A10 Vascular Smooth Muscle Cells Research Articles

Preventive Effect and Mechanism of Crossostephium chinense Extract on Balloon Angioplasty-Induced Neointimal Hyperplasia.

Balloon angioplasty-induced neointimal hyperplasia remains a clinical problem that must be resolved. The bioactivities of the Crossostephium chinense extract (CCE) have demonstrated potential in preventing the progression of restenosis. The present study evaluated whether CCE can suppress balloon angioplasty-induced neointima formation and elucidated its possible pharmacological mechanisms. A rat model of carotid arterial balloon angioplasty was established to evaluate the inhibitory effect of CCEs on neointimal hyperplasia. Two cell lines, A10 vascular smooth muscle cells (VSMCs) and RAW264.7 macrophages, were used to investigate the potential regulatory activities and pharmacological mechanisms of CCEs in cell proliferation and migration and in inflammation. Our in vitro results indicated that CCE3, the ethanolic extract of C. chinense, exerted the strongest growth inhibitory and antimigratory effects on VSMCs. CCE3 blocked the activation of focal adhesion kinase, platelet-derived growth factor receptor-β (PDGFRB), and its downstream molecules (AKT and mTOR) and reduced the expression of matrix metalloproteinase-2. In addition, our findings revealed that CCE3 significantly increased the expression of miRNA-132, an inhibitory regulator of inflammation and restenosis, and suppressed the expression of inflammation-related molecules (inducible nitric oxide synthase, cyclooxygenase-2, interleukin- (IL-) 1β, and IL-6). Our in vivo study results indicated that balloon injury-induced neointimal hyperplasia was inhibited by CCE3. CCE3 could reduce neointima formation in balloon-injured arteries, and this effect may be partially attributed to the CCE3-induced suppression of PDGFRB-mediated downstream pathways and inflammation-related molecules.

Angiotensin II-induced histone deacetylase 5 phosphorylation, nuclear export, and Egr-1 expression are mediated by Akt pathway in A10 vascular smooth muscle cells.

Angiotensin II (ANG II) regulates an array of physiological and pathological responses in vascular smooth muscle cells (VSMCs) by activating ERK1/2 and phosphoinositide 3-kinase (PI3K)/Akt signaling pathways. We have demonstrated that ANG II and insulin-like growth factor-1 (IGF-1) induce the expression of early growth response protein-1 (Egr-1), a zinc finger transcription factor, which regulates the transcription of cell cycle regulatory genes network in VSMCs. We have reported that IGF-1 induces the phosphorylation of histone deacetylase 5 (HDAC5), which has been implicated in the expression of genes linked to VSMC growth and hypertrophy, via a PI3K/Akt-dependent pathway in VSMCs. However, the involvement of PI3K/Akt pathways in ANG II-induced HDAC5 phosphorylation and the contribution of HDAC5 in Egr-1 expression and hypertrophy in VSMCs remain unexplored. Here, we show that pharmacological blockade of the PI3K/Akt pathway either by wortmannin/SC66 or siRNA-induced silencing of Akt attenuated ANG II-induced HDAC5 phosphorylation and its nuclear export. Moreover, SC66 or Akt knockdown also suppressed ANG II-induced Egr-1 expression. Furthermore, pharmacological inhibition of HDAC5 by MC1568 or TMP-195 or knockdown of HDAC5 and the blockade of the nuclear export of HDAC5 by leptomycin B or KPT-330 significantly reduced ANG II-induced Egr-1 expression. In addition, depletion of either HDAC5 or Egr-1 by siRNA attenuated VSMC hypertrophy in response to ANG II. In summary, our results demonstrate that ANG II-induced HDAC5 phosphorylation and its nuclear exclusion are mediated by PI3K/Akt pathway and HDAC5 is an upstream regulator of Egr-1 expression and hypertrophy in VSMCs.NEW & NOTEWORTHY ANG II-induced histone deacetylase 5 (HDAC5) phosphorylation and nuclear export occurs via the phosphoinositide 3-kinase/Akt pathway. Akt, through HDAC5, regulates ANG II-induced expression of early growth response protein-1 (Egr-1), which is a transcription factor linked with vascular dysfunction. Inhibition of HDAC5 exclusion by nuclear export inhibitors suppresses ANG II-induced Egr-1 expression. HDAC5 is an upstream mediator of Egr-1 expression and cell hypertrophy in response to ANG II in vascular smooth muscle cells.

