Induced Smooth Muscle Cells Proliferation Research Articles

Fibulin-1 promotes intimal hyperplasia after venous stent implantation through ACE mediated angiotensin II signaling

ObjectiveStent intimal hyperplasia leads to in stent restenosis and thrombosis. This study determined whether Fibulin-1 activity in smooth muscle cells (SMCs) contributes to stent restenosis or thrombosis. MethodsStent implantation was conducted in a pig model. Target vessel samples were stained and analyzed by protein mass spectrometry. Cell experiments and Fibulin-1 SMC specific knockout mice (Fbln1SMKO) were used to investigate the mechanism of Fibulin-1 induced SMC proliferation and thrombosis. ResultsSMC proliferation and phenotypic transition are the main pathological changes of intimal hyperplasia in venous stents. Protein mass spectrometry analysis revealed a total of 67 upregulated proteins and 39 downregulated proteins in intimal hyperplasia after stent implantation compared with normal iliac vein tissues. Among them, Fibulin-1 ranked among the top proteins altered. Fibulin-1 overexpressing human SMCs (Fibulin-1-hSMCs) showed increased migration and phenotypic switching from contractile to secretory type and Fibulin-1 inhibition decreased the activity of SMCs. Mechanistically, Fibulin-1-hSMCs displayed increased levels of angiotensin converting enzyme (ACE) expression and angiotensin II signaling. Inhibition of ACE or angiotensin II signaling alleviated the migration of Fibulin-1-hSMCs. Using Fibulin-1 SMC specific knockout mice (Fbln1SMKO) and venous thrombosis model, we demonstrated that Fibulin-1 deletion attenuated intimal SMCs proliferation and thrombosis. Further, Fibulin-1 concentration was high in iliac vein compression syndrome (IVCS) patients treated with stent and was an independent predictor of venous insufficiency. ConclusionsFibulin-1 promotes SMC proliferation partially through ACE secretion and angiotensin II signaling after stent implantation. Fibulin-1 plays a role in venous insufficiency syndrome, implicating the protein in the detection and treatment of IVCS.

Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation

Aims: Chronic intermittent hypoxia (IH), a characteristic feature of obstructive sleep apnea (OSA), contributes to cardiovascular diseases, including atherosclerosis, potentially through persistent oxidative stress and inflammation. TRAF3IP2 (TRAF3 Interacting Protein 2) is an oxidative stress-responsive proinflammatory adapter molecule and plays a causal role in a preclinical model of atherosclerosis. Since SGLT2 (Sodium/Glucose Cotransporter 2) inhibitors have shown protective effects in CVD by inhibiting oxidative stress and inflammation, we hypothesized that IH promotes the crosstalk between oxidative stress and TRAF3IP2, resulting in IL-6-dependent human aortic smooth muscle cell (SMC) proliferation, and that these effects are inhibited by the SGLT2 inhibitor empagliflozin. Materials and methods: Primary human aortic SMC were exposed to various cycles of IH. Normoxia served as a control. To understand the molecular mechanisms underlying IH-induced nitroxidative stress, TRAF3IP2 and IL-6 induction, and SMC proliferation and those targeted by empagliflozin were determined by treating SMC with various pharmacological inhibitors and viral vectors. Results: IH upregulated TRAF3IP2 expression, TRAF3IP2-dependent superoxide, hydrogen peroxide and nitric oxide generation, NF-kB and HIF-1a activation, IL-6 induction, and SMC proliferation. Exposure to IL-6 by itself induced SMC proliferation in part via TRAF3IP2, IL-6R, gp130, JAK, and STAT3. Further, SMC express SGLT2 at basal conditions, and is upregulated by both IH and IL-6. Importantly, empagliflozin inhibited IH-induced TRAF3IP2 upregulation, reactive oxygen and nitrogen species generation, TRAF3IP2-dependent HIF-1a and NF-kB activation, IL-6 induction, and IL-6-dependent JAK-STAT3-mediated SMC proliferation. Moreover, empagliflozin inhibited IL-6-induced STAT3-dependent SMC proliferation. Conclusions: These results suggest the therapeutic potential of empagliflozin in IH and inflammatory vascular proliferative diseases associated with OSA.

CircSOD2: A Novel Regulator for Smooth Muscle Proliferation and Neointima Formation.

