Vascular Smooth Muscle Cell Hypertrophy Research Articles

Novel signaling axis of FHOD1-RNF213-Col1α/Col3α in the pathogenesis of hypertension-induced tunica media thickening

Hypertension-induced tunica media thickening (TMT) is the most important fundamental for the subsequent complications like stroke and cardiovascular diseases. Pathogenically, TMT originates from both vascular smooth muscle cells (VSMCs) hypertrophy due to synthesizing more amount of intracellular contractile proteins and excess secretion of extracellular matrix. However, what key molecules are involved in the pathogenesis of TMT is unknown. We hypothesize that formin homology 2 domain-containing protein 1 (FHOD1), an amply expressed mediator for assembly of thin actin filament in VSMCs, is a key regulator for the pathogenesis of TMT. In this study, we found that FHOD1 expression and its phosphorylation/activation were both upregulated in the arteries of three kinds of hypertensive rats. Ang-II induced actin filament formation and hypertrophy through activation and upregulation of FHOD1 in VSMCs. Active FHOD1-mediated actin filament assembly and secretions of collagen-1α/collagen-3α played crucial roles in Ang-II-induced VSMCs hypertrophy in vitro and hypertensive TMT in vivo. Proteomics demonstrated that activated FL-FHOD1 or its C-terminal diaphanous-autoregulatory domain significantly upregulated RNF213 (ring finger protein 213), a 591-kDa cytosolic E3 ubiquitin ligase with its loss-of-functional mutations being a susceptibility gene for Moyamoya disease which has prominent tunica media thinning in both intracranial and systemic arteries. Mechanistically, activated FHOD1 upregulated its downstream effector RNF213 independently of its classical pathway of decreasing G-actin/F-actin ratio, transcription, and translation, but dependently on its C-terminus-mediated stabilization of RNF213 protein. FHOD1-RNF213 signaling dramatically promoted collagen-1α/collagen-3α syntheses in VSMCs. Our results discovered a novel signaling axis of FHOD1-RNF213-collagen-1α/collagen-3α and its key role in the pathogenesis of hypertensive TMT.

Hibiscus attenuates renovascular hypertension-induced aortic remodeling dose dependently: the oxidative stress role and Ang II/cyclophilin A/ERK1/2 signaling.

Introduction: The high levels of angiotensin II (Ang II) can modify the vascular tone, enhance vascular smooth muscle cells (VSMCs) proliferation and hypertrophy and increase the inflammatory cellular infiltration into the vessel wall. The old herbal nonpharmacological agent, Hibiscus (HS) sabdariffa L has multiple cardioprotective impacts; thus, we investigated the role of HS extract in amelioration of renovascular hypertension (RVH)-induced aortic remodeling. Materials and methods: Thirty-five rats (7/group) were randomly allocated into 5 groups; group: I: Control-sham group, and RVH groups; II, III, IV, and V. The rats in RVH groups were subjected to the modified Goldblatt two-kidneys, one clip (2K1C) for induction of hypertension. In group: II, the rats were left untreated whereas in group III, IV, and V: RVH-rats were treated for 6weeks with low dose hibiscus (LDH), medium dose hibiscus (MDH), and high dose hibiscus (HDH) respectively. Results: We found that the augmented pro-contractile response of the aortic rings was ameliorated secondary to the in-vivo treatment with HS dose dependently. The cyclophilin A (CyPA) protein levels positively correlated with the vascular adhesion molecule-1 (VCAM-1) and ERK1/2, which, in turn, contribute to the reactive oxygen species (ROS) production. Daily HS intake modified aortic renovation by enhancing the antioxidant capacity, restraining hypertrophy and fibrosis, downregulation of the metastasis associated lung adenocarcinoma transcript (MALAT1), and cyclophilin A (CyPA)/ERK1/2 levels. Discussion: Adding to the multiple beneficial effects, HS aqueous extract was able to inhibit vascular smooth muscle cell proliferation induced by 2K1C model. Thus, adding more privilege for the utilization of the traditional herbal extracts to attenuate RVH-induced aortopathy.

CEBPB/POU2F2 modulates Endothelin1 expression in prehypertensive SHR vascular smooth muscle cells.

