Medial Smooth Muscle Cells Research Articles

Abstract 104: EBI3 Deficiency Exacerbates Experimental Abdominal Aortic Aneurysms

Background: Abdominal aortic aneurysms (AAAs) are an age-and gender-related chronic vascular disease. Ebi3 (EB virus-induced gene 3) encodes a cytokine subunit protein shared by two anti-inflammatory cytokines IL-27 and IL-35. Given the importance of inflammation in AAA pathogenesis, we evaluated the influence of Ebi3 deficiency on experimental AAAs. Methods and Results: AAAs were created in10 week old male Ebi3-knoctout (KO) and wild type (WT) mice via the intra-aortic infusion of porcine pancreatic elastase (PPE), and evaluated using in vivo transabdominal ultrasonography and histology at sacrifice (Figure). Following PPE infusion, KO mice experienced more significant and rapid aneurysmal aortic enlargement. All KO and WT mice developed AAAs (≥50% diameter increase) within 7 and 14 days, respectively. Medial elastin and smooth muscle cell attenuation were more pronounced in aneurysmal aortae of KO mice, as were both aortic macrophage and T cell infiltration. However, no difference in aortic B cell infiltration or mural angiogenesis was noted between groups. Conclusions: Ebi3 deficiency promotes the onsets and progression of experimental AAAs in association with increased aortic accumulation of macrophages and T cells. Our study suggests a protective role of Ebi3 in experimental AAAs, potentially related to IL-27 and/or IL-35 production.

Direct visualisation of smooth muscle cell phenotypic plasticity and migration by long‐term imaging (867.12)

An abnormal increase in the number and distribution of SMCs is a key feature of the vascular remodelling that underlies cardiovascular disease. The predominant explanation for these changes is that medial SMCs undergo phenotypic modulation followed by migration to the intima and subsequent proliferation. However, there has been no direct demonstration of this dedifferentiation process by tracking the fate of contractile SMCs and the very existence of phenotypic plasticity has been questioned. We therefore employed high‐resolution, multi‐wavelength fluorescence and simultaneous phase contrast, time‐lapse microscopy to monitor the fate of unambiguously identified, freshly isolated, mature SMCs. Phenotypic modulation of the initially contractile SMCs was clearly observed in response to serum‐containing culture media. Extensive cellular remodelling occurred, though a consistent sequence of events was observed in which spindle‐shaped SMCs initially rounded up following exposure to media. The cells remained round for varying lengths of time (1‐3 days) before spreading outwards and, at this stage, repetitive contractions occurred (measured via bursts of high speed imaging). Finally, the cells flattened further and migration began. Surprisingly, after the onset of migration, the extrusion of subcellular structures (e.g. mitochondria) from one cell and their subsequent uptake by another cell was observed. The phenotypically modulated SMCs also formed direct connections with other cells (tunnelling nanotubes). These results highlight the complexity of the remodelling process, the highly dynamic cellular interactions involved and the significant cell‐cell variation in response and will enable a detailed picture of SMC remodelling to be built.Grant Funding Source: Supported by the Wellcome Trust (092292/Z/10/Z) and the British Heart Foundation (PG/11/70/29086)

PPARδ reduces abdominal aortic aneurysm formation in angiotensin II-infused apolipoprotein E-deficient mice by regulating extracellular matrix homeostasis and inflammatory responses

