Activation of PPARδ counteracts angiotensin II-induced ROS generation by inhibiting rac1 translocation in vascular smooth muscle cells

Angiotensin II (Ang II)-mediated modification of the redox milieu of vascular smooth muscle cells (VSMCs) has been implicated in several pathophysiological processes, including cell proliferation, migration and differentiation. In this study, we demonstrate that the peroxisome proliferator-activated receptor (PPAR) δ counteracts Ang II-induced production of reactive oxygen species (ROS) in VSMCs. Activation of PPARδ by GW501516, a specific ligand for PPARδ, significantly reduced Ang II-induced ROS generation in VSMCs. This effect was, however, reversed in the presence of small interfering (si)RNA against PPARδ. The marked increase in ROS levels induced by Ang II was also eliminated by the inhibition of phosphatidylinositol 3-kinase (PI3K) but not of protein kinase C, suggesting the involvement of the PI3K/Akt signalling pathway in this process. Accordingly, ablation of Akt with siRNA further enhanced the inhibitory effects of GW501516 in Ang II-induced superoxide production. Ligand-activated PPARδ also blocked Ang II-induced translocation of Rac1 to the cell membrane, inhibiting the activation of NADPH oxidases and consequently ROS generation. These results indicate that ligand-activated PPARδ plays an important role in the cellular response to oxidative stress by decreasing ROS generated by Ang II in vascular cells.     

PPARδ coordinates angiotensin II-induced senescence in vascular smooth muscle cells through PTEN-mediated inhibition of superoxide generation

Cellular senescence-associated changes in blood vessels have been implicated in aging and age-related cardiovascular disorders. Here, we demonstrate that peroxisome proliferator-activated receptor (PPAR) δ coordinates angiotensin (Ang) II-induced senescence of human vascular smooth muscle cells (VSMCs). Activation of PPARδ by GW501516, a specific ligand for PPARδ, significantly attenuated Ang II-induced generation of superoxides and suppressed senescence of VSMCs. A marked increase in the levels of p53 and p21 induced by Ang II was blunted by the treatment with GW501516. Ligand-activated PPARδ up-regulated expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and suppressed the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Knockdown of PTEN with siRNA abrogated the effects of PPARδ on cellular senescence, on PI3K/Akt signaling, and on generation of ROS in VSMCs treated with Ang II. Finally, administration of GW501516 to apoE-deficient mice treated with Ang II significantly reduced the number of senescent cells in the aorta, where up-regulation of PTEN with reduced levels of phosphorylated Akt and ROS was demonstrated. Thus, ligand-activated PPARδ confers resistance to Ang II-induced senescence by up-regulation of PTEN and ensuing modulation of the PI3K/Akt signaling to reduce ROS generation in vascular cells.     

PGC-1α limits angiotensin II-induced rat vascular smooth muscle cells proliferation via attenuating NOX1-mediated generation of reactive oxygen species

AngII (angiotensin II)-induced excessive ROS (reactive oxygen species) generation and proliferation of VSMCs (vascular smooth muscle cells) is a critical contributor to the pathogenesis of atherosclerosis. PGC-1α [PPARγ (peroxisome-proliferator-activated receptor γ) co-activator-1α] is involved in the regulation of ROS generation, VSMC proliferation and energy metabolism. The aim of the present study was to investigate whether PGC-1α mediates AngII-induced ROS generation and VSMC hyperplasia. Our results showed that the protein content of PGC-1α was negatively correlated with an increase in cell proliferation and migration induced by AngII. Overexpression of PGC-1α inhibited AngII-induced proliferation and migration, ROS generation and NADPH oxidase activity in VSMCs. Conversely, Ad-shPGC-1α (adenovirus-mediated PGC-1α-specific shRNA) led to the opposite effects. Furthermore, the stimulatory effect of Ad-shPGC-1α on VSMC proliferation was significantly attenuated by antioxidant and NADPH oxidase inhibitors. Analysis of several key subunits of NADPH oxidase (Rac1, p22^phox, p40^phox, p47^phox and p67^phox) and mitochondrial ROS revealed that these mechanisms were not responsible for the observed effects of PGC-1α. However, we found that overexpression of PGC-1α promoted NOX1 degradation through the proteasome degradation pathway under AngII stimulation and consequently attenuated NOX1 (NADPH oxidase 1) expression. These alterations underlie the inhibitory effect of PGC-1α on NADPH oxidase activity. Our data support a critical role for PGC-1α in the regulation of proliferation and migration of VSMCs, and provide a useful strategy to protect vessels against atherosclerosis.     