Synthesis and characterization of electrospun nanofibrous tissue engineering scaffolds generated from in situ polymerization of ionomeric polyurethane composites

Tissue scaffolds need to be engineered to be cell compatible, have timely biodegradable character, be functional with respect to providing niche cell support for tissue repair and regeneration, readily accommodate multiple cell types, and have mechanical properties that enable the simulation of the native tissue. In this study, electrospun degradable polar hydrophobic ionic polyurethane (D-PHI) scaffolds were generated in order to yield an extracellular matrix-like structure for tissue engineering applications. D-PHI oligomers were synthesized, blended with a degradable linear polycarbonate polyurethane (PCNU), and electrospun with simultaneous in situ UV cross-linking in order to generate aligned nanofibrous scaffolds in the form of elastomeric composite materials. The D-PHI/PCNU scaffold fibre morphology, cross-linking efficiency, surface nature, mechanical properties, in vivo degradation and integration, as well as in vitro cell compatibility were characterized. The results showed that D-PHI/PCNU scaffolds had a high cross-linking efficiency, stronger polar nature, and lower stiffness relative to PCNU scaffolds. In vivo, the D-PHI/PCNU scaffold degraded relatively slowly, thereby enabling new tissue time to form and yielding very good integration with the latter tissue. Based on a study with A10 vascular smooth muscle cells, the D-PHI/PCNU scaffold was able to support high cell viability, adhesion, and expression of typical smooth muscle cell markers after a 7-day culture period, which was comparable to PCNU scaffolds. These characterization results demonstrate that the unique properties of a D-PHI/PCNU scaffold, combined with the benefits of electrospinning, could allow for the generation of a tissue engineered scaffold that mimics important aspects of the native extracellular matrix and could be used for functional tissue regeneration. Statement of SignificanceTissue engineered scaffolds should recapitulate native extracellular matrix features. This study investigates the processing of a classical polycarbonate polyurethane (PCNU) with a cross-linked and degradable ionomeric polyurethane (D-PHI), polymerized via in situ rapid light curing to yield a 3-dimensional co-electrospun nanofibre matrix with chemical diversity and low modulus character. This research advances the use of D-PHI for tissue engineering applications by providing a facile means of changing physical and chemical properties in classical PCNUs without the need to adjust spinning viscosities of the base polymer. Further, the in vivo and cell culture findings set the stage for introducing unique elastic materials which inherently support wound healing, repair, and regeneration in tissues, for applications that require the recapitulation of native extracellular matrix physical features.

Synthesis and Characterization of Electrospun Nanofibrous Tissue Engineering Scaffolds Generated from in Situ Polymerization of Ionomeric Polyurethane Composites

Tissue scaffolds need to be engineered to be cell compatible, have timely biodegradable character, be functional with respect to providing niche cell support for tissue repair and regeneration, readily accommodate multiple cell types, and have mechanical properties that enable the simulation of the native tissue. In this study, electrospun degradable polar hydrophobic ionic polyurethane (D-PHI) scaffolds were generated in order to yield an extracellular matrix-like structure for tissue engineering applications. D-PHI oligomers were synthesized, blended with a degradable linear polycarbonate polyurethane (PCNU), and electrospun with simultaneous in situ UV cross-linking in order to generate aligned nanofibrous scaffolds in the form of elastomeric composite materials. The D-PHI/PCNU scaffold fibre morphology, cross-linking efficiency, surface nature, mechanical properties, in vivo degradation and integration, as well as in vitro cell compatibility were characterized. The results showed that D-PHI/PCNU scaffolds had a high cross-linking efficiency, stronger polar nature, and lower stiffness relative to PCNU scaffolds. In vivo, the D-PHI/PCNU scaffold degraded relatively slowly, thereby enabling new tissue time to form and yielding very good integration with the latter tissue. Based on a study with A10 vascular smooth muscle cells, the D-PHI/PCNU scaffold was able to support high cell viability, adhesion, and expression of typical smooth muscle cell markers after a 7-day culture period, which was comparable to PCNU scaffolds. These characterization results demonstrate that the unique properties of a D-PHI/PCNU scaffold, combined with the benefits of electrospinning, could allow for the generation of a tissue engineered scaffold that mimics important aspects of the native extracellular matrix and could be used for functional tissue regeneration.

Ang‐II‐Induced Phosphorylation and Nuclear Export of Histone Deacetylase 5 is Mediated by an AKT‐Dependent Pathway in Vascular Smooth Muscle Cells