Vascular smooth muscle cell (SMC) proliferation contributes to neointima formation following vascular injury. Circular RNA-a novel type of noncoding RNA with closed-loop structure-exhibits cell- and tissue-specific expression patterns. However, the role of circular RNA in SMC proliferation and neointima formation is largely unknown. The objective of this study is to investigate the role and mechanism of circSOD2 in SMC proliferation and neointima formation. Approach and Results: Circular RNA profiling of human aortic SMCs revealed that PDGF (platelet-derived growth factor)-BB up- and downregulated numerous circular RNAs. Among them, circSOD2, derived from back-splicing event of SOD2 (superoxide dismutase 2), was significantly enriched. Knockdown of circSOD2 by short hairpin RNA blocked PDGF-BB-induced SMC proliferation. Inversely, circSOD2 ectopic expression promoted SMC proliferation. Mechanistically, circSOD2 acted as a sponge for miR-206, leading to upregulation of NOTCH3 (notch receptor 3) and NOTCH3 signaling, which regulates cyclin D1 and CDK (cyclin-dependent kinase) 4/6. In vivo studies showed that circSOD2 was induced in neointima SMCs in balloon-injured rat carotid arteries. Importantly, knockdown of circSOD2 attenuated injury-induced neointima formation along with decreased neointimal SMC proliferation. CircSOD2 is a novel regulator mediating SMC proliferation and neointima formation following vascular injury. Therefore, circSOD2 could be a potential therapeutic target for inhibiting the development of proliferative vascular diseases.

Abstract 777: Dual-Specificity Phosphatase 6 Deficiency Protects Against Intimal Hyperplasia After Arterial Injury

Dysregulation of MAPK activation cascades has been implicated in various diseases. Dual-specificity phosphatases (DUSPs) are a subfamily of protein tyrosine phosphatases that dephosphorylate MAPKs and modulate critical signaling pathways. Studies have shown that DUSP6 plays an important role in the heart and diet-induced obesity. However, the role of DUSP6 in vascular disease is largely unclear. Our goal is to investigate whether DUSP6 has a role in occlusive vascular disease. To this end, we used a neointima formation model by denuding endothelium of mouse femoral arteries with a guide wire. We showed that DUSP6 was expressed at very low levels in the normal uninjured mouse arteries; however, DUSP6 expression was increased in the arterial wall of injured blood vessels, suggesting a potential role of DUSP6 in vascular disease. Compared with wild-type mice, DUSP6 deficiency reduced neointima formation 4 weeks after arterial injury in mice. BrdU incorporation assays revealed that lack of DUSP6 resulted in decreased smooth muscle cell (SMC) proliferation in the neointima and media 2 weeks after injury, indicating DUSP6 may promote SMC proliferation in response to injury. Indeed, overexpressing DUSP6 in primary SMCs increased cellular proliferation, similar to that exerted by inflammatory cytokine IL-1β while knocking down DUSP6 expression with siRNA abrogated IL-1β-induced SMC proliferation. Pathway analysis showed that IL-1β-induced ERK1/2 activation preceded DUSP6 induction in SMCs. Intriguingly, ERK1/2 inhibitor U0126 abolished IL-1β-mediated ERK1/2 activation, DUSP6 induction, and SMC proliferation. Taken together, our results indicate that DUSP6 induction promotes vascular remodeling under pathological conditions. DUSP6 might be a therapeutic target for preventing/treating occlusive vascular disease.

Abstract 507: Dedicator of Cytokinesis 2 Regulates Smooth Muscle Cell Proliferation via p38 Mitogen-Activated Protein Kinase Signaling Pathway

Smooth muscle cell (SMC) proliferation is an important process during vascular development and involved in vascular remodeling of a number of cardiovascular diseases including atherosclerosis, hypertension, and restenosis after angioplasty. However, the underlying mechanisms are not completely understood. In the present study, we identified dedicator of cytokinesis 2 (DOCK2) as one of the factors mediating the SMC proliferation. Knockout of DOCK2 (DOCK2-/-) caused abnormal blood vessel development in yolk sac and hemorrhage in mouse embryos. In addition, dorsal aorta wall structure was disrupted in DOCK2-/- embryos, and the SMC numbers in the dorsal aorta were decreased by 49% compared with wild type (WT). Consistently, proliferating cell nuclear antigen (PCNA)-positive cells in DOCK2-/- dorsal aorta media were much less the WT. These data suggest that the abnormal vascular development in DOCK2-/- embryos was due to the defective SMC proliferation. In vitro, platelet-derived growth factor (PDGF)-BB and serum upregulated DOCK2 along with PCNA expression in SMCs. Knockout or knockdown of DOCK2 by its shRNA inhibited the PDGF-BB-induced PCNA expression while forced expression of DOCK2 induced PCNA expression in SMCs. 5-ethynyl-2'-deoxyuridine (EdU) assay showed that knockdown of DOCK2 blocked PDGF-BB-induced SMC proliferation. Mechanistically, DOCK2 induced SMC proliferation by activating p38 mitogen-activated protein kinase (MAPK). Overexpression of DOCK2 induced while knockdown of DOCK2 inhibited p38 phosphorylation. Blockade of p38 MAPK signaling by its pathway-specific inhibitor diminished DOCK2-induced SMC proliferation. In a rat carotid artery balloon-injury model, knockdown of DOCK2 by adenoviral delivery of its shRNA blocked the injury-induced neointima formation and attenuated the PCNA expression. Furthermore, deletion of DOCK2 blocked ligation-induced neointima formation and PCNA expression in mouse arteries. Taken together, our studies demonstrated that DOCK2 is an important factor regulating SMC proliferation during embryo development as well as vascular remodeling in pathological conditions.