The pathogenesis of hypertension is not fully understood; ET1 is involved in developing essential hypertension. ET1 can promote VSMC proliferation or hypertrophy through autocrine and paracrine effects. Proliferating smooth muscle cells in the aorta are "dedifferentiated" cells that cause increased arterial stiffness and remodeling. Male SHRs had higher aortic stiffness than normal control male WKY rats. Male SHR VSMCs expressed high levels of the ET1 gene, but endothelial cells did not. Therefore, it is necessary to understand the molecular mechanism of enhanced ET1 expression in SHR VSMCs. We identified POU2f2 and CEBPB as the main molecules that enhance ET1 expression in male SHR VSMCs. A promoter activity analysis confirmed that the enhancer region of the ET1 promoter in male SHR VSMCs was from -1309 to -1279 bp. POU2f2 and CEBPB exhibited an additive role in the enhancer region of the ET1 promoter. POU2f2 or CEBPB overexpression sufficiently increased ET1 expression, and co-transfection with the CEBPB and POU2f2 expression plasmids had additive effects on the activity of the ET1 promoter and ET1 secretion level of male WKY VSMCs. In addition, knockdown of POU2f2 also revealed that POU2f2 is necessary to enhance ET1 expression in SHR VSMCs. The enhancer region of the ET1 promoter is highly conserved in rats, mice, and humans. POU2f2 and CEBPB mRNA levels were significantly increased in remodeled human VMSCs. In conclusion, the novel regulation of POU2f2 and CEBPB in VSMCs will help us understand the pathogenesis of hypertension and support the development of future treatments for hypertension.

Downregulation of natriuretic peptide receptor-C in vascular smooth muscle cells from spontaneously hypertensive rats contributes to vascular remodeling

Hypertension is associated with vascular remodeling due to hyperproliferation and hypertrophy of vascular smooth muscle cells (VSMC). VSMC from several animal models of hypertensive rats including spontaneously hypertensive rats (SHR) exhibit hyperproliferation, hypertrophy and decreased expression of natriuretic peptide receptor-C (NPR-C). In addition, angiotensin II (Ang II) and growth factors that promotes vascular remodeling have also been shown to attenuate the expression of NPR-C in VSMC. The present study investigates the relationship between the decreased expression of NPR-C and vascular remodeling in SHR and the underlying molecular mechanisms. Aortic VSMC from SHR and their control Wistar Kyoto (WKY) rats were transfected with cDNA of NPR-C and used for the vascular remodeling studies. Transfection of VSMC with cDNA of NPR-C augmented the expression of NPR-C in both VSMC from SHR and WKY rats and resulted in the attenuation of hyperproliferation and hypertrophy of VSMC from SHR. The overexpression of NPR-C also resulted in the attenuation of increased expression of epidermal growth factor receptor (EGFR), platelet derived growth factor receptor (PDGFR), cell cycle proteins, cyclin D1, cyclin-dependent kinase 4 (Cdk4), phospho-retinoblastoma (pRb) and Giα-2 proteins, all these signaling molecules implicated in the hyperproliferation/hypertrophy of VSMC from SHR. In summary, these results indicate that augmenting the decreased expression of NPR-C in VSMC from SHR improves vascular remodeling by attenuating hyperproliferation and hypertrophy through decreasing the overexpression of several signaling molecules. It may be suggested that NPR-C plays a vasculoprotective role and that the downregulation of NPR-C contributes to the vascular remodeling in SHR.

Distinct Spatial Conformations and Trafficking Patterns of β‐arrestin Isoforms in AT1R Signaling