Acetylshikonin attenuates angiotensin II-induced proliferation and motility of human brain smooth muscle cells by inhibiting Wnt/β-catenin signaling

Cerebrovascular smooth muscle cell proliferation and migration contribute to hyperplasia in case of cerebrovascular remodeling and stroke. In the present study, we investigated the effects of acetylshikonin, the main ingredient of a Chinese traditional medicine Zicao, on human brain vascular smooth muscle cell (HBVSMCs) proliferation and migration induced by angiotensin II (AngII), and the underlying mechanisms. We found that acetylshikonin treatment significantly inhibited AngII-induced HBVSMCs proliferation and cell cycle transition from G1 to S phase. Wound-healing assay and Transwell assay showed that AngII-induced cell migration and invasion were markedly attenuated by acetylshikonin. In addition, AngII challenge significantly induced Wnt/β-catenin signaling activation, as evidenced by increased β-catenin phosphorylation and nuclear translocation and GSK-3β phosphorylation. However, acetylshikonin treatment inhibited the activation of Wnt/β-catenin signaling. Consequently, western blotting analysis revealed that acetylshikonin effectively reduced the expression of downstream target genes in AngII-treated cells, including c-myc, survivin and cyclin D1, which contributed to the inhibitory effect of acetylshikonin on HBVSMCs proliferation. Further, stimulation with recombinant Wnt3a dramatically reversed acetylshikonin-mediated inhibition of proliferation and cell cycle transition in HBVSMCs. Our study demonstrates that acetylshikonin prevents AngII-induced cerebrovascular smooth muscle cells proliferation and migration through inhibition of Wnt/β-catenin pathway, indicating that acetylshikonin may present a potential option for the treatment of cerebrovascular remodeling.     

iPLA2β overexpression in smooth muscle exacerbates angiotensin II-induced hypertension and vascular remodeling

Objectives

Calcium independent group VIA phospholipase A₂ (iPLA₂β) is up-regulated in vascular smooth muscle cells in some diseases, but whether the up-regulated iPLA₂β affects vascular morphology and blood pressure is unknown. The current study addresses this question by evaluating the basal- and angiotensin II infusion-induced vascular remodeling and hypertension in smooth muscle specific iPLA₂β transgenic (iPLA₂β -Tg) mice.

Method and Results

Blood pressure was monitored by radiotelemetry and vascular remodeling was assessed by morphologic analysis. We found that the angiotensin II-induced increase in diastolic pressure was significantly higher in iPLA₂β-Tg than iPLA₂β-Wt mice, whereas, the basal blood pressure was not significantly different. The media thickness and media∶lumen ratio of the mesenteric arteries were significantly increased in angiotensin II-infused iPLA₂β-Tg mice. Analysis revealed no difference in vascular smooth muscle cell proliferation. In contrast, adenovirus-mediated iPLA₂β overexpression in cultured vascular smooth muscle cells promoted angiotensin II-induced [³H]-leucine incorporation, indicating enhanced hypertrophy. Moreover, angiotensin II infusion-induced c-Jun phosphorylation in vascular smooth muscle cells overexpressing iPLA2β to higher levels, which was abolished by inhibition of 12/15 lipoxygenase. In addition, we found that angiotensin II up-regulated the endogenous iPLA₂β protein in-vitro and in-vivo.

Conclusion

The present study reports that iPLA₂β up-regulation exacerbates angiotensin II-induced vascular smooth muscle cell hypertrophy, vascular remodeling and hypertension via the 12/15 lipoxygenase and c-Jun pathways.     

Protein kinase Cδ mediates MCP-1 mRNA stabilization in vascular smooth muscle cells