Reduced Expression of Glutathione S-Transferase α 4 Promotes Vascular Neointimal Hyperplasia in CKD

Neointima formation is the leading cause of arteriovenous fistula (AVF) failure. We have shown that CKD accelerates this process by transforming the vascular smooth muscle cells (SMCs) lining the AVF from a contractile to the synthetic phenotype. However, the underlying mechanisms affecting this transformation are not clear. Previous studies have shown that the α-class glutathione transferase isozymes have an important role in regulating 4-hydroxynonenal (4-HNE)-mediated proliferative signaling of cells. Here, using both the loss- and gain-of-function approaches, we investigated the role of glutathione S-transferase α4 (GSTA4) in modulating cellular 4-HNE levels for the transformation and proliferation of SMCs. Compared with non-CKD controls, mice with CKD had downregulated expression of GSTA4 at the mRNA and protein levels, with concomitant increase in 4-HNE in arteries and veins. This effect was associated with upregulated phosphorylation of MAPK signaling pathway proteins in proliferating SMCs. Overexpressing GSTA4 blocked 4-HNE-induced SMC proliferation. Additionally, inhibitors of MAPK signaling inhibited the 4-HNE-induced responses. Compared with wild-type mice, mice lacking GSTA4 exhibited increased CKD-induced neointima formation in AVF. Transient expression of an activated form of GSTA4, achieved using a combined Tet-On/Cre induction system in mice, lowered levels of 4-HNE and reduced the proliferation of SMCs. Together, these results demonstrate the critical role of GSTA4 in blocking CKD-induced neointima formation and AVF failure.

Platelet extracellular vesicles induce a pro-inflammatory smooth muscle cell phenotype

ABSTRACTExtracellular vesicles (EVs) are mediators of cell communication during health and disease, and abundantly released by platelets upon activation or during ageing. Platelet EVs exert modulatory effects on immune and vascular cells. Platelet EVs may modulate the function of vascular smooth muscle cells (SMC). Platelet EVs were isolated from platelet-rich plasma and incubated with SMC in order to assess binding, proliferation, migration and pro-inflammatory phenotype of the cells. Platelet EVs firmly bound to resting SMC through the platelet integrin αIIbβ3, while binding also occurred in a CX3CL1–CX3CR1-dependent manner after cytokine stimulation. Platelet EVs increased SMC migration comparable to platelet derived growth factor or platelet factor 4 and induced SMC proliferation, which relied on CD40- and P-selectin interactions. Flow-resistant monocyte adhesion to platelet EV-treated SMC was increased compared with resting SMC. Again, this adhesion depended on integrin αIIbβ3 and P-selectin, and to a lesser extent on CD40 and CX3CR1. Treatment of SMC with platelet EVs induced interleukin 6 secretion. Finally, platelet EVs induced a synthetic SMC morphology and decreased calponin expression. Collectively, these data indicate that platelet EVs exert a strong immunomodulatory activity on SMC. In particular, platelet EVs induce a switch towards a pro-inflammatory phenotype, stimulating vascular remodelling.

Eukaryotic elongation factor 2 kinase controls proliferation and migration of vascular smooth muscle cells.

Eukaryotic elongation factor 2 kinase (eEF2K), also known as calmodulin (CaM)-dependent protein kinase (CaMK) III, is a unique member of CaMK family protein. We have recently found that expression of eEF2K protein increased in mesenteric artery from spontaneously hypertensive rats. As pathogenesis of hypertension is in part regulated by vascular structural remodelling via proliferation and migration of vascular smooth muscle cells (SMCs), we tested the hypothesis that eEF2K controls SMCs proliferation and migration. In rat mesenteric arterial SMCs, an eEF2K inhibitor, A-484954 (10 μm), significantly inhibited platelet-derived growth factor (PDGF)-BB (10 ng mL(-1) )-induced SMCs proliferation as determined by a cell counting and bromodeoxyuridine incorporation assay. PDGF-BB (10 ng mL(-1) )-induced SMCs migration was significantly inhibited by A-484954 (10 μm) as determined by a Boyden chamber assay. A-484954 (10 μm) significantly inhibited PDGF-BB (10 ng mL(-1) )-induced phosphorylation of eEF2K, extracellular signal-regulated kinase (ERK), Akt, p38 and heat-shock protein (HSP) 27 as determined by Western blotting. It was confirmed that a CaM inhibitor, W-7 (50 μm), inhibited PDGF-BB (10 ng mL(-1) )-induced phosphorylation of eEF2K. In an ex vivo mesenteric arterial ring assay, 10% foetal bovine serum-induced SMCs outgrowth was significantly inhibited by A-484954 (10 μm). We for the first time revealed that eEF2K mediates PDGF-BB-induced SMCs proliferation and migration through activating ERK, Akt, p38 and HSP27 signals in a CaM-dependent manner. Our results suggest eEF2K as a novel pharmaceutical target for the prevention of hypertensive cardiovascular diseases.