Excessive activation of the AT1R by the hormone Angiotensin II (AngII) is implicated in the pathogenesis of hypertension by promoting vascular smooth muscle cell migration, hypertrophy, and cardiac remodeling. A limitation of current drug development to target this receptor is the complex pattern of its downstream signaling pathways, the majority of which are mediated by G proteins and β‐arrestins. Although the two β‐arrestin isoforms, β‐arrestin 1 and 2, share ~78% amino acid sequence homology, they have been shown to differentially regulate signaling pathways such as receptor internalization and the mitogen‐activated protein kinase pathways, leading to distinct physiologic and pathophysiologic outcomes. In our study, we investigated the functional divergence of the β‐arrestin isoforms at the AT1R and its link to physiologic function. We hypothesized that β‐arrestin isoforms adopt distinct recruitment patterns and spatial conformations that lead to differential regulation of downstream signaling and physiologic outcomes and “biased responses” by different agonists. Using the split NanoLuciferase complementation assay (NanoBiT), we determined that β‐arrestin 1 and 2 are recruited differentially to the receptor, the plasma membrane and the endosomes following AT1R stimulation with AngII and the β‐arrestin‐biased agonist TRV023. Compared to TRV023, AngII was found to be more potent and efficacious in inducing the recruitment of both β‐arrestin isoforms to the receptor. We also developed a new conformational biosensor, called the complex Fluorescent Arsenical Hairpin (FlAsH) assay, to assess the spatial conformational signatures of the β‐arrestin isoforms when they are bound to the AT1R or localized at the plasma membrane and endosomes. β‐arrestins 1 and 2 were found to adopt distinct conformations at all three cellular locations following agonist treatment. Our finding provides new insights into the mechanisms underlying the functional differences between the β‐arrestin isoforms at the AT1R. Further studies will be necessary to elucidate the link between the trafficking and conformational differences of the β‐arrestin isoforms and their distinct physiological roles in AT1R signaling.

Regulation of total LC3 levels by angiotensin II in vascular smooth muscle cells

Hypertension is associated with high circulating angiotensin II (Ang II). We have reported that autophagy regulates Ang II‐induced vascular smooth muscle cell (VSMC) hypertrophy, but the mechanism mediating this effect is still unknown. Therefore, we studied how Ang II regulates LC3 levels in VSMCs and whether Bag3, a co‐chaperone known to regulate LC3 total levels, may be involved in the effects elicited by Ang II. A7r5 cell line or rat aortic smooth muscle cell (RASMC) primary culture were stimulated with Ang II 100 nM for 24 h and LC3 I, LC3 II and Bag3 protein levels were determined by Western blot. MAP1LC3B mRNA levels were assessed by RT‐qPCR. Ang II increased MAP1LC3B mRNA levels and protein levels of LC3 I, LC3 II and total LC3 (LC3 I + LC3 II). Cycloheximide, but not actinomycin D, abolished LC3 II and total LC3 increase elicited by Ang II in RASMCs. In A7r5 cells, cycloheximide prevented the Ang II‐mediated increase of LC3 I and total LC3, but not LC3 II. Moreover, Ang II increased Bag3 levels, but this increase was not observed upon co‐administration with either losartan 1 μM (AT1R antagonist) or Y‐27632 10 μM (ROCK inhibitor). These results suggest that Ang II may regulate total LC3 content through transcriptional and translational mechanisms. Moreover, Bag3 is increased in response to Ang II by a AT1R/ROCK signalling pathway. These data provide preliminary evidence suggesting that Ang II may stimulate autophagy in VSMCs by increasing total LC3 content and LC3 processing.

Vascular smooth muscle cell-specific miRNA-214 knockout inhibits angiotensin II-induced hypertension through upregulation of Smad7.

Vascular remodeling is a prominent trait during the development of hypertension, attributable to the phenotypic transition of vascular smooth muscle cells (VSMCs). Increasing studies demonstrate that microRNA plays an important role in this process. Here, we surprisingly found that smooth muscle cell-specific miR-214 knockout (miR-214 cKO) significantly alleviates angiotensin II (Ang II)-induced hypertension, which has the same effect as that of miR-214 global knockout mice in response to Ang II stimulation. Under the treatment of Ang II, miR-214 cKO mice exhibit substantially reduced systolic blood pressure. The vascular medial thickness and area in miR-214 cKO blood vessels were obviously reduced, the expression of collagen I and proinflammatory factors were also inhibited. VSMC-specific deletion of miR-214 blunts the response of blood vessels to the stimulation of endothelium-dependent and -independent vasorelaxation and phenylephrine and 5-HT induced vasocontraction. In vitro, Ang II-induced VSMC proliferation, migration, contraction, hypertrophy, and stiffness were all repressed with miR-214 KO in VSMC. To further explore the mechanism of miR-214 in the regulation of the VSMC function, it is very interesting to find that the TGF-β signaling pathway is mostly enriched in miR-214 KO VSMC. Smad7, the potent negative regulator of the TGF-β/Smad pathway, is identified to be the target of miR-214 in VSMC. By which, miR-214 KO sharply enhances Smad7 levels and decreases the phosphorylation of Smad3, and accordingly alleviates the downstream gene expression. Further, Ang II-induced hypertension and vascular dysfunction were reversed by antagomir-214. These results indicate that miR-214 in VSMC established a crosstalk between Ang II-induced AT1R signaling and TGF-β induced TβRI /Smad signaling, by which it exerts a pivotal role in vascular remodeling and hypertension and imply that miR-214 has the potential as a therapeutic target for the treatment of hypertension.