Augmented levels of angiotensin‐II (Ang‐II) are associated with the development of cardiovascular disorders such as atherosclerosis and hypertension. Exaggerated activation of Ang‐II‐induced signaling events and expression of genes linked to cell proliferation, hypertrophy and migration have been demonstrated to contribute to the development of these vascular pathologies. Ang‐II induces the phosphorylation of class II histone deacetylases (HDACs), particularly HDAC4 and HDAC5. HDACs regulate gene transcription by their ability to modify the acetylation status of the lysine residues in histone and non‐histone proteins. A heightened activation of HDACs, notably HDAC5, has been associated with vascular disorders such as atherosclerosis. We have shown earlier that Ang‐II induces the expression of early growth response factor‐1 (Egr‐1), a zinc finger transcription factor which is upregulated in atherosclerotic lesions and in animal models of vascular injury. We have also reported that the PI3Kinase/AKT pathway plays a key role in inducing the expression of Egr‐1 in vascular smooth muscle cells (VSMC). However, the role of the AKT pathway in HDAC5 phosphorylation and the contribution of HDAC5 in Egr‐1 expression in VSMC remains unexplored. Here, we show that Ang‐II induced the phosphorylation of HDAC5 in a time and dose‐dependent manner, and pharmacological blockade of the PI3K/AKT pathway by wortmannin or SC66 attenuated Ang‐II‐induced HDAC5 phosphorylation in A10 VSMC. In addition, Ang‐II treatment of VSMC resulted in the translocation of HDAC5 from the nucleus to the cytosol, and the inhibition of AKT by SC66 almost completely attenuated the nuclear export of HDAC5 induced by Ang‐II. In contrast, pharmacological blockade of the MAPK pathway by UO126 was ineffective in reducing Ang‐II‐induced HDAC5 phosphorylation. Furthermore, pharmacological inhibition of HDAC5 by MC1568 or siRNA‐induced silencing of HDAC5 suppressed Ang‐II‐induced Egr‐1 expression. In summary, our results suggest that Ang‐II induces the phosphorylation and nuclear export of HDAC5 via the PI3K/AKT‐dependent signaling pathway and that HDAC5 is an upstream modulator of Egr‐1 expression in VSMC.Support or Funding InformationSupported by Canadian Institutes of Health ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protein kinase B/AKT mediates insulin-like growth factor 1-induced phosphorylation and nuclear export of histone deacetylase 5 via NADPH oxidase 4 activation in vascular smooth muscle cells.

Insulin-like growth factor 1 (IGF-1) mediates the generation of reactive oxygen species (ROS) and the activation of growth promoting signaling pathways. Histone deacetylases (HDACs) regulate gene transcription by deacetylating lysine residues in histone and nonhistone proteinsand a heightened HDAC activation, notably of HDAC5, is associated with vascular disorders, such as atherosclerosis. Although the contribution of IGF-1 in these pathologies is well documented, its role in HDAC phosphorylation and activation remains unexplored. Here, we examined the effect of IGF-1 on HDAC5 phosphorylation in vascular smooth muscle cells (VSMCs) and identified the signaling pathways involved in controlling HDAC5 phosphorylation and nuclear export. Treatment of A10 VSMCs with IGF-1 enhanced HDAC5 phosphorylation. Blockade of the IGF-1 receptor tyrosine kinase (TK) activity with the specific pharmacological inhibitor, AG1024, significantly inhibited IGF-1-induced HDAC5 phosphorylation, whereas the epidermal growth factor receptor (EGFR) TK antagonist, AG1478, had no effect. Inhibition of the mitogen-activated protein kinase pathway with U0126, SP600125, or SB203580, did not affect HDAC5 phosphorylation, whereas two inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT pathways, wortmannin and SC66, almost completely attenuated IGF-1-induced responses as confirmed by immunoblotting of phospho-HDAC5 and by small interfering RNA (siRNA)-induced AKT silencing. Moreover, the NAD(P)H oxidase (Nox) inhibitor, diphenyleneiodonium (DPI), and Nox4 siRNA, attenuated IGF-1-induced phosphorylation of HDAC5 and AKT. The HDAC5 phosphorylation resulted in its nuclear export, which was reversed by SC66 and DPI. Our results indicate that IGF-1-induced phosphorylation and nuclear export of HDAC5 involve Nox4-dependent ROS generation and PI3K/AKT signaling pathways.

Involvement of CGRP-RCP in the caveolin-1/ERK1/2 signal pathway in the static pressure-induced proliferation of vascular smooth muscle cells.

Previous study suggested that the receptor component protein (RCP), one of the components of calcitonin gene-related peptide (CGRP) receptor, plays a multiple role in the cellular signal transduction. The study was designed to investigate whether or not the RCP involved in the regulation of caveolin-1/extracellular signal-regulated kinases-1 and -2 (ERK1/2) signal pathway in the vascular smooth muscle cells (VSMCs) proliferation induced by static pressure. Mouse-derived VSMCs line A10 (A10 VSMCs) was served as project in this experiment. Results showed that the A10 VSMCs viability and proliferating cell nuclear antigen (PCNA) expression which were increased by static pressure were inhibited by pretreatment of CGRP. In like manner, the expressions of the decreased-caveolin-1 and the increased-phosphorylated ERK1/2 (p-ERK1/2) induced by static pressure were significantly reversed by pretreatment of CGRP, respectively. Meanwhile, the expression of RCP was up-regulated by the static pressure. Silence of RCP gene with the small interrupt RNA (siRNA) not only significantly increased A10 VSMC proliferation but also increased the expression of p-ERK1/2 in response to static pressure. When treatment of A10 VSMCs with 120-mmHg static pressure for different time, however, the protein band of caveolin-1 and RCP was the least at time point of 10 min, but the p-ERK1/2 expression was the most maximum. In conclusion, RCP maybe involved in the static pressure-induced A10 VSMCs proliferation by regulation of caveolin-1/ERK1/2 signal pathway.