Abstract 97: High mobility group box-1 Promotes Restenosis Via Toll like receptor-4 Signal Pathway

Background: High-mobility group box 1 (HMGB1) is an endogenous molecule released during cell stress and death termed damage-associated molecular patterns (DAMPs). HMGB1 activates the pattern recognition receptor, toll-like receptor 4 (TLR4), and induces sterile inflammation. However, how HMGB1 and TLR4 affect restenosis, the major complication following balloon and stent intervention clinically, remains unknown. We tested the hypothesis that HMGB1 released following acute arterial injury promotes intimal hyperplasia (IH), a hallmark of restenosis, via TLR4 signaling pathway. Methods and Results: Wire injury of the carotid artery in C57BL/6 wild-type (WT) mice significantly increased intima-to-media ratio in 4 weeks. Global deletion of HMGB1 using an inducible knockout mouse strain prevented IH and vessel remodeling. IH decreased by over 50% in WT mice treated with a HMGB1 neutralizing antibody. Of the mouse strains deficient in putative receptors and co-regulator for HMGB1 (TLR4-/-, TLR2-/-, RAGE-/- and CD14-/-), TLR4-/- mice showed the greatest inhibition of IH after injury. Both TLR4 adaptors MYD88 and TRIF synergistically participated in the inflammatory response to vascular injury. HMGB1 antibody-treated mice and TLR4-/- mice showed a marked decrease in monocytic recruitment following injury. Mice with selective depletion of TLR4 from macrophages (TLR4-/--Mø) exhibited similar level of IH inhibition and macrophage infiltration, compared to the global TLR4-/- mice. In vitro, disulfide HMGB1 concentration-dependently promoted smooth muscle cell (SMC) migration and MCP-1/CCR2 expression, which were abolished by treatment with TLR4 inhibitory peptide. Moreover, conditioned media from HMGB1-treated macrophage induced SMC proliferation, which was blunted by blocking TLR4 on macrophage, but not SMCs. Finally, HMGB1 increased cytokine (TNF-α and IL-6), chemokine (MCP-1) and mitogen (PDGF-A) levels in macrophage in a TLR4-dependent manner. Conclusion: These findings demonstrate, for the first time, that HMGB1 released following acute arterial injury promotes restenosis via SMC migration and MCP-1/CCR2 production as well as macrophage-released TNF-α, IL-6, MCP-1 and PDGF-A in SMC through the TLR4-MyD88/TRIF signal cascade.

Abstract 467: PCSK6 Is Upregulated in Vascular Diseases Characterized by Inflammation and Smooth Muscle Cell Proliferation

Recently we demonstrated upregulation of the proprotein convertase PCSK6 in a large cohort of human carotid atherosclerotic plaques (n=127) compared to normal arteries and in symptomatic vs. asymptomatic lesions. PCSK6 was localized to smooth muscle cells (SMCs) in the fibrous cap and showed a positive correlation to markers of inflammation, extracellular matrix remodeling and cytokines. Here, we aimed at elucidating the role of this protease in vascular disease by examining its expression in different human pathologies and in animal models. Increased expression of PCSK6 in vascular diseases was validated in public microarray datasets and other available human cohorts. PCSK6 was upregulated in carotid atherosclerotic plaques vs. controls (n=32 patients, p<0.0001), as well as in abdominal aortic aneurysm (AAA) vs. normal tissue (n=14, p<0.0001) and in thoracic aortic aneurysm (TAA) tissue from bicuspid vs. tricuspid patients (n=244, p=0.012). By eQTL analyses, several SNPs in the PCSK6 genomic region were shown to influence its expression in carotid plaques and TAA tissue. Among these, rs6598465 showed a mild association to progression of maximum intima-media thickness in the left and right arteries in a separate cohort of high-risk coronary artery disease subjects (n=3400, p=0.037). By immunohistochemistry, PCSK6 localized mainly to SMCs in carotid plaques, AAA and TAA tissue, but was also found to be expressed by CD68 and CD163+ macrophages. Investigation of mouse, rat and human artery tissues with pronounced intimal hyperplasia revealed strong expression of PCSK6 in proliferating SMCs. In rat carotid artery balloon injury, PCSK6 was downregulated in the early phases after injury mostly defined by inflammatory response, while upregulated in later phases with prominent SMC activation, and consistently localized in SMCs. Expression of PCSK6 in this model was strongly positively correlated solely to PDGFB and IGF1 (p<0.0001), cytokines known to induce SMC proliferation. We established a functional link between elevated expression of PCSK6 and vascular diseases characterized by inflammation and SMC proliferation. Further investigations in vitro are necessary to provide mechanistic insight into the role of this protease in vascular disease.