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.

Role of cyclic AMP response element binding protein (CREB) in angiotensin II-induced responses in vascular smooth muscle cells.

Cyclic AMP response element (CRE) binding protein (CREB) is a nuclear transcription factor that regulates the transcription of several genes containing the CRE sites on their promoters. CREB is activated by phosphorylation on a key serine residue, Ser311, in response to a wide variety of extracellular stimuli including angiotensin II (Ang II). Ang II is an important vasoactive peptide and mitogen for vascular smooth muscle cells (VSMC) that in addition to regulating the contractile response in VSMC also plays an important role in phenotypic switch of VSMC from contractile to a synthetic state. The synthetic VSMC are known to exhibit proliferative and migratory properties due to hyperactivation of Ang II-induced signaling events. Ang II has been shown to induce CREB phosphorylation/activation and transcription of genes implicated in proliferation, growth, and migration. Here, we have highlighted some key studies that have demonstrated an important role of CREB in Ang II-mediated gene transcription, proliferation, hypertrophy, and migration of VSMC.

Molecular Mechanisms of Adiponectin-Induced Attenuation of Mechanical Stretch-Mediated Vascular Remodeling.

Hypertension induces vascular hypertrophy, which changes blood vessels structurally and functionally, leading to reduced tissue perfusion and further hypertension. It is also associated with dysregulated levels of the circulating adipokines leptin and adiponectin (APN). Leptin is an obesity-associated hormone that promotes vascular smooth muscle cell (VSMC) hypertrophy. APN is a cardioprotective hormone that has been shown to attenuate hypertrophic cardiomyopathy. In this study, we investigated the molecular mechanisms of hypertension-induced VSMC remodeling and the involvement of leptin and APN in this process. To mimic hypertension, the rat portal vein (RPV) was mechanically stretched, and the protective effects of APN on mechanical stretch-induced vascular remodeling and the molecular mechanisms involved were examined by using 10 μg/ml APN. Mechanically stretching the RPV significantly decreased APN protein expression after 24 hours and APN mRNA expression in a time-dependent manner in VSMCs. The mRNA expression of the APN receptors AdipoR1, AdipoR2, and T-cadherin significantly increased after 15 hours of stretch. The ratio of APN/leptin expression in VSMCs significantly decreased after 24 hours of mechanical stretch. Stretching the RPV for 3 days increased the weight and [3H]-leucine incorporation significantly, whereas APN significantly reduced hypertrophy in mechanically stretched vessels. Stretching the RPV for 10 minutes significantly decreased phosphorylation of LKB1, AMPK, and eNOS, while APN significantly increased p-LKB1, p-AMPK, and p-eNOS in stretched vessels. Mechanical stretch significantly increased p-ERK1/2 after 10 minutes, whereas APN significantly reduced stretch-induced ERK1/2 phosphorylation. Stretching the RPV also significantly increased ROS generation after 1 hour, whereas APN significantly decreased mechanical stretch-induced ROS production. Exogenous leptin (3.1 nM) markedly increased GATA-4 nuclear translocation in VSMCs, whereas APN significantly attenuated leptin-induced GATA-4 nuclear translocation. Our results decipher molecular mechanisms of APN-induced attenuation of mechanical stretch-mediated vascular hypertrophy, with the promising potential of ultimately translating this protective hormone into the clinic.

Calcitonin gene-related peptide inhibits angiotensin II-induced NADPH oxidase-dependent ROS via the Src/STAT3 signalling pathway.