Natriuretic peptide receptor-C-mediated attenuation of vascular smooth muscle cell hypertrophy involves Gqα/PLCβ1 proteins and ROS-associated signaling.

Hypertension is associated with vascular remodeling due to hyperproliferation and hypertrophy of vascular smooth muscle cells (VSMC). Recently, we showed the implication of enhanced expression of Gqα and PLCβ1 proteins in hypertrophy of VSMCs from 16‐week‐old spontaneously hypertensive rats (SHR). The aim of this study was to investigate whether C‐ANP4‐23, a natriuretic peptide receptor‐C (NPR‐C) ligand that was shown to inhibit vasoactive peptide‐induced enhanced protein synthesis in A10 VSMC could also attenuate hypertrophy of VSMC isolated from rat model of cardiac hypertrophy and to further explore the possible involvement of Gqα/PLCβ1 proteins and ROS‐mediated signaling in this effect. The protein synthesis and cell volume, markers of hypertrophy were significantly enhanced in VSMC from 16‐week‐old SHR compared with age‐matched WKY rats and C‐ANP4‐23 treatment attenuated both to WKY levels. In addition, C‐ANP4‐23 treatment also attenuated the enhanced expression of AT1 receptor, Gqα, PLCβ1, Nox4, and p47phox proteins, the enhanced activation of EGFR, PDGFR, IGF‐1R, enhanced phosphorylation of ERK1/2/AKT and c‐Src in VSMC from SHR. Furthermore, the enhanced levels of superoxide anion and NADPH oxidase activity exhibited by VSMC from SHR were also attenuated to control levels by C‐ANP4‐23 treatment. These results indicate that C‐ANP4‐23 via the activation of NPR‐C attenuates VSMC hypertrophy through decreasing the overexpression of Gqα/PLCβ1 proteins, enhanced oxidative stress, increased activation of growth factor receptors, and enhanced phosphorylation of MAPK/AKT signaling pathways. Thus, it can be suggested that C‐ANP4‐23 may be used as a therapeutic agent for the treatment of vascular complications associated with hypertension and atherosclerosis.

WNK1 is required for proliferation induced by hypotonic challenge in rat vascular smooth muscle cells.

Hypotonic challenge evoked vascular cell proliferation through activation of volume-regulated Cl- channel (VRCC), leading to a decrease in the intracellular Cl- concentration ([Cl-]i). We hypothesize that the decrease in [Cl-]i may activate one or several Cl--sensitive kinases, resulting in a subsequent signaling cascade. In this study we demonstrated that WNK1, a Cl--sensitive kinase, was involved in VRCC-induced proliferative signaling pathway in A10 vascular smooth muscle cells in vitro. A10 cells were exposed to a hypotonic challenge (225 mosmol·kg-1·H20), which caused significantly increase in WNK1 phosphorylation without altering WNK1 protein expression. WNK1 overexpression significantly increased hypotonic-induced A10 cell proliferation, whereas silencing of WNK1 caused an opposite action. WNK1 mutation did not affect hypotonic-induced WNK1 phosphorylation and cell proliferation. Silencing of WNK1 caused cell cycle arrest at G0/G1 phase and prevented transition from G1 to S phase, whereas the WNK1 overexpression accelerated cell cycle transition from G1 to S phase. Silencing of WNK1 significantly inhibited cyclin D1/cyclin E1 expression and increased p27kip/p21cip expression. WNK1 overexpression significantly increased cyclin D1/cyclin E1 expression and reduced p27KIP/p21CIP expression. In addition, WNK1 knockdown or overexpression significantly attenuated or increased the hypotonic-induced phosphorylation of Akt and PI3K respectively.In conclusion, the reduction in [Cl-]i caused by hypotonic challenge-induced VRCC opening evokes WNK1 phosphorylation in A10 VSMCs, which mediates cell cycle transition from G0/G1 to S phase and proliferation through the PI3K-Akt signaling pathway.