Histone Deacetylase 4 Controls Neointimal Hyperplasia via Stimulating Proliferation and Migration of Vascular Smooth Muscle Cells

Histone deacetylases (HDACs) are transcriptional coregulators. Recently, we demonstrated that HDAC4, one of class IIa family members, promotes reactive oxygen species-dependent vascular smooth muscle inflammation and mediates development of hypertension in spontaneously hypertensive rats. Pathogenesis of hypertension is, in part, modulated by vascular structural remodeling via proliferation and migration of vascular smooth muscle cells (SMCs). Thus, we examined whether HDAC4 controls SMC proliferation and migration. In rat mesenteric arterial SMCs, small interfering RNA against HDAC4 inhibited platelet-derived growth factor (PDGF)-BB-induced SMC proliferation as determined by a cell counting and bromodeoxyuridine incorporation assay as well as migration as determined by Boyden chamber assay. Expression and activity of HDAC4 were increased by PDGF-BB. HDAC4 small interfering RNA inhibited phosphorylation of p38 mitogen-activated protein kinase and heat shock protein 27 and expression of cyclin D1 as measured by Western blotting. HDAC4 small interfering RNA also inhibited PDGF-BB-induced reactive oxygen species production as measured fluorometrically using 2', 7'-dichlorofluorescein diacetate and nicotinamide adenine dinucleotide phosphate oxidase activity as measured by lucigenin assay. A Ca(2+)/calmodulin-dependent protein kinase II inhibitor, KN93, inhibited PDGF-BB-induced SMC proliferation and migration as well as phosphorylation of HDAC4. In vivo, a class IIa HDACs inhibitor, MC1568 prevented neointimal hyperplasia in mice carotid ligation model. MC1568 also prevented increased activation of HDAC4 in the neointimal lesions. The present results for the first time demonstrate that HDAC4 controls PDGF-BB-induced SMC proliferation and migration through activation of p38 mitogen-activated protein kinase/heat shock protein 27 signals via reactive oxygen species generation in a Ca(2+)/calmodulin-dependent protein kinase-dependent manner, which may lead to the neointimal hyperplasia in vivo.

Role of class IA PI 3-kinase isoforms in neointima formation following balloon angioplasty: only p110alpha is important

Growth factor induced proliferation and migration of vascular smooth muscle cells (SMCs) are essential for neointima formation following balloon angioplasty. Growth factors such as platelet-derived growth factor (PDGF) activate receptor tyrosine kinases (RTKs). RTK-induced responses are largely mediated by activation of class IA phosphatidylinositol 3'-kinase (PI3K) isoforms. Class IA PI3Ks comprise one of three different catalytic isoforms (p110alpha, p110beta, p110delta). Here, we sought to clarify, which p110 subunits contribute to neointima formation following balloon injury. Western blot analysis of lysates from cultured human, mouse and rat SMCs revealed that p110alpha, p110beta, and p110delta are expressed in these cells. However, co-immunoprecipitation experiments using an antibody against the PDGF beta receptor indicate that only p110alpha and p110delta bind to activated RTKs. p110beta signals downstream of G-protein coupled receptors (GPCR) as p110beta inhibition by TGX-221 (1μM) completely abrogated LPA (GPCR agonist) induced AKT activation downstream of PI3K. TGX-221 had no influence on RTK induced AKT activation. Inhibition (PIK75, 500 nM) or siRNA mediated knockdown of p110alpha completely abrogated PDGF mediated proliferation (BrdU incorporation assay) and migration (modified Boyden-chamber) of SMCs. p110beta (TGX-221, 3μM) and p110delta (IC87114, 3μM) inhibition had no influence. However, knockdown of p110delta using a specific siRNA significantly reduced PDGF induced SMC proliferation without effecting SMC migration. As expected, siRNA against p110beta was noneffective. By means of SMC-specific p110alpha deficient mice and p110delta knockout mice, we analysed in vivo the relevance of p110alpha and p110delta for restenosis formation following balloon angioplasty. The extent of neointima formation was quantified 4 weeks following balloon angioplasty of carotid arteries from wild-type (WT) and p110alpha ko as well as from WT and p110delta ko mice (n=7, respectively). Whereas p110alpha deficiency largely suppressed neointima formation (p<0.05), p110delta deficiency was noneffective. In conclusion, our results indicate that the p110alpha subunit of PI3K, is crucial for growth factor-mediated proliferation and migration of SMCs in restonosis following balloon angioplasty. Therefore, p110alpha represents a promising therapeutic target.