We had previously demonstrated that the calcitonin gene‐related peptide (CGRP) suppresses the oxidative stress and vascular smooth muscle cell (VSMC) proliferation induced by vascular injury. A recent study also indicated that CGRP protects against the onset and development of angiotensin II (Ang II)‐induced hypertension, vascular hypertrophy and oxidative stress. However, the mechanism behind the effects of CGRP on Ang II‐induced oxidative stress is unclear. CGRP significantly suppressed the level of reactive oxygen species (ROS) generated by NADPH oxidase in Ang II‐induced VSMCs. The Ang II‐stimulated activation of both Src and the downstream transcription factor, STAT3, was abrogated by CGRP. However, the antioxidative effect of CGRP was lost following the expression of constitutively activated Src or STAT3. Pre‐treatment with H‐89 or CGRP8–37 also blocked the CGRP inhibitory effects against Ang II‐induced oxidative stress. Additionally, both in vitro and in vivo analyses show that CGRP treatment inhibited Ang II‐induced VSMC proliferation and hypertrophy, accompanied by a reduction in ROS generation. Collectively, these results demonstrate that CGRP exhibits its antioxidative effect by blocking the Src/STAT3 signalling pathway that is associated with Ang II‐induced VSMC hypertrophy and hyperplasia.

Adiponectin Reduces Angiotensin II‐Induced Vascular Hypertrophy by Targeting ROS Formation and Actin Cytoskeleton Remodeling

Background and aimsAdiponectin (APN), an adipocytokine, exerts cardioprotective effects, while angiotensin II (Ang II) plays a critical role in the pathogenesis of hypertrophy of vascular smooth muscle cells (VSMCs) induces hypertension and vascular remodeling. Ang II induces VSMC hypertrophy in part via activation of RhoA/ROCK pathway. In this study, we investigated the molecular mechanisms associated with Ang II‐induced RhoA activation and translocation and the ability of ANP to counteract Ang II‐hypertrophic effect through inhibition of RhoA activation, actin cytoskeleton remolding and reactive oxygen species (ROS) formation.MethodsStudies were carried out using rat aortic tissues and VSMCs. The effect of APN on Ang II‐induced RhoA translocation was assessed by Western blotting (WB) and Immunohistochemistry. Leucine incorporation was used to examine NO effect on Ang II induced hypertrophy. The phosphorylation of cofilin‐2 and ERK1/2 was detected by WB. Moreover, immunohistochemistry was performed on aortic frozen sections to detect ROS, F‐actin and G‐actin levels, RhoA‐Cav 1 co‐localization and eNOS translocation.ResultsAng II‐induced VSMCs hypertrophy was prevented by supplementing APN. The hypertrophic response to Ang II was associated with RhoA translocation to caveolae‐rich membrane, cofilin‐2 and ERK1/2 phosphorylation, in addition to a significant decrease in G/F actin ratio. Moreover, ROS production significantly increased with Ang II treatment as revealed by confocal microscopy. However, these effects were inhibited by APN and caveolae disruption. Taken together, these results suggest that Ang II hypertrophic effect is due, in part, to RhoA translocation to caveolae rich micro‐domains and its downstream effectors which is counteracted by APN.ConclusionThese findings suggest the ability of APN to attenuate Angiotensin II‐induced vascular hypertrophy.

Role of Histone Deacetylases 5 (HDAC5) in Ang II‐Induced the Early Growth Response Protein‐1 (Egr‐1) Expression and Vascular Hypertrophy

Angiotensin II (Ang II), a key vasoactive peptide, is known to play an important role in the pathophysiology of vascular diseases. A heightened activation of Ang II‐induced signaling pathways that promote proliferation, hypertrophy and migration of vascular smooth muscle cells (VSMCs) has been suggested to contribute to vascular dysfunctions. We have shown earlier that Ang II enhances the expression of early growth response protein‐1 (Egr‐1), a zinc transcription factor, which is upregulated in animal models of vascular diseases. Histone deacetylases (HDACs) remove acetyl groups of lysine residues from histone and non‐histone proteins and elevated activation of class IIa HDACs is reported in several vascular pathologies. Recent studies have demonstrated that the phosphorylation and the nuclear export of HDAC5, a member of class IIa HDACs, is associated with vascular remodeling through their ability to interact with transcription factors linked to cell proliferation, migration and hypertrophy. However, the involvement of HDAC5 in Ang II‐induced Egr‐1 expression remain unexplored. Here, we showed that the specific pharmacological inhibition of class IIa HDACs by MC1568 or TMP‐195 as well as siRNA‐mediated silencing of HDAC5 attenuated Ang II induced Egr‐1 expression in VSMCs. In addition, the blockade of the nuclear export of HDAC5 by using nuclear transport inhibitors leptomycin B or KPT‐330 decreased Ang II‐induced Egr‐1 expression. We also demonstrated that siRNA‐induced silencing of HDAC5 or Egr‐1 prevented VSMC hypertrophy in response to Ang II stimulation. In summary, our results show that Ang II‐induced Egr‐1 expression is regulated by the activation of HDAC5 and Ang II‐induced cell hypertrophy is mediated by HDAC5 and Egr‐1.Support or Funding InformationSupported by the Canadian Institutes of Health Research