BackgroundAbdominal aortic aneurysm (AAA) is an inflammatory disorder characterized by a localized degradation of connective tissue and apoptosis of vascular smooth muscle cells. This study examined whether the ligand-activated peroxisome proliferator-activated receptor (PPAR) δ can directly antagonize angiotensin II (Ang II)-induced AAA formation in apoE-deficient mice. Methods and resultsSix-month-old male apoE-deficient mice were infused with Ang II and/or GW501516 (1.44 and 3.3mg/kg/day, respectively) via osmotic mini-pumps. At day 28, aortic size was measured and tissues were collected for analyses. Co-infusion of GW501516, an activator of PPARδ, attenuated both the incidence and the severity of Ang II-induced AAA in apoE-deficient mice. Ligand-activated PPARδ also reduced infiltration of macrophages, resulting in significant decreases in chemotactic proteins such as monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, and inducible nitric oxide synthase. The anti-inflammatory effect of GW501516 was associated with the suppression of apoptotic cell death, along with the inhibition of medial smooth muscle cell loss and focal elastin destruction, which leads to a medial dissection and aortic rupture. These ameliorative effects of GW501516 on Ang II-induced aneurysm were correlated with increased expression of extracellular matrix (ECM) proteins, such as types I and III collagen, fibronectin, and elastin, along with the up-regulation of transforming growth factor-β1. In addition, ligand-activated PPARδ also increased the expression of tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-3, while it strongly suppressed that of matrix metalloproteinase-2. ConclusionsPPARδ attenuates Ang II-induced AAA formation by regulating ECM homeostasis and inflammatory responses, suggesting a novel strategy for the treatment of AAA.

Recent Highlights of ATVB

Aortic diseases are common in many populations and are receiving increasing research focus. There are a broad spectrum of aortic diseases that occur in specific regions and appear to have different causes. For example, abdominal aortic aneurysms (AAAs) are most common in aged men.1 In contrast, many forms of thoracic aortic aneurysms (TAAs) occur early in life with a strong genetic basis and no sex discrimination.2 Both aortic aneurysms are amenable to surgical repair. Although surgical approaches have become increasingly sophisticated and less invasive,3 there remains an urgent need to determine factors that predispose to susceptibility and to divert treatment from surgical to medical approaches.4,5 This switch to medical treatment will require an increased knowledge of the mechanisms for several facets of aneurysms that cover the span of initiation, progression, and rupture. In this regard, many recent publications in ATVB have provided further insight into established pathways contributing to aneurysm development such as proteolysis, inflammation, and attenuation of the medial smooth muscle cell population, and a few publications have raised the possibility of new pathways such as adipokines and mineralocorticoid signaling. This article highlights these recent publications within a brief context of the literature. Cigarette smoking remains the major risk factor for development and progression of AAAs.6,7 Several experimental studies have demonstrated that smoke exposure augments AAA induced in mice by either subcutaneous angiotensin II (AngII) infusion or intra-aortic elastase perfusion.8,9 However, it is unclear whether cessation of smoking impacts the development of AAAs. The study of Jin et al10 demonstrated that cessation of cigarette smoking exposure did not immediately decrease the augmentation of AAAs. This sustained effect was attributable to regulation of leukocytic metabolism. Also of note is that cigarette smoking–induced augmentation of AAAs was unaffected …

Monocyte Chemoattractant Protein-1 (MCP-1) Regulates Macrophage Cytotoxicity in Abdominal Aortic Aneurysm

AimsIn abdominal aortic aneurysm (AAA), macrophages are detected in the proximity of aortic smooth muscle cells (SMCs). We have previously demonstrated in a murine model of AAA that apoptotic SMCs attract monocytes and other leukocytes by producing MCP-1. Here we tested whether infiltrating macrophages also directly contribute to SMC apoptosis.Methods and ResultsUsing a SMC/RAW264.7 macrophage co-culture system, we demonstrated that MCP-1-primed RAWs caused a significantly higher level of apoptosis in SMCs as compared to control macrophages. Next, we detected an enhanced Fas ligand (FasL) mRNA level and membrane FasL protein expression in MCP-1-primed RAWs. Neutralizing FasL blocked SMC apoptosis in the co-culture. In situ proximity ligation assay showed that SMCs exposed to primed macrophages contained higher levels of receptor interacting protein-1 (RIP1)/Caspase 8 containing cell death complexes. Silencing RIP1 conferred apoptosis resistance to SMCs. In the mouse elastase injury model of aneurysm, aneurysm induction increased the level of RIP1/Caspase 8 containing complexes in medial SMCs. Moreover, TUNEL-positive SMCs in aneurysmal tissues were frequently surrounded by CD68+/FasL+ macrophages. Conversely, elastase-treated arteries from MCP-1 knockout mice display a reduction of both macrophage infiltration and FasL expression, which was accompanied by diminished apoptosis of SMCs.ConclusionOur data suggest that MCP-1-primed macrophages are more cytotoxic. MCP-1 appears to modulate macrophage cytotoxicity by increasing the level of membrane bound FasL. Thus, we showed that MCP-1-primed macrophages kill SMCs through a FasL/Fas-Caspase8-RIP1 mediated mechanism.