Reactive Oxygen Species‐Dependent PKB Activation Contributes to Insulin‐Like Growth Factor 1‐Induced Phosphorylation of Histone Deacetylase 5 in Vascular Smooth Muscle Cells

Insulin‐like growth factor 1 (IGF‐1), a potent mitogenic and vasoactive factor, has been shown to play an important role in the development of cardiovascular diseases. This occurs through the generation of reactive oxygen species (ROS) as well as through the hyperactivation of mitogenic and growth promoting signaling pathways and the subsequent alteration in gene expression. Histone deacetylases (HDACs), by their ability to modify the acetylation status of lysine residues in histone and non‐histone proteins, regulate gene transcription. Recent studies have demonstrated that a heightened activation of HDACs, notably HDAC5, is associated with vascular disorders such as atherosclerosis. However, a role of IGF‐1 in HDAC activation by phosphorylation remains poorly characterized. Therefore, in the present studies, we examined the effect of IGF‐1 on the phosphorylation of HDAC5 in vascular smooth muscle cells (VSMCs) and identified the signalling pathways involved in this process. Treatment of A10 VSMCs with IGF‐1 enhanced the phosphorylation of HDAC5 at serine 498 in a time and dose‐dependent fashion. Pretreatment of cells with AG1024, a selective pharmacological inhibitor of IGF‐1 receptor, significantly inhibited IGF‐1‐induced HDAC5 phosphorylation in A10 VSMCs whereas AG1478, a selective inhibitor of epidermal growth factor receptor, did not have an inhibitory effect on the levels of phospho‐HDAC5. Pharmacological blockade of the MAPK pathway with PD98059 and UO126 had no effect on HDAC5 phosphorylation, however wortmannin and SC‐66, inhibitors of the PI3K/PKB pathways, almost completely attenuated IGF‐1‐induced HDAC5 phosphorylation. In addition, pretreatment of A10 VSMCs with Diphenyleneiodonium (DPI) and apocynin, two NAD(P)H oxidase inhibitors, as well as the antioxidant N‐Acetyl‐Cysteine (NAC), resulted in an attenuation of IGF‐1‐induced phosphorylation of HDAC5 as well as PKB. In summary, these data demonstrate that IGF‐1 induces the phosphorylation of HDAC5 in a ROS‐ and PKB‐dependent fashion in VSMCs.Support or Funding InformationSupported by a grant from the Canadian Institutes of Health Research.

Novel Fabrication of MicroRNA Nanoparticle-Coated Coronary Stent for Prevention of Post-Angioplasty Restenosis.

Background and ObjectivesMicroRNA 145 is known to be responsible for cellular proliferation, and its enhanced expression reportedly inhibits the retardation of vascular smooth muscle cell growth specifically. In this study, we developed a microRNA 145 nanoparticle immobilized, hyaluronic acid (HA)-coated stent.Materials and MethodsFor the gene therapy, we used disulfide cross-linked low molecular polyethylenimine as the carrier. The microRNA 145 was labeled with YOYO-1 and the fluorescent microscopy images were obtained. The release of microRNA 145 from the stent was measured with an ultra violet spectrophotometer. The downstream targeting of the c-Myc protein and green fluorescent protein was determined by Western blotting. Finally, we deployed microRNA 145/ssPEI nanoparticles immobilized on HA-coated stents in the balloon-injured external iliac artery in a rabbit restenosis model.ResultsCellular viability of the nanoparticle-immobilized surface tested using A10 vascular smooth muscle cells showed that MSN exhibited negligible cytotoxicity. In addition, microRNA 145 and downstream signaling proteins were identified by western blots with smooth muscle cell (SMC) lysates from the transfected A10 cell, as the molecular mechanism for decreased SMC proliferation that results in the inhibition of in-stent restenosis. MicroRNA 145 released from the stent suppressed the growth of the smooth muscle at the peri-stent implantation area, resulting in the prevention of restenosis at the post-implantation. We investigated the qualitative analyses of in-stent restenosis in the rabbit model using micro-computed tomography imaging and histological staining.ConclusionMicroRNA 145-eluting stent mitigated in-stent restenosis efficiently with no side effects and can be considered a successful substitute to the current drug-eluting stent.

TROGLITAZONE INHIBITS VASCULAR SMOOTH MUSCLE CELL PROLIFERATION IN A PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA (PPARγ)-INDEPENDENT MANNER

Re-occlusion of blood vessels following percutaneous coronary intervention with stenting (in stent stenosis) occurs in 5–10% of patients receiving drug-eluting stents. Re-occlusion of the treated vessel is observed when vascular smooth muscle cells (VSMCs) proliferate and migrate in response to vessel injury, inflammation and impaired healing of the endothelial cell layer. Novel drug eluting stents that reduce inflammation and permit endothelial repair whilst preventing detrimental VSMC proliferation and migration might reduce the occurrence of in stent restenosis. We previously have shown that ibuprofen has differential effects on vascular smooth muscle and endothelial cell proliferation and migration and, thus, might be a promising drug to deliver in a novel drug eluting stent platform. We do not fully understand why vascular smooth muscle and endothelial cells respond differently to ibuprofen but hypothesise that ibuprofen is regulating VSM C proliferation and migration, at least in part, via a peroxisome proliferator-activated receptor gamma (PPARγ)-dependent mechanism. In this study, we have used PPARγ agonists, troglitazone, pioglitazone, and rosiglitazone, to establish the role of PPARγ in the regulation of cell proliferation in the A10 VSMC cell line. Interestingly, whilst 50 µM and 100 µM troglitazone inhibited A10 VSMC proliferation by 58.9±4.6% and 21.6±4.8%, respectively, pioglitazone and rosiglitazone had no effect on VSMC cell proliferation at any concentrations tested. The observations that only one PPARγ agonist tested inhibited VSMC proliferation and that high concentrations of troglitazone were required to observe a significant effect (EC50=54.6 µM) suggest that troglitazone might be acting in a PPARγ-independent manner.