Interleukin-17A induces vascular remodeling of small arteries and blood pressure elevation.

An important link exists between hypertension and inflammation. Hypertensive patients present elevated circulating levels of proinflammatory cytokines, including interleukin-17A (IL-17A). This cytokine participates in host defense, autoimmune and chronic inflammatory pathologies, and cardiovascular diseases, mainly through the regulation of proinflammatory factors. Emerging evidence also suggests that IL-17A could play a role in regulating blood pressure and end-organ damage. Here, our preclinical studies in a murine model of systemic IL-17A administration showed that increased levels of circulating IL-17A raised blood pressure induced inward remodeling of small mesenteric arteries (SMAs) and arterial stiffness. In IL-17A-infused mice, treatment with hydralazine and hydrochlorothiazide diminished blood pressure elevation, without modifying mechanical and structural properties of SMA, suggesting a direct vascular effect of IL-17A. The mechanisms of IL-17A seem to involve an induction of vascular smooth muscle cell (VSMC) hypertrophy and phenotype changes, in the absence of extracellular matrix (ECM) proteins accumulation. Accordingly, treatment with an IL-17A neutralizing antibody diminished SMA remodeling in a model of angiotensin II (Ang II) infusion. Moreover, in vitro studies in VSMCs reported here, provide further evidence of the direct effects of IL-17A on cell growth responses. Our experimental data suggest that IL-17A is a key mediator of vascular remodeling of the small arteries, which might contribute, at least in part, to blood pressure elevation.

Heteromerization fingerprints between bradykinin B2 and thromboxane TP receptors in native cells.

Bradykinin (BK) and thromboxane-A2 (TX-A2) are two vasoactive mediators that modulate vascular tone and inflammation via binding to their cognate “class A” G-protein coupled receptors (GPCRs), BK-B2 receptors (B2R) and TX-prostanoid receptors (TP), respectively. Both BK and TX-A2 lead to ERK1/2-mediated vascular smooth muscle cell (VSMC) proliferation and/or hypertrophy. While each of B2R and TP could form functional dimers with various GPCRs, the likelihood that B2R-TP heteromerization could contribute to their co-regulation has never been investigated. The main objective of this study was to investigate the mode of B2R and TP interaction in VSMC, and its possible impact on downstream signaling. Our findings revealed synergistically activated ERK1/2 following co-stimulation of rat VSMC with a subthreshold dose of BK and effective doses of the TP stable agonist, IBOP, possibly involving biased agonist signaling. Single detection of each of B2R and TP in VSMC, using in-situ proximity ligation assay (PLA), provided evidence of the constitutive expression of nuclear and extranuclear B2R and TP. Moreover, inspection of B2R-TP PLA signals in VSMC revealed agonist-modulated nuclear and extranuclear proximity between B2R and TP, whose quantification varied substantially following single versus dual agonist stimulations. B2R-TP interaction was further verified by the findings of co-immunoprecipitation (co-IP) analysis of VSMC lysates. To our knowledge, this is the first study that provides evidence supporting the existence of B2R-TP heteromerization fingerprints in primary cultured VSMC.

Vildagliptin Reduces Stenosis of Injured Carotid Artery in Diabetic Mouse Through Inhibiting Vascular Smooth Muscle Cell Proliferation via ER Stress/NF-κB Pathway.