Monocyte chemoattractant protein-1 (MCP-1) is an inflammatory chemokine that promotes atherosclerosis and is a mediator of the response to arterial injury. We previously demonstrated that platelet-derived growth factor (PDGF) and angiotensin II (Ang) induce the accumulation of MCP-1 mRNA in vascular smooth muscle cells mainly by increasing mRNA stability. In the present study, we have examined the signaling pathways involved in this stabilization of MCP-1 mRNA. The effect of PDGF (BB isoform) and Ang on MCP-1 mRNA stability was mediated by the PDGF β and angiotensin II receptor AT1R, respectively, and did not involve transactivation between the two receptors. The effect of PDGF-BB was blocked by inhibitors of protein kinase C (PKC), but not by inhibitors of phosphoinositol 3-kinase (PI3K), Src, or NADPH oxidase (NADPHox). In contrast, the effect of Ang was blocked by inhibitors of Src, and PKC, but not by inhibitors of PI3 K, or NADPHox. The effect of PDGF BB on MCP-1 mRNA stability was blocked by siRNA directed against PKCδ and protein kinase D (PKD), whereas the effect of Ang was blocked only by siRNA directed against PKCδ. These results suggest that the enhancement of MCP-1 mRNA stability by PDGF-BB and Ang are mediated by distinct “proximal” signaling pathways that converge on activation of PKCδ. This study identifies a novel role for PKCδ in mediating mRNA stability in smooth muscle cells.     

Suppression of angiotensin Ⅱ stimulated responses in aortic vascular smooth muscle cells of experimental cirrhotic rats

INTRODUCTIONLivercirrhosisisoftencomplicatedbyhyperdynamiccirculation,whichischaracterizedbyadecreaseinarterialbloodpressureandperipheralvascularresistanceandanincreaseincardiacoutputandsplanchnicbloodnow[1].Thesehaemodynamicdisturbancesareproposedtocontributetothedevelopmelltofportalhypertension,retentionofsodiumandwater,ascitesformationandcorrelatewithprognosisoflivercirrhosis[2].Ithasbeensuggestedthatprimaryarterialvasodilationmayinducethishyperdynamiccirculation[3].Buttheunderlyingmec…     

PPARδ modulates oxLDL-induced apoptosis of vascular smooth muscle cells through a TGF-β/FAK signaling axis

The peroxisome proliferator-activated receptor delta (PPARδ) has been implicated in the modulation of vascular homeostasis. However, its roles in the apoptotic cell death of vascular smooth muscle cells (VSMCs) are poorly understood. Here, we demonstrate that PPARδ modulates oxidized low-density lipoprotein (oxLDL)-induced apoptosis of VSMCs through the transforming growth factor-β (TGF-β) and focal adhesion kinase (FAK) signaling pathways. Activation of PPARδ by GW501516, which is a specific ligand, significantly inhibited oxLDL-induced cell death and generation of reactive oxygen species in VSMCs. These inhibitory effects were significantly reversed in the presence of small interfering (si)RNA against PPARδ, or by blockade of the TGF-β or FAK signaling pathways. Furthermore, PPARδ-mediated recovery of FAK phosphorylation suppressed by oxLDL was reversed by SB431542, a specific ALK5 receptor inhibitor, indicating that a TGF-β/FAK signaling axis is involved in the action of PPARδ. Among the protein kinases activated by oxLDL, p38 mitogen-activated protein kinase was suppressed by ligand-activated PPARδ. In addition, oxLDL-induced expression and translocation of pro-apoptotic or anti-apoptotic factors were markedly affected in the presence of GW501516. Those effects were reversed by PPARδ siRNA, or inhibitors of TGF-β or FAK, which also suggests that PPARδ exerts its anti-apoptotic effect via a TGF-β/FAK signaling axis. Taken together, these findings indicate that PPARδ plays an important role in the pathophysiology of disease associated with apoptosis of VSMC, such as atherosclerosis and restanosis.     

c-Jun N-terminal kinase attenuates TNFα signaling by reducing Nox1-dependent endosomal ROS production in vascular smooth muscle cells

Tumor necrosis factor-α (TNFα), a proinflammatory cytokine, causes vascular smooth muscle cell (VSMC) proliferation and migration and promotes inflammatory vascular lesions. Nuclear factor-kappa B (NF-κB) activation by TNFα requires endosomal superoxide production by Nox1. In endothelial cells, TNFα stimulates c-Jun N-terminal kinase (JNK), which inhibits NF-κB signaling. The mechanism by which JNK negatively regulates TNFα-induced NF-κB activation has not been defined. We hypothesized that JNK modulates NF-κB activation in VSMC, and does so via a Nox1-dependent mechanism. TNFα-induced NF-κB activation was TNFR1- and endocytosis-dependent. Inhibition of endocytosis with dominant-negative dynamin (DynK44A) potentiated TNFα-induced JNK activation, but decreased ERK activation, while p38 kinase phosphorylation was not altered. DynK44A attenuated intracellular, endosomal superoxide production in wild-type (WT) VSMC, but not in NADPH oxidase 1 (Nox1) knockout (KO) cells. siRNA targeting JNK1 or JNK2 potentiated, while a JNK activator (anisomycin) inhibited, TNFα-induced NF-κB activation in WT, but not in Nox1 KO cells. TNFα-stimulated superoxide generation was enhanced by JNK1 inhibition in WT, but not in Nox1 KO VSMC. These data suggest that JNK suppresses the inflammatory response to TNFα by reducing Nox1-dependent endosomal ROS production. JNK and endosomal superoxide may represent novel targets for pharmacologic modulation of TNFα signaling and vascular inflammation.     