ZIPK is critical for the motility and contractility of VSMCs through the regulation of nonmuscle myosin II isoforms

Migration of medial vascular smooth muscle cells (VSMCs) into the intimal layer contributes to pathological remodeling of the blood vessel in arterial hypertension and atherosclerosis. It is well established that reorganization of cytoskeletal networks is an essential component of cellular motile events. However, there is currently a lack of insight into the cellular characteristics of VSMC migration under three-dimensional environments. Here, we investigated the mechanisms of VSMC migration and remodeling using two different collagen matrix assays as in vitro models: migration of VSMCs within a collagen matrix for VSMC invasion and contraction of a collagen gel by VSMCs for VSMC remodeling and contraction. We found that nonmuscle myosin IIA (NMIIA) and nonmuscle myosin IIB (NMIIB) differentially contribute to the migratory capacity of VSMCs via NMII isoform-dependent cytoskeletal reorganization. Depletion of NMIIA by short hairpin RNA revealed a unique interplay between actomyosin and microtubules during VSMC migration. On the other hand, NMIIB was required for the structural maintenance of migrating VSMC. Interestingly, there was a significant difference between NMIIA and NMIIB knockdown in the VSMC migration but not in the VSMC-mediated collagen gel contraction. Furthermore, depletion of zipper-interacting protein kinase by short hairpin RNA resulted in an impairment of VSMC migration and a substantial decrease of VSMC-mediated collagen gel contraction. These results suggest that NMIIA and NMIIB uniquely control VSMC migration and may contribute to vascular remodeling, which are both regulated by zipper-interacting protein kinase.

Regulatory effect of AMP-activated protein kinase on pulmonary hypertension induced by chronic hypoxia in rats: in vivo and in vitro studies

Activation of AMP-activated protein kinase (AMPK) plays an important role in cardiovascular protection. It can inhibit arterial smooth muscle cell proliferation and cardiac fibroblast collagen synthesis induced by anoxia. However, the role of AMPK-dependent signalling cascades in the pulmonary vascular system is currently unknown. This study aims to determine the effects of AMPK on pulmonary hypertension and pulmonary vessel remodelling induced by hypoxia in rats using in vivo and in vitro studies. In vivo study: pulmonary hypertension, right ventricular hypertrophy and pulmonary vascular remodelling were found in hypoxic rats. Meanwhile, AMPKα1 and phosphorylated AMPKα1 were increased markedly in pulmonary arterioles and lung tissues. Mean pulmonary arterial pressure, index of right ventricular hypertrophy and parameters of pulmonary vascular remodelling, including vessel wall area/total area, density of nuclei in medial smooth muscle cells, and thickness of the medial smooth muscle cell layer were markedly suppressed by AICAR, an AMPK agonist. In vitro study: the expression of AMPKα1 and phosphorylated AMPKα1 was increased in pulmonary artery smooth muscle cells (PASMCs) under hypoxic conditions. The effects of PASMC proliferation stimulated by hypoxia were reinforced by treatment with Compound C, an AMPK inhibitor. AICAR inhibited the proliferation of PASMCs stimulated by hypoxia. These findings suggest that AMPK is involved in the formation of hypoxia-induced pulmonary hypertension and pulmonary vessel remodelling. Up-regulating AMPK can contribute to decreasing pulmonary vessel remodelling and pulmonary hypertension induced by hypoxia.