Sonic hedgehog promotes autophagy of vascular smooth muscle cells

Sonic hedgehog (Shh) is a morphogen critically involved in development that is reexpressed in atherosclerotic lesions. It also stimulates proliferation of vascular smooth muscle cells (SMCs). Autophagy in vascular SMCs is known to promote SMC survival and increase plaque stability. The aim of this study was to investigate whether Shh induces autophagy of vascular SMCs. Our study showed that both Shh protein and microtubule-associated protein 1 light chain 3 (LC3)-II were increased in SMCs within neointimal lesions of mouse common carotid arteries. In cultured mouse aortic SMCs, recombinant mouse Shh stimulated LC3-II levels. Overexpression of wild-type mouse Shh through the tetracycline-regulated expression-inducible system in human aortic SMCs time-dependently increased the levels of LC3-II and also stimulated protein kinase B (AKT) phosphorylation. Pretreatment with AKT inhibitor IV (AKTI IV) inhibited AKT phosphorylation and the increase in LC3-II. Shh-induced autophagy was further confirmed by the formation of autophagosomes as detected by immunostaining and transmission electron microscopy, which was inhibited by AKTI IV. Shh further increased SMC LC3-II in the presence of bafilomycin A1, (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester, and pepstatin A or siRNA for the autophagy-related gene 7 (ATG7). In addition, Shh induced SMC proliferation, which was inhibited not only by AKTI IV but also by cyclopamine, an inhibitor of Shh receptor. Inhibition of autophagy with 3-methyladenine (3-MA), bafilomycin A1, or ATG7 siRNA resulted in inhibition of cell proliferation. Treatment with 3-MA, AKTI IV, or cyclopamine inhibited neointima formation in mouse common carotid arteries. Taken together, our results have shown that Shh induces autophagy of vascular SMCs involving AKT activation, suggesting a role of autophagy in Shh-induced cellular responses.

Hypertension impairs myocardin function: a novel mechanism facilitating arterial remodelling

Hypertension evokes detrimental changes in the arterial vessel wall that facilitate stiffening and thus lead to a further rise in mean blood pressure, eventually causing heart failure. The underlying pathophysiological remodelling process is elicited by an increase in wall stress (WS) and is strictly dependent on the activation of vascular smooth muscle cells (SMC). However, it remains unclear as to why these cells fail to maintain their contractile and quiescent phenotype in a hypertensive environment. In this context, we reveal that the knockdown of myocardin--a pivotal transcriptional determinant of the contractile SMC phenotype--is sufficient to induce SMC proliferation. In line with this observation, immunofluorescence analysis of the media of remodelling arteries from hypertensive mice demonstrated a significant decrease in the abundance of myocardin and an increase in SMC proliferation. Subsequent analyses of isolated perfused mouse arteries and human cultured SMCs exposed to cyclic stretch (i.e. mimicking one component of WS) suggested that this biomechanical force facilitates serine phosphorylation of myocardin. Furthermore, this biomechanical stimulus promotes rapid translocation of myocardin from the nucleus to the cytoplasm, inhibits its mRNA expression, and causes proteasomal degradation of the cytoplasmic protein. Collectively, these findings suggest that hypertension negates the activity of myocardin in SMCs on multiple levels, hence eliminating a crucial determinant of SMC quiescence. This mechanism may control the initial switch from the contractile towards the synthetic SMC phenotype during hypertension and may offer an interesting novel approach to prevent cardiovascular disease.