Dipeptidyl peptidase-4 (DPP-4) inhibitors are novel anti-hyperglycemic drugs for type 2 diabetes. It has been reported that DDP-4 inhibitor could exert pleiotropic effects on cardiovascular system. This study was to explore the effect and mechanism of vildagliptin on the stenosis of injured carotid artery in diabetic mouse. Twenty six-week-old male db/db mice (BKS) were randomized into vildagliptin treated and vehicle control groups. Ligation injury was first performed in left carotid arteries of all diabetic mice, then oral vildagliptin or equal amount of PBS was correspondingly administered to the mice from the next day to ligation injury for 4 weeks. Effects on proliferation were detected via histological and morphometric analysis. Endoplasmic reticulum (ER) stress and nuclear factor kappa B (NF-κB) markers were determined by immunoblot analysis. After 4 weeks of vildagliptin delivery, it was observed that the intimal area and neointimal thickness of the ligated carotid arteries were significantly reduced as compared to the control group. In vivo, vildagliptin suppressed the expressions of PCNA and α-SMA, phospho-p65, phospho-IKKα/β, GRP78 and CHOP, as well as IRE-1 in vascular smooth muscle cells (VSMCs). In vitro, the proliferation and hypertrophy of VSMCs were significantly inhibited by blocking the IRE-1 pathway, and the inhibition of phospho-IRE-1 expression down-regulated the expression of phospho-IKKα/β in VSMCs. Vildagliptin reduced the stenosis of injured carotid arteries in diabetic mice, and this effect was achieved via inhibiting the activation of ER stress/NF-κB pathway.

Angiotensin II-Regulated Autophagy Is Required for Vascular Smooth Muscle Cell Hypertrophy

Hypertension is a disease associated to increased plasma levels of angiotensin II (Ang II). Ang II can regulate proliferation, migration, ROS production and hypertrophy of vascular smooth muscle cells (VSMCs). However, the mechanisms by which Ang II can affect VSMCs remain to be fully elucidated. In this context, autophagy, a process involved in self-digestion of proteins and organelles, has been described to regulate vascular remodeling. Therefore, we sought to investigate if Ang II regulates VSMC hypertrophy through an autophagy-dependent mechanism. To test this, we stimulated A7r5 cell line and primary rat aortic smooth muscle cells with Ang II 100 nM and measured autophagic markers at 24 h by Western blot. Autophagosomes were quantified by visualizing fluorescently labeled LC3 using confocal microscopy. The results showed that treatment with Ang II increases Beclin-1, Vps34, Atg-12–Atg5, Atg4 and Atg7 protein levels, Beclin-1 phosphorylation, as well as the number of autophagic vesicles, suggesting that this peptide induces autophagy by activating phagophore initiation and elongation. These findings were confirmed by the assessment of autophagic flux by co-administering Ang II together with chloroquine (30 μM). Pharmacological antagonism of the angiotensin type 1 receptor (AT1R) with losartan and RhoA/Rho Kinase inhibition prevented Ang II-induced autophagy. Moreover, Ang II-induced A7r5 hypertrophy, evaluated by α-SMA expression and cell size, was prevented upon autophagy inhibition. Taking together, our results suggest that the induction of autophagy by an AT1R/RhoA/Rho Kinase-dependent mechanism contributes to Ang II-induced hypertrophy in VSMC.

Ligand-activated PPARδ inhibits angiotensin II-stimulated hypertrophy of vascular smooth muscle cells by targeting ROS.

We investigated the effect of peroxisome proliferator-activated receptor δ (PPARδ) on angiotensin II (Ang II)-triggered hypertrophy of vascular smooth muscle cells (VSMCs). Activation of PPARδ by GW501516, a specific ligand of PPARδ, significantly inhibited Ang II-stimulated protein synthesis in a concentration-dependent manner, as determined by [3H]-leucine incorporation. GW501516-activated PPARδ also suppressed Ang II-induced generation of reactive oxygen species (ROS) in VSMCs. Transfection of small interfering RNA (siRNA) against PPARδ significantly reversed the effects of GW501516 on [3H]-leucine incorporation and ROS generation, indicating that PPARδ is involved in these effects. By contrast, these GW501516-mediated actions were potentiated in VSMCs transfected with siRNA against NADPH oxidase (NOX) 1 or 4, suggesting that ligand-activated PPARδ elicits these effects by modulating NOX-mediated ROS generation. The phosphatidylinositol 3-kinase inhibitor LY294002 also inhibited Ang II-stimulated [3H]-leucine incorporation and ROS generation by preventing membrane translocation of Rac1. These observations suggest that PPARδ is an endogenous modulator of Ang II-triggered hypertrophy of VSMCs, and is thus a potential target to treat vascular diseases associated with hypertrophic changes of VSMCs.