Contribution of Intimal Smooth Muscle Cells to Cholesterol Accumulation and Macrophage-Like Cells in Human Atherosclerosis

Intimal smooth muscle cells (SMCs) contribute to the foam cell population in arterial plaque, and express lower levels of the cholesterol exporter ATP-binding cassette transporter A1 (ABCA1) in comparison with medial arterial SMCs. The relative contribution of SMCs to the total foam cell population and their expression of ABCA1 in comparison with intimal monocyte-derived macrophages, however, are unknown. Although the expression of macrophage markers by SMCs following lipid loading has been described, the relevance of this phenotypic switch by SMCs in human coronary atherosclerosis has not been determined. Human coronary artery sections from hearts explanted at the time of transplantation were processed to clearly delineate intracellular and extracellular lipids and allow costaining for cell-specific markers. Costaining for oil red O and the SMC-specific marker SM α-actin of foam cell-rich lesions revealed that 50±7% (average±standard error of the mean, n=14 subjects) of total foam cells were SMC derived. ABCA1 expression by intimal SMCs was significantly reduced between early and advanced atherosclerotic lesions, with no loss in ABCA1 expression by myeloid lineage cells. Costaining with the macrophage marker CD68 and SM α-actin revealed that 40±6% (n=15) of CD68-positive cells originated as SMCs in advanced human coronary atherosclerosis. These findings suggest SMCs contain a much larger burden of the excess cholesterol in human coronary atherosclerosis than previously known, in part, because of their relative inability to release excess cholesterol via ABCA1 in comparison with myeloid lineage cells. Our results also indicate that many cells identified as monocyte-derived macrophages in human atherosclerosis are in fact SMC derived.

Delayed inhaled carbon monoxide mediates the regression of established neointimal lesions

Intimal hyperplasia (IH) contributes to the failure of vascular interventions. While many investigational therapies inhibit the development of IH in animal models, few of these potential therapies can reverse established lesions. Inhaled carbon monoxide (CO) dramatically inhibits IH in both rats and pigs when given perioperatively. It also prevented the development of pulmonary arterial hypertension in rodents. Interestingly, CO could reverse pulmonary artery structural changes and right heart hemodynamic changes when administered after the establishment of pulmonary hypertension. Thus, we hypothesize that inhaled CO may mediate the regression of established neointimal lesions. Rats underwent carotid artery balloon angioplasty injury. Carotid arteries were collected at 2 and 4 weeks after injury for morphometric analysis of the neointima. Another group was treated with inhaled CO (250 parts per million) for 1 hour daily from week 2 until week 4. Additional rats were sacrificed 3 days after initiating CO treatment, and the carotid arteries were examined for apoptosis by terminal deoxynucleotidyl transferase dUTP nick end-labeling, proliferation by Ki67 staining, and autophagy by microtubule-associated protein light chain 3 I/II staining. At 2 weeks following injury, sizable neointimal lesions had developed (intimal/media = 0.92 ± 0.22). By 4 weeks, lesion size remained stable (0.80 ± 0.09). Delayed inhaled CO treatment greatly reduced neointimal lesion size vs the 2- and 4-week control mice (0.38 ± 0.05; P < .05). Arteries from the CO-treated rats exhibited significantly reduced apoptosis compared with control vessels (3.18% ± 1.94% vs 16.26% ± 5.91%; P = .036). Proliferation was also dramatically reduced in the CO-treated animals (2.98 ± 1.55 vs 10.37 ± 2.80; P = .036). No difference in autophagy between control and CO-treated rats was detected. Delayed administration of inhaled CO reduced established neointimal lesion size. This effect was mediated by the antiproliferative effect of CO on medial and intimal smooth muscle cells without increases in arterial wall apoptosis or autophagy. Future studies will examine additional time points to determine if there is temporal variation in the rates of apoptosis and autophagy.

Previously differentiated medial vascular smooth muscle cells contribute to neointima formation following vascular injury.