The effects of PPARγ agonist rosiglitazone on neointimal hyperplasia in rabbit carotid anastomosis model

BackgroundNeointimal hyperplasia involving smooth muscle cell (SMC) proliferation, migration and extracellular matrix (ECM) degradation is an important component of atherosclerosis. It develops as a response to vascular injury after balloon angioplasty and vascular graft placement. Matrix metalloproteinases (MMPs) induce SMC proliferation, migration and contribute to intimal hyperplasia by degrading ECM. PPARγ agonists inhibit SMC proliferation, migration and lesion formation. In this study, we aimed to investigate the effects of PPARγ agonist rosiglitazone on neointimal hyperplasia and gelatinase (MMP-2 and MMP-9) expressions in rabbit carotid anastomosis model.MethodsNew Zealand white rabbits (n = 13, 2.7–3.2 kg) were divided into placebo and treatment groups. Right carotid artery (CA) was transected and both ends were anastomosed. Treatment group (n = 6) received rosiglitazone (3 mg/kg/day/p.o.) and placebo group (n = 7) received PBS (phosphate buffered saline, 2.5 ml/kg/day/p.o.) for 4 weeks postoperatively. After the sacrification, right and left CAs were isolated. Morphometric analyses and immunohistochemical examinations for gelatinases were performed.ResultsIntimal area (0.055 ± 0.005 control vs 0.291 ± 0.020 μm2 anastomosed, p < 0,05) and index (0.117 ± 0.002 control vs 0.574 ± 0.013 anastomosed, p < 0,01) significantly increased in anastomosed arteries compared to control arteries from placebo group. However, in rosiglitazone-treated group, intimal area (0.291 ± 0.020 PBS vs 0.143 ± 0.027 rosiglitazone, p < 0,05) and index (0.574 ± 0.013 PBS vs 0.263 ± 0.0078 rosiglitazone, p < 0,01) significantly decreased. Furthermore, gelatinase immunopositivity was found to have significantly increased in anastomosed arteries from placebo group and decreased with rosiglitazone treatment.ConclusionsThese results suggest that rosiglitazone may prevent neointimal hyperplasia, which is the most important factor involved in late graft failure, by inhibiting gelatinase enzyme expression.

Fibronectin glycation increases IGF-I induced proliferation of human aortic smooth muscle cells

The advanced glycation end products, namely AGEs, contribute to long-termed complications of diabetes mellitus, including macroangiopathy, where smooth muscle cells (SMC) proliferation stimulated by platelet-derived growth factor (PDGF) isoforms and insulin-like growth factor-I (IGF-I) plays an important role. The objective of the present study was to investigate the effect of an AGE-modified extracellular matrix protein on IGF-I induced SMC proliferation and on the IGF-I-IGF binding protein 4 (IGFBP-4) axis under basal conditions and after stimulation with PDGF-BB. IGF-I resulted in significantly higher thymidine incorporation in SMC seeded on AGE-modified fibronectin (AGE-FN) in comparison to cells seeded on fibronectin (FN). This augmented proliferation could not be accounted for by increased expression of IGF-IR, by decreased secretion of IGFBP-4, a binding protein that inhibits IGF-I mitogenic effects or by increased IGF-IR autophosphorylation. PDGF-BB did not modulate IGF-IR and IGFBP-4 mRNA expression in any of the substrata, however, this growth factor elicited opposite effects on the IGFBP-4 content in the conditioned media, increasing it in cells plated on FN and diminishing it in cells plated on AGE-FN. These findings suggest that one mechanism by which AGE-modified proteins is involved in the pathogenesis of diabetes-associated atherosclerosis might be by increasing SMC susceptibility to IGF-I mitogenic effects.

Effects of Fluid Shear Stress on a Distinct Population of Vascular Smooth Muscle Cells

Vascular smooth muscle cells (SMCs) are a major cell type involved in vascular remodeling. The various developmental origins of SMCs such as neural crest and mesoderm result in heterogeneity of SMCs, which plays an important role in the development of vascular remodeling and diseases. Upon vascular injury, SMCs are exposed to blood flow and subjected to fluid shear stress. Previous studies have shown that fluid shear stress inhibits SMC proliferation. However, the effect of shear stress on the subpopulation of SMCs from specific developmental origin and vascular bed is not well understood. Here we investigated how shear stress regulates human aortic SMCs positive for neural crest markers. DNA microarray analysis showed that shear stress modulates the expression of genes involved in cell proliferation, matrix synthesis, cell signaling, transcription and cytoskeleton organization. Further studies demonstrated that shear stress induced SMC proliferation and cyclin D1, downregulated cell cycle inhibitor p21, and activated Akt pathway. Inhibition of PI-3 kinase blocked these shear stress-induced changes. These results suggest that SMCs with neural crest characteristics may respond to shear stress in a different manner. This finding has significant implications in the remodeling and diseases of blood vessels.

Reduction of reactive oxygen species prevents hypoxia-induced CREB depletion in pulmonary artery smooth muscle cells.