C-Ski inhibits autophagy of vascular smooth muscle cells induced by oxLDL and PDGF.

Autophagy is increasingly being recognized as a critical determinant of vascular smooth muscle cell (VSMC) biology. Previously, we have demonstrated that c-Ski inhibits VSMC proliferation stimulated by transforming growth factor β (TGF-β), but it is not clear whether c-Ski has the similar protective role against other vascular injury factors and whether regulation of autophagy is involved in its protective effects on VSMC. Accordingly, in this study, rat aortic A10 VSMCs were treated with 40 µg/ml oxidized low-density lipoprotein (oxLDL) or 20 ng/ml platelet-derived growth factor (PDGF), both of which were autophagy inducers and closely related to the abnormal proliferation of VSMCs. Overexpression of c-Ski in A10 cells significantly suppressed the oxLDL- and PDGF- induced autophagy. This action of c-Ski resulted in inhibiting the cell proliferation, the decrease of contractile phenotype marker α-SMA expression while the increase of synthetic phenotype marker osteopontin expression stimulated by oxLDL or PDGF. Inversely, knockdown of c-Ski by RNAi enhanced the stimulatory effects of oxLDL or PDGF on A10 cell growth and phenotype transition. And further investigation found that inhibition of AKT phosphorylation to downregulate proliferating cell nuclear antigen (PCNA) expression, was involved in the regulation of autophagy and associated functions by c-Ski in the oxLDL- and PDGF-stimulated VSMCs. Collectively, c-Ski may play an important role in inhibiting autophagy to protect VSMCs against some harsh stress including oxLDL and PDGF.

Nitric oxide attenuates the enhanced expression of Giα proteins in vascular smooth muscle cells from spontaneously hypertensive rats: molecular mechanisms (1065.15)

We have previously shown that the nitric oxide donor, SNAP, decreased the expression of Giα proteins and associated functions in A10 vascular smooth muscle cells. The present study was undertaken to investigate if SNAP can also decrease the expression of Giα proteins and associated signaling in aortic vascular smooth muscle cells (VSMC) from 12‐week‐old Wistar‐Kyoto (WKY) and Spontaneously Hypertensive rats (SHR) and to further explore the underlying mechanism(s). Treatment of VSMC with SNAP (100 µM) for 24h decreased the expression of Giα‐2, ‐3 proteins. This decrease was subverted by inhibiting peroxynitrite with wither Mn (III) tetrakis (4‐benzoic acid) porphyrin (MnTBAP) or uric acid, and by inhibiting MEK with PD98059. However, inhibition of guanylate cyclase by 1H‐(1, 2, 4) oxadiazolo (4, 3‐a) quinoxalin‐1‐one (ODQ) was unable to restore the SNAP‐induced decrease in Giα‐2, ‐3. In addition, the enhanced activity of NADPH oxidase and protein levels of its subunits (Nox‐4, p47phox and p22phox) were decreased by SNAP. The decreased expression of NADPH oxidase subunits was restored towards control levels by MnTBAP. Furthermore, SNAP treatment decreased the enhanced levels of O2‐, TBARS and protein carbonyl in SHR to control levels. SNAP also attenuated the increased phospholyration of PDGFR, EGFR and IGF‐1R and c‐Src. These results suggest that SNAP decreased the enhanced expression of Giα proteins in VSMC from SHR by attenuating the increased oxidative stress, growth factor receptor activation and ensuing ERK1/2 signaling and not by a cGMP‐dependent mechanism.Grant Funding Source: CIHR

CAMP attenuates the enhanced expression of Gi proteins and hyperproliferation of vascular smooth muscle cells from SHR: role of ROS and ROS-mediated signaling