ROS directly activates transforming growth factor β type 1 receptor signalling in human vascular smooth muscle cells

BackgroundWidely used NAPDH oxidase (Nox) inhibitor, apocynin is a prodrug that needs to be converted to its pharmacologically active form by myeloperoxidase. In myeloperoxidase deficient non phagocytic cells such as vascular smooth muscle cells (VSMCs) apocynin stimulates the production of ROS. ROS is generated by the activation of many signalling pathways, thus we have used apocynin as a pharmacological tool to characterise the role of endogenous ROS in activating the transforming growth factor beta receptor (TGFBR1) without the activation of other pathways.MethodsThe in vitro study utilized human VSMCs. Western blotting and quantitative real time PCR were performed to assess signalling pathways and gene expression, respectively. Intracellular ROS levels was measured using fluorescence detection assay.ResultsTreatment with apocynin of human VSMCs stimulated ROS production and the phosphorylation of TGFBR1 and subsequent activation of TGFBR1 signalling leading to the formation of phosphorylated Smad2 which consequently upregulates the mRNA expression of glycosaminoglycan synthesizing enzyme.ConclusionsThese findings outline a specific involvement of ROS production in TGFBR1 activation. Furthermore, because apocynin stimulates Nox and ROS production, apocynin must be used with considerable care in vitro as its actions clearly extend beyond the stimulation of Nox enzymes and it has consequences for cellular signalling.General significanceApocynin can stimulate Nox leading to the production of ROS and the outcome is completely dependent upon the redox properties of the cell.     

Activation of the non-canonical NF-κB/p52 pathway in vascular endothelial cells by RANKL elicits pro-calcific signalling in co-cultured smooth muscle cells

BackgroundThe intimal endothelium is known to condition the underlying medial smooth muscle cell (SMC) layer of the vessel wall, and is highly responsive to receptor-activator of nuclear factor-κB ligand (RANKL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), pro-calcific and anti-calcific agents, respectively. In this paper, we tested the hypothesis that RANKL-induced activation of endothelial NF-κB signalling is essential for pro-calcific activation of the underlying SMCs.MethodsFor these studies, human aortic endothelial and smooth muscle cell mono-cultures (HAECs, HASMCs) were treated with RANKL (0–25 ng/ml ± 5 ng/ml TRAIL) for 72 h. Non-contact transwell HAEC:HASMC co-cultures were also employed in which the luminal HAECs were treated with RANKL (± 5 ng/ml TRAIL), followed by analysis of pro-calcific markers in the underlying subluminal HASMCs.ResultsTreatment of either HAECs or HASMCs with RANKL activated the non-canonical NF-κB/p52 and canonical NF-κB/p65 pathways in both cell types. In RANKL ± TRAIL-treated HAECs, recombinant TRAIL, previously demonstrated by our group to strongly attenuate the pro-calcific signalling effects of RANKL, was shown to specifically block the RANKL-mediated activation of non-canonical NF-κB/p52, clearly pointing to the mechanistic relevance of this specific pathway to RANKL function within endothelial cells. In a final series of HAEC:HASMC transwell co-culture experiments, RANKL treatment of HAECs that had been genetically silenced (via siRNA) for the NF-κB2 gene (the molecular forerunner to NF-κB/p52 generation) exhibited strongly attenuated pro-calcific activation of underlying HASMCs relative to scrambled siRNA controls.SummaryThese in vitro observations provide valuable mechanistic insights into how RANKL may potentially act upon endothelial cells through activation of the alternative NF-κB pathway to alter endothelial paracrine signalling and elicit pro-calcific responses within underlying vascular smooth muscle cells.     