Abstract P243: Vascular Drp1 Mediates Angiotensin II-Induced Cardiovascular Hypertrophy via Global as Well as Selective de novo Protein Synthesis.

Mitochondrial fission has been implicated in various cardiovascular and metabolic diseases but the role of the mitochondrial fission inducer Drp1 in regulating hypertension and cardiovascular remodeling remains unclear. To study the involvement of Drp1 in cardiovascular hypertrophy, rat aortic vascular smooth muscle cells (VSMCs) are infected with dominant negative (dn) Drp1 adenovirus and stimulated with 100 nM angiotensin II (AngII). In vivo, tamoxiphen-inducible SMMHC-Cre+/- Drp1 floxed mice and control mice are infused with AngII (1000ng/kg/min) for 2 weeks. In VSMCs, AngII-induced mitochondrial fission was attenuated with AT1 blocker RNH6270 (ARB) as well as with dnDrp1 (100 moi). ARB and dnDrp1 treatment also inhibited AngII-enhanced global as well as specific de novo protein synthesis evaluated by SunSet assay. To identify potential mediators downstream of Drp1-mitochondrial fission contributing to VSMC hypertrophy, shotgun proteomic analysis was performed on proteins extracted from AngII or vehicle treated VSMC with dnDrp1 or control adenovirus infection. 22 exclusively regulated proteins induced by AngII and attenuated by dnDrp1 were identified including previously known AngII targets, HMGB1 and PAIRB. In addition, ARB and dnDrp1 inhibited AngII-induced enhancement of mitochondrial ox-phosphorylation and oxidative stress (assessed with mito-timer adenovirus). In vivo, VSMC specific DRP1 silencing suppressed AngII-induced cardiac hypertrophy assessed with heart weight/body weight ratio Mean±SEM: 6.33±0.14 vs 5.25±0.07 (p<0.001) as well as with echocardiogram: LVPWd 1.46±0.02 vs 0.81±0.03 (p<0.001). However, both control and the Drp1 silenced mice developed hypertension when infused with AngII as assessed by DSI radio-telemetry system. In conclusion, these data suggest that Drp1 mediates AngII-induced global as well as specific de novo protein synthesis thereby contributing to cardiovascular hypertrophic remodeling.

IGF-1 Deficiency Promotes Pathological Remodeling of Cerebral Arteries: A Potential Mechanism Contributing to the Pathogenesis of Intracerebral Hemorrhages in Aging.

Clinical and experimental studies show that age-related decline in circulating insulin-like growth factor-1 (IGF-1) levels promotes the pathogenesis of intracerebral hemorrhages, which critically contribute to the development of vascular cognitive impairment and disability in older adults. Yet, the mechanisms by which IGF-1 deficiency compromises structural integrity of the cerebral vasculature are not completely understood. To determine the role of IGF-1 deficiency in pathological remodeling of middle cerebral arteries (MCAs), we compared alterations in vascular mechanics, morphology, and remodeling-related gene expression profile in mice with liver-specific knockdown of IGF-1 (Igf1f/f + TBG-Cre-AAV8) and control mice with or without hypertension induced by angiotensin-II treatment. We found that IGF-1 deficiency resulted in thinning of the media and decreased wall-to-lumen ratio in MCAs. MCAs of control mice exhibited structural adaptation to hypertension, manifested as a significant increase in wall thickness, vascular smooth muscle cell (VSMC) hypertrophy, decreased internal diameter and up-regulation of extracellular matrix (ECM)-related genes. IGF-1 deficiency impaired hypertension-induced adaptive media hypertrophy and dysregulated ECM remodeling, decreasing elastin content and attenuating adaptive changes in ECM-related gene expression. Thus, circulating IGF-1 plays a critical role in maintenance of the structural integrity of cerebral arteries. Alterations of VSMC phenotype and pathological remodeling of the arterial wall associated with age-related IGF-1 deficiency have important translational relevance for the pathogenesis of intracerebral hemorrhages and vascular cognitive impairment in elderly hypertensive patients.