BackgroundThe origins of neointimal smooth muscle cells that arise following vascular injury remains controversial. Studies have suggested that these cells may arise from previously differentiated medial vascular smooth muscle cells, resident stem cells or blood born progenitors. In the current study we examined the contribution of the previously differentiated vascular smooth muscle cells to the neointima that forms following carotid artery ligation.MethodsWe utilized transgenic mice harboring a cre recombinase-dependent reporter gene (mTmG). These mice express membrane targeted tandem dimer Tomato (mTomato) prior to cre-mediated excision and membrane targeted EGFP (mEGFP) following excision. The mTmG mice were crossed with transgenic mice expressing either smooth muscle myosin heavy chain (Myh11) or smooth muscle α-actin (Acta2) driven tamoxifen regulated cre recombinase. Following treatment of adult mice with tamoxifen these mice express mEGFP exclusively in differentiated smooth muscle cells. Subsequently vascular injury was induced in the mice by carotid artery ligation and the contribution of mEGFP positive cells to the neointima determined.ResultsAnalysis of the cellular composition of the neointima that forms following injury revealed that mEGFP positive cells derived from either Mhy11 or Acta2 tagged medial vascular smooth muscle cells contribute to the majority of neointima formation (79 ± 17% and 81 ± 12%, respectively).ConclusionThese data demonstrate that the majority of the neointima that forms following carotid ligation is derived from previously differentiated medial vascular smooth muscle cells.

Pathogenesis of focal cytoplasmic necrosis of the smooth muscle cells in hypertensive rat arterial media.

Hypertensive rat arteries exhibited severe medial smooth muscle cell injury and necrosis. Electron microscopic observations showed the smooth muscle cells of these arteries exhibited characteristics of focal cytoplasmic necrosis forming new cytodemarcating membrane between the healthy cytoplasm and necrotic cytoplasm. When the focal necrotic cytoplasm disappeared from the injured smooth muscle cells, it left it with a moth-eaten leaf-like appearance (moth-eaten necrosis). At an advanced stage of injury, smooth muscle cells changed to islet-like cell bodies with newly formed basement membranes around them, and further islet-like cell bodies and cell debris disappeared leaving lamellar and reticular basement membranes.In hypertensive rats injected with nitroblue tetrazolium (NBT), formazan deposits were observed in the medial cells and nitrotyrosine, a biomarker of peroxynitrite, were immunohistochemically observed in the arterial media. Nick-end positive extranuclear small granular bodies, which might have derived from focal necrotic cytoplasm and nucleus, were detected in the arterial media using DNA nick-end labeling method. Based on electron microscopical and histochemical findings, we conjectured that the focal cytoplasmic necrosis of the smooth muscle cells in the arterial media depended on injury arising from mitochondria-derived oxidants.

The small GTPases regulate HMC05-induced NQO-1 expression with an antioxidant effect in smooth muscle cells.

Recently, Banhabackchulchunmatang (HMC05) has been implicated as a preventive and/or therapeutic candidate for cardiovascular diseases due to its inhibition of atherosclerosis lesions and its reduction of neointima formation. Knowledge of the mechanism of HMC05 in smooth muscle cells (SMC) is limited. However, SMC may be a potential target for HMC05 therapy because they are supported by the HMC05-mediated preservation of medial smooth muscle cell layers in pathogenic progression. Therefore, in the present study, we hypothesized that the effect of HMC05 is associated with reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H):quinone oxidoreductase-1 (NQO-1) gene regulation, which precipitates an antioxidant effect in SMC. HMC05 significantly increased NQO-1 gene expression in a dose- and time-dependent manner. The reactive oxygen species-mediated toxicity that was generated by xanthine/xanthine oxidase was suppressed by HMC05. The knockdown of the NQO-1 gene abrogated the HMC05-mediated cytoprotection. Interestingly, pretreatment with a chemical inhibitor of geranylgeranyl transferase 1 or farnesyl transferase abolished the NQO-1 gene induction and cytoprotection by HMC05. The transfection of dominant negative RhoA or Ras suppressed HMC05-induced gene expression. Berberine and hesperidin, which are found in large quantities in HMC05, also induced NQO-1 gene expression. Taken together, this is the first study to demonstrate that HMC05 is efficacious in protection against oxidative stress through NOQ-1 gene induction via the regulation of RhoA and/or Ras, and that berberine and hesperidin are major components of NQO-1 gene induction. This study provides mechanistic targets of HMC05 in reducing atherosclerotic lesions in atherosclerosis.