PDGF-β receptor and PKC have no effect on angiotensin II-induced JAK2 and STAT1 phosphorylation in vascular smooth muscle cells under high glucose condition

Background: The mechanisms responsible for the accelerated cardiovascular disease in diabetes, as well as the increased hypertrophic effects of angiotensin II (Ang II) under hyperglycemic condition, are not very clear. Evidences show that platelet-derived growth factor (PDGF) and protein kinase C (PKC) play a critical role in this effect. In our study, we examined the role of PKC and PDGF receptor on JAK2 and STAT1 phosphorylation under high glucose (HG) condition (25 mmol/L) in response to Ang II in cultured vascular smooth muscle cells (VSMC).

Methods: VSMCs were isolated from the thoracic aorta of male Wistar rats and were cultured. Growth-arrested VSMCs were placed in either normal glucose (NG) or HG condition for 48?h and then VSMCs were stimulated with agonists and antagonists. The tyrosine phosphorylation of JAK2 or STAT were determined by immunoblotting using specific antibodies.

Results: High glucose markedly increased the phosphorylation of tyrosine residues of JAK2 and serine residues of STAT 1 compared with cells cultured in NG (5.5 mmol/L) with and without Ang II stimulation. Experiments made with specific PDGF-β receptor inhibitor AG1295 and PKC inhibitor GF109203X showed that there were no changes in Ang II-stimulated JAK2 and STAT1 phosphorylation under NG and HG conditions compared with experiments without inhibitors.

Conclusion: According to our findings, Ang II-stimulated JAK2 and STAT1 phosphorylation under either NG or HG condition do not proceed via a different pathway rather than PKC and PDGF-β receptor.     

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.     

Osteopontin regulates α-smooth muscle actin and calponin in vascular smooth muscle cells

vSMCs (vascular smooth muscle cells) lose differentiation markers and gain uncontrolled proliferative activity during the early stages of atherosclerosis. Previous studies have shown that OPN (osteopontin) mRNA and protein levels increase significantly on induction of proliferative activity by allylamine (an atherogenic amine) and that this response can be inhibited by OPN antibodies. We have investigated the role of OPN in vSMC differentiation. Primary cultures of aortic mouse vSMCs were transfected with an OPN expression plasmid and several vSMC differentiation markers including α-SM actin (α-smooth muscle actin), SM22-α, tropomyosin and calponin were monitored in this cellular model. α-SM actin and calponin protein levels were significantly decreased by OPN overexpression. Down-regulation of α-SM actin and calponin was also observed on extracellular treatment of mouse vSMCs with recombinant OPN. In addition, calponin mRNA was significantly decreased under serum-restricted conditions when OPN mRNA was dramatically increased, while α-SM actin mRNA remained unchanged. These data indicate that OPN down-regulates α-SM actin and calponin expression through an extracellular signalling pathway. Functional connectivity between OPN and vSMC differentiation markers has been established. Since vSMCs lose differentiation features during early atherosclerosis, a mechanistic basis for OPN functions as a critical regulator of proliferative cardiovascular disease has been presented.     

GLP-1 promotes mitochondrial metabolism in vascular smooth muscle cells by enhancing endoplasmic reticulum–mitochondria coupling

Incretin GLP-1 has important metabolic effects on several tissues, mainly through the regulation of glucose uptake and usage. One mechanism for increasing cell metabolism is modulating endoplasmic reticulum (ER)–mitochondria communication, as it allows for a more efficient transfer of Ca²⁺ into the mitochondria, thereby increasing activity. Control of glucose metabolism is essential for proper vascular smooth muscle cell (VSMC) function. GLP-1 has been shown to produce varied metabolic actions, but whether it regulates glucose metabolism in VSMC remains unknown. In this report, we show that GLP-1 increases mitochondrial activity in the aortic cell line A7r5 by increasing ER–mitochondria coupling. GLP-1 increases intracellular glucose and diminishes glucose uptake without altering glycogen content. ATP, mitochondrial potential and oxygen consumption increase at 3 h of GLP-1 treatment, paralleled by increased Ca²⁺ transfer from the ER to the mitochondria. Furthermore, GLP-1 increases levels of Mitofusin-2 (Mfn2), an ER-mitochondria tethering protein, via a PKA-dependent mechanism. Accordingly, PKA inhibition and Mfn2 down-regulation prevented mitochondrial Ca²⁺ increases in GLP-1 treated cells. Inhibiting both Ca²⁺ release from the ER and Ca²⁺ entry into mitochondria as well as diminishing Mfn2 levels blunted the increase in mitochondrial activity in response to GLP-1. Altogether, these results strongly suggest that GLP-1 increases ER–mitochondria communication in VSMC, resulting in higher mitochondrial activity.