We previously showed that angiotensin II (ANG II)-induced overexpression of inhibitory G proteins (Gi) was attenuated by dibutyryl-cAMP (db-cAMP) in A10 vascular smooth muscle cells (VSMC). Since enhanced levels of endogenous ANG II contributed to the overexpression of Gi protein and hyperproliferation of VSMC from spontaneously hypertensive rats (SHR), the present study was therefore undertaken to examine if cAMP could also attenuate the overexpression of Gi proteins and hyperproliferation of VSMC from SHR and to explore the underlying molecular mechanisms responsible for this response. The enhanced expression of Giα proteins in VSMC from SHR and Nω-nitro-L-arginine methyl ester hypertensive rats was decreased by db-cAMP. In addition, enhanced inhibition of adenylyl cyclase by inhibitory hormones and forskolin-stimulated adenylyl cyclase activity by low concentration of GTPγS in VSMC from SHR was also restored to Wistar-Kyoto (WKY) levels by db-cAMP. Furthermore, db-cAMP also attenuated the hyperproliferation and the increased production of superoxide anion, NAD(P)H oxidase activity, overexpression of Nox1/Nox2/Nox4 and p47phox proteins, increased phosphorylation of PDGF-receptor (R), EGF-R, c-Src, and ERK1/2 to control levels. In addition, the protein kinase A (PKA) inhibitor reversed the effects of db-cAMP on the expression of Nox4 and Giα proteins and hyperproliferation of VSMC from SHR to WKY levels, while stimulation of the exchange protein directly activated by cAMP did not have any effect on these parameters. These results suggest that cAMP via PKA pathway attenuates the overexpression of Gi proteins and hyperproliferation of VSMC from SHR through the inhibition of ROS and ROS-mediated transactivation of EGF-R/PDGF-R and MAPK signaling pathways.

Quercetin Attenuates Warfarin-induced Vascular Calcification in Vitro Independently from Matrix Gla Protein

Warfarin can stimulate vascular calcification in vitro via activation of β-catenin signaling and/or inhibition of matrix Gla protein (MGP) carboxylation. Calcification was induced in vascular smooth muscle cells (VSMCs) with therapeutic levels of warfarin in normal calcium and clinically acceptable phosphate levels. Although TGF/BMP and PKA pathways are activated in calcifying VSMCs, pharmacologic analysis reveals that their activation is not contributory. However, β-catenin activity is important because inhibition of β-catenin with shRNA or bioflavonoid quercetin prevents calcification in primary human VSMCs, rodent aortic rings, and rat A10 VSMC line. In the presence of quercetin, reactivation of β-catenin using the glycogen synthase kinase-3β (GSK-3β) inhibitor LiCl restores calcium accumulation, confirming that quercetin mechanism of action hinges on inhibition of the β-catenin pathway. Calcification in VSMCs induced by 10 μm warfarin does not associate with reduced levels of carboxylated MGP, and inhibitory effects of quercetin do not involve induction of MGP carboxylation. Further, down-regulation of MGP by shRNA does not alter the effect of quercetin. These results suggest a new β-catenin-targeting strategy to prevent vascular calcification induced by warfarin and identify quercetin as a potential therapeutic in this pathology.

Role of epidermal growth factor receptor transactivation in endothelin-1-induced enhanced expression of Gi protein and proliferation in A10 vascular smooth muscle cells

We have recently shown that vasoactive peptides such as angiotensin II (Ang II) and endothelin-1 (ET-1) increase the expression of Gi proteins and the proliferation of A10 vascular smooth muscle cells (VSMC) through mitogen-activated protein (MAP) kinase-phosphoinositide (PI) 3-kinase pathways. This study was intended to examine the implication of epidermal growth factor receptor (EGFR) activation in ET-1-induced enhanced expression of Gi proteins and proliferation of A10 VSMC, and to further investigate the underlying mechanisms responsible for these increases. Cell proliferation was determined by [(3)H]thymidine incorporation and the expression of Gi proteins; extracellular signal-regulated kinases 1 and 2 (ERK1/2) and EGFR phosphorylation was determined by Western blotting. Treatment of A10 VSMC with ET-1 enhanced the expression of Gi proteins, which was attenuated by BQ123 and BQ788, antagonists of ET(A) and ET(B) receptor respectively. In addition, ET-1 enhanced the phosphorylation of EGFR in A10 VSMC, which was restored to the control levels by EGFR inhibitor and ETA and ETB receptor antagonists. Furthermore, ET-1 also augmented the proliferation and ERK1/2 phosphorylation of A10 VSMC, which were restored to the control levels by inhibition of EGFR. These data suggest that ET-1 transactivates EGFR, which, through MAP kinase signaling, may contribute to the enhanced expression of Gi proteins and thus increased proliferation of A10 VSMC.