Cathepsin K Activity Controls Injury-Related Vascular Repair in Mice

Cathepsin K (CatK) is one of the most potent mammalian collagenases. We showed previously the increased expression of CatK in human and animal atherosclerotic lesions. Here, we hypothesized that ablation of CatK mitigates injury-induced neointimal hyperplasia. Male wild-type (CatK(+/+)) and CatK-deficient (CatK(-/-)) mice underwent ligation or a combination of ligation and polyethylene cuff-replacement injuries to the right common carotid artery just proximal to its bifurcation, and they were then processed for morphological and biochemical studies at specific time points. On operative day 28, CatK(-/-) significantly reduced neointimal formation and neovessel formation in both single- and combination-injured arteries compared with the Cat K(+/+) mice. At early time points, CatK(-/-) reduced the lesion macrophage contents and medial smooth muscle cell proliferation, the mRNA levels of monocyte chemoattractant protein-1, toll-like receptor-2, toll-like receptor-4, chemokine ligand-12, and the gelatinolytic activity related to matrix metalloproteinase-2/-9. An aorta-explant assay revealed that smooth muscle cell movement was impaired in the CatK(-/-) mice compared with the CatK(+/+) mice. In addition, the smooth muscle cells and macrophages from CatK(-/-) mice had less invasive ability through a reconstituted basement membrane barrier. This vasculoprotective effect was mimicked by Cat inhibition with trans-epoxysuccinyl-L-leucylamido-{4-guanidino} butane (E64d). These results demonstrate an essential role of CatK in neointimal lesion formation in response to injury, possibly via the reduction of toll-like receptor-2/-4-mediated inflammation and smooth muscle cell proliferation, suggesting a novel therapeutic strategy for the control of endovascular treatment-related restenosis by regulating CatK activity.

The CCR5 chemokine receptor mediates vasoconstriction and stimulates intimal hyperplasia in human vessels in vitro

AimsThe chemokine receptor CCR5 and its inflammatory ligands have been linked to atherosclerosis, an accelerated form of which occurs in saphenous vein graft disease. We investigated the function of vascular smooth muscle CCR5 in human coronary artery and saphenous vein, vascular tissues susceptible to atherosclerosis, and vasospasm.Methods and resultsCCR5 ligands were vasoconstrictors in saphenous vein and coronary artery. In vein, constrictor responses to CCL4 were completely blocked by CCR5 antagonists, including maraviroc. CCR5 antagonists prevented the development of a neointima after 14 days in cultured saphenous vein. CCR5 and its ligands were expressed in normal and diseased coronary artery and saphenous vein and localized to medial and intimal smooth muscle, endothelial, and inflammatory cells. [125I]-CCL4 bound to venous smooth muscle with KD = 1.15 ± 0.26 nmol/L and density of 22 ± 9 fmol mg−1 protein.ConclusionsOur data support a potential role for CCR5 in vasoconstriction and neointimal formation in vitro and imply that CCR5 chemokines may contribute to vascular remodelling and augmented vascular tone in human coronary artery and vein graft disease. The repurposing of maraviroc for the treatment of cardiovascular disease warrants further investigation.

