, a potent antioxidant. AM also inhibited Ang II-induced up-regulation of Nox1, a critical membrane-bound component of reduced nicotinamide adenine dinucleotide phosphate oxidase in VSMCs, in the same degree as N-acetyl-l-cysteine. Our study demonstrates for the first time that AM directly inhibits intracellular ROS generation via an AM receptor-mediated and c-AMP-protein kinase A-dependent mechanism in VSMCs and that AM with its potent antioxidant action inhibits redox-sensitive JNK activation and gene expression induced by Ang II. These data suggest that AM plays a protective role as an endogenous antioxidant in Ang II-induced vascular injury.