Prolonged exposure to methylglyoxal causes disruption of vascular KATP channel by mRNA instability

Diabetes mellitus is characterized by hyperglycemia and excessive production of intermediary metabolites including methylglyoxal (MGO), a reactive carbonyl species that can lead to cell injuries. Interacting with proteins, lipids, and DNA, excessive MGO can cause dysfunction of various tissues, especially the vascular walls where diabetic complications often take place. However, the potential vascular targets of excessive MGO remain to be fully understood. Here we show that the vascular Kir6.1/SUR2B isoform of ATP-sensitive K(+) (K(ATP)) channels is likely to be disrupted with an exposure to submillimolar MGO. Up to 90% of the Kir6.1/SUR2B currents were suppressed by 1 mM MGO with a time constant of ∼2 h. Consistently, MGO treatment caused a vast reduction of both Kir6.1 and SUR2B mRNAs endogenously expressed in the A10 vascular smooth muscle cells. In the presence of the transcriptional inhibitor actinomycin-D, MGO remained to lower the Kir6.1 and SUR2B mRNAs to the same degree as MGO alone, suggesting that the MGO effect is likely to compromise the mRNA stability. Luciferase reporter assays indicated that the 3'-untranslated regions (UTRs) of the Kir6.1 but not SUR2 mRNA were targeted by MGO. In contrast, the SUR2B mRNAs obtained with in vitro transcription were disrupted by MGO directly, while the Kir6.1 transcripts were unaffected. Consistent with these results, the constriction of mesenteric arterial rings was markedly augmented with an exposure to 1 mM MGO for 2 h, and such an MGO effect was totally eliminated in the presence of glibenclamide. These results therefore suggest that acting on the 3'-UTR of Kir6.1 and the coding region of SUR2B, MGO causes instability of Kir6.1 and SUR2B mRNAs, disruption of vascular K(ATP) channels, and impairment of arterial function.

Suppression of post-angioplasty restenosis with an Akt1 siRNA-embedded coronary stent in a rabbit model

Restenosis is the formation of blockages occurring at the site of angioplasty or stent placement. In order to avoid such blockages, the suppression of smooth muscle cells near the implanted stent is required. The Akt1 protein is known to be responsible for cellular proliferation, and specific inhibition of Akt1 gene expression results in the retardation of cell growth. To take advantage of these benefits, we developed a new delivery technique for Akt1 siRNA nanoparticles from a hyaluronic acid (HA)-coated stent surface. For this purpose, the disulfide cross-linked low molecular polyethyleneimine (PEI) (ssPEI) was used as a gene delivery carrier because disulfide bonds are stable in an oxidative extracellular environment but degrade rapidly in reductive intracellular environments. In this study, Akt1 siRNA showed efficient ionic interaction with the ssPEI carrier, which was confirmed by polyacrylamide gel electrophoresis. Akt1 siRNA/ssPEI nanoparticles (ASNs) were immobilized on the HA-coated stent surface and exhibited stable binding and localization, followed by time-dependent sustained release for intracellular uptake. Cellular viability on the nanoparticle-immobilized surface was assessed using A10 vascular smooth muscle cells, and the results revealed that immobilized ASNs exhibited negligible cytotoxicity against the adhering A10 cells. Transfection efficiency was quantified using a luciferase assay; the transgene expression of Akt1 suppression through the delivered Akt1 siRNA was measured using RT-PCR and western blot, demonstrating higher gene silencing efficiency when compared to other carriers. ASN coated on HA stents were deployed in the balloon-injured external iliac artery in rabbits in vivo. It was shown that the Akt1 released from the stent suppressed the growth of the smooth muscle at the peri-stent implantation area, resulting in the prevention of restenosis in the post-implantation phase.

Implication of multiple signaling pathways in the regulation of angiotensin II induced enhanced expression of Giα proteins in vascular smooth muscle cells

We have previously shown that A10 vascular smooth muscle cells (VSMC) exposed to angiotensin II (Ang II) exhibited overexpression of Giα proteins. In the present study, we examined the involvement of different signaling pathways in regulating Ang II induced enhanced expression of Giα proteins in VSMC by using pharmacological inhibitors. Ang II induced increased expression of Giα proteins in A10 VSMC was markedly attenuated by actinomycin D, losartan (an AT(1) receptor antagonist), dibutyryl cAMP, phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitors staurosporine and GP109203X, but not by PD123319 (an AT(2) receptor antagonist). In addition, BAPTA-AM and TMB-8 (chelators of intracellular Ca(2+)); and nifedipine (a blocker of L-type Ca(2+) channels) significantly inhibited Ang II induced enhanced expression of Giα proteins. On the other hand, extracellular Ca(2+) chelation using EGTA did not affect the Ang II evoked enhanced levels of Giα proteins. Furthermore, pretreatment of A10 VSMC with calmidazolium (an inhibitor of calmodulin), or KN93 (an inhibitor of CaM kinase), or genistein (an inhibitor of protein tyrosine kinase, PTK), also attenuated the increased levels of Giα proteins induced by Ang II. These results suggest that Ang II induced enhanced expression of Giα proteins may be regulated by different signaling pathways through AT(1) receptors in A10 VSMC.