Lysophosphatidic Acid Induces Shear Stress–dependent Contraction in Mouse Aortic Strip In Situ

We previously reported that lysophosphatidic acid (LPA) regulates Ca²⁺ influx of fluid flow in stimulated endothelial cells and that LPA and shear stress showed increment and suppressive effects on phenylephrine-induced vasoconstriction and acetylcholine-induced vasodilatation, respectively. However, a vasoconstrictive effect of LPA alone in the presence of shear stress was not found. The present study examined the effect of LPA alone in the presence of shear stress on Ca²⁺ responses in endothelial and smooth muscle cells and contraction in mouse aortic strip using real-time 2-photon laser scanning microscopy and a custom-made parallel-plate flow chamber. Application of micromolar LPA and high shear stress elicited movement of endothelial cells after Ca²⁺ responses. The endothelial cells moved along the major axis of smooth muscle cells, a direction that was identical to that found during vasoconstriction evoked by the application of phenylephrine. The frequency of Ca²⁺ oscillations in smooth muscle cells was highest according to endothelial movement. Vasoconstriction evoked by LPA and shear stress was significantly reduced by the application of a thromboxane A₂ receptor antagonist, a cyclooxygenase inhibitor, and a thromboxane synthase inhibitor. These results suggest that micromolar LPA and high shear stress elicit vasoconstriction that is caused by Ca²⁺-dependent contraction in medial smooth muscle cells. Thromboxane A₂ may be involved in that response.

Inhibition of Phosphatidylinositol 3-kinase/Akt Signaling Attenuates Hypoxia-induced Pulmonary Artery Remodeling and Suppresses CREB Depletion in Arterial Smooth Muscle Cells

Hypoxia-induced pulmonary hypertension is characterized by progressive remodeling of the pulmonary artery (PA) system and loss of the transcription factor, cAMP response element binding protein (CREB) in PA smooth muscle cells (SMCs). Previous in vitro studies suggested that platelet-derived growth factor, a mitogen produced in the hypoxic arterial wall, elicits loss of CREB in medial SMCs via the PI3K/Akt pathway. These events trigger switching of SMCs from a quiescent, contractile phenotype to a proliferative, migratory, dedifferentiated, and synthetic phenotype, which contributes to PA thickening. Here, we investigated whether inhibition of PI3K or Akt could attenuate arterial remodeling in the lung and prevent CREB loss in PA medial SMCs in rats subjected to chronic hypoxia. Inhibition of either enzyme-blunted hypoxia-induced PA remodeling and SMC CREB depletion and diminished SMC proliferation and collagen deposition. Inhibition of Akt, but not PI3K, suppressed muscularization of distal arterioles and blunted right ventricular hypertrophy. Interestingly, mean PA pressure was elevated equally by hypoxia in untreated and inhibitor-treated groups but was normalized acutely by the Rho kinase inhibitor, Fasudil. We conclude that PI3K and Akt inhibitors can attenuate hypoxia-induced PA remodeling and SMC CREB depletion but fail to block the development of pulmonary hypertension because of their inability to repress Rho kinase-mediated vasoconstriction.

The 14q32 MicroRNA-487b Targets the Antiapoptotic Insulin Receptor Substrate 1 in Hypertension-Induced Remodeling of the Aorta

To study the role of microRNAs in hypertension-induced vascular pathology before the onset of symptoms of severe cardiovascular disease. MicroRNAs play a crucial role in cardiovascular disease. However, microRNAs are often studied in full-blown cardiovascular disease models, not during development of cardiovascular pathology. Angiotensin II was infused into healthy adult rats, inducing chronic hypertension, and microRNA expression profiles were obtained. The most prominently regulated microRNA, miR-487b, was further investigated, using primary cultures of rat aortic and human umbilical cord arterial cells. MiR-487b is predicted to target insulin receptor substrate 1 (IRS1). IRS1 plays an important role in both insulin signaling and cell proliferation and survival. IRS1 mRNA and protein levels were downregulated in aortae of hypertensive rats. MiR-487b binds directly to both rat and human IRS1 3'UTR and inhibits reporter gene expression in vitro. In primary rat and human arterial adventitial fibroblasts, inhibition of miR-487b leads to upregulation of IRS1 expression. Upregulation of miR-487b had the opposite effect, confirming direct targeting of IRS1 by miR-487b.Immunohistochemistry of aortic cross sections and rt/qPCR analyses of the separate aortic wall layers showed that both IRS1 and miR-487b were present mainly in the adventitia and less or not at all in the intima and tunica media. IRS1 expression in adventitial fibroblasts was predominantly nuclear and nuclear IRS1 is known to have antiapoptotic effects. Indeed, inhibition of miR-487b protected adventitial fibroblasts, and also medial smooth muscle cells, against serum starvation-induced apoptosis and increased cell survival. Angiotensin II-induced hypertension leads to upregulation of miR-487b, which targets IRS1. Via downregulation of IRS1, miR-487b can contribute to cell death and loss of adventitial and medial integrity during hypertension-induced vascular pathology.