Hypoxia-induced pulmonary arterial hypertension (PAH) is a deadly disease characterized by progressive remodeling and persistent vasoconstriction of the pulmonary arterial system. Remodeling of the pulmonary artery (PA) involves smooth muscle cell (SMC) proliferation, hypertrophy, migration, and elevated extracellular matrix (ECM) production elicited by mitogens and oxidants produced in response to hypoxic insult. We previously reported that the transcription factor cAMP response element binding protein (CREB) is depleted in medial PA SMCs in remodeled, hypertensive vessels in rats or calves exposed to chronic hypoxia. In culture, CREB loss can be induced in PA SMCs by exogenous oxidants or platelet-derived growth factor. Forced depletion of CREB with small interfering RNA (siRNA) in PA SMCs is sufficient to induce their proliferation, hypertrophy, migration, dedifferentiation, and ECM production. This suggests that oxidant and/or mitogen-induced loss of CREB in medial SMCs is, in part, responsible for PA thickening. Here, we tested whether oxidant scavengers could prevent the loss of CREB in PA SMCs and inhibit SMC proliferation, migration, and ECM production using in vitro and in vivo models. Exposure of PA SMCs to hypoxia induced hydrogen peroxide (H2O2) production and loss of CREB. Treatment of SMCs with exogenous H2O2 or a second oxidant, Sin-1, elicited CREB depletion under normoxic conditions. Exogenous H2O2 also induced SMC proliferation, migration, and increased elastin levels as did forced depletion of CREB. In vivo, hypoxia-induced thickening of the PA wall was suppressed by the superoxide dismutase mimetic, Tempol, which also prevented the loss of CREB in medial SMCs. Tempol also reduced hypoxia-induced SMC proliferation and elastin deposition in the PA. The data indicate that CREB levels in the arterial wall are regulated in part by oxidants produced in response to hypoxia and that CREB plays a crucial role in regulating SMC phenotype and PA remodeling.

Requirement for p38 Mitogen-Activated Protein Kinase Activity in Neointima Formation After Vascular Injury

Angioplasty and stent delivery are performed to treat atherosclerotic vascular disease but often cause deleterious neointimal lesion formation. Previously, growth factor receptor-bound protein 2 (Grb2), an intracellular linker protein, was shown to be essential for neointima formation and for p38 mitogen-activated protein kinase (MAPK) activation in vascular smooth muscle cells (SMCs). In this study, the role of vascular SMC p38alpha MAPK in neointimal development was examined. Compound transgenic mice were generated with doxycycline-inducible SMC-specific expression of dominant-negative p38alpha MAPK (DN-p38alpha). Doxycycline treatment resulted in the expression of DN-p38alpha mRNA and protein in transgenic arteries. Doxycycline-treated compound transgenic mice were resistant to neointima formation 21 days after carotid injury and showed reduced arterial p38 MAPK activation. To explore the mechanism by which p38alpha MAPK promotes neointima formation, an in vitro SMC culture system was used. Inhibition of p38alpha MAPK in cultured SMCs by treatment with SB202190 or small interfering RNA blocked platelet-derived growth factor-induced SMC proliferation, DNA replication, phosphorylation of the retinoblastoma protein, and induction of minichromosome maintenance protein 6. SMC p38alpha MAPK activation is required for neointima formation, perhaps because of its ability to promote retinoblastoma protein phosphorylation and minichromosome maintenance protein 6 expression.

Cysteinyl-Leukotrienes in Cerebrovascular Disease

Although the initial interest in the field of leukotriene (LT) research was focused on their potent bronchoconstrictive effects, subsequent studies have suggested LTs as key mediators in several inflammatory diseases. Genetic variations within the enzymes transforming arachidonic acid into LTs (Figure) have for example been associated with atherosclerosis and an increased risk of cardiovascular events.1 Figure Metabolism of arachidonic acid into leukotrienes (LTs) by 5-lipoxygenase (5-LO), 5-LO activating protein (FLAP), LTA4 hydrolase (LTA4H), and LTC4 synthase (LTC4S). Cysteinyl-LTs (LTC4, LTD4, LTE4) activate CysLT receptors, which can be targeted by antiasthmatic CysLT1 receptor antagonists. Inset shows the 5 exons (I-V) of the LTC4S gene and its 5′-flanking untranscribed region (UTR). The 2 reported single nucleotide polymorphisms (SNP) are located at 444 and 1072 bp upstream, respectively. The −444 A to C substitution has been associated with increased LTC4 synthesis in eosinophils,6 aspirin intolerant asthma,5 increased risk for coronary calcium,10 increased carotid artery intima media thickness (IMT),10 and a decreased risk of ischemic cerebrovascular events.4 See accompanying article on page 990 Leukotriene C4 synthase (LTC4S; Figure) is a microsomal glutathione-S-transferase (mGST), which conjugates LTA4 with glutathione.2 The product, LTC4, shares its following enzymatic steps with glutathione to …