A Deficiency of Herp, an Endoplasmic Reticulum Stress Protein, Suppresses Atherosclerosis in ApoE Knockout Mice by Attenuating Inflammatory Responses

Herp was originally identified as an endoplasmic reticulum (ER) stress protein in vascular endothelial cells. ER stress is induced in atherosclerotic lesions, but it is not known whether Herp plays any role in the development of atherosclerosis. To address this question, we generated Herp- and apolipoprotein E (apoE)-deficient mice (Herp−/−; apoE−/− mice) by crossbreeding Herp−/− mice and apoE−/− mice. Herp was expressed in the endothelial cells and medial smooth muscle cells of the aorta, as well as in a subset of macrophages in the atherosclerotic lesions in apoE−/− mice, while there was no expression of Herp in the Herp−/−; apoE−/− mice. The doubly deficient mice developed significantly fewer atherosclerotic lesions than the apoE−/− mice at 36 and 72 weeks of age, whereas the plasma levels of cholesterol and triglycerides were not significantly different between the strains. The plasma levels of non-esterified fatty acids were significantly lower in the Herp−/−; apoE−/− mice when they were eight and 16 weeks old. The gene expression levels of ER stress response proteins (GRP78 and CHOP) and inflammatory cytokines (IL-1β, IL-6, TNF-α and MCP-1) in the aorta were significantly lower in Herp−/−; apoE−/− mice than in apoE−/− mice, suggesting that Herp mediated ER stress-induced inflammation. In fact, peritoneal macrophages isolated from Herp-deficient mice and RAW264.7 macrophages in which Herp was eliminated with a siRNA expressed lower levels of mRNA for inflammatory cytokines when they were treated with tunicamycin. Herp deficiency affected the major mediators of the unfolded protein response, including IRE1 and PERK, but not ATF6. These findings suggest that a deficiency of Herp suppressed the development of atherosclerosis by attenuating the ER stress-induced inflammatory reactions.

MiR-663 and the miRaculous Vascular Smooth Muscle Phenotypic Switch

Cardiovascular diseases, such as ischemic heart disease and stroke, represent the number one cause of death worldwide.1 In most cases, atherosclerosis and hypertension constitute the underlying causes for cardiovascular disease because it leads to arterial occlusion and impaired cardiac function, respectively. Therapeutic compensation of atherosclerotic artery occlusion by bypass grafting, angioplasty, or stenting bears the risk of intimal hyperplasia and subsequent restenosis.2 In general, a prerequisite for any pathological and physiological vascular remodeling process is the phenotypic switch of vascular smooth muscle cells (VSMCs).3 Even in adult blood vessels, VSMCs retain a remarkable plasticity that is essential for any changes in the vessel wall architecture. Contractile VSMCs representing the majority of VSMCs in healthy vessels guarantee maintenance of vascular tone and thereby the systemic blood pressure and blood flow by controlling the blood vessel diameter. As outlined in the Figure, a plethora of humoral factors, such as platelet-derived growth factor-BB and biomechanical stimuli, especially chronic changes in blood flow and wall stress, is capable to trigger the phenotypic switch of VSMCs from a quiescent, contractile differentiated state to a synthetic, proliferative, and dedifferentiated state.3,4 Figure. The effect of microRNA (miR)-633 on vascular homeostasis and remodeling. Major stimuli, transcription factors, and miRs that are involved in the differentiation and phenotypic switch of vascular smooth muscle cells (VSMCs) are depicted. miR-143/miR-145 promotes the contractile state by inhibiting suppressors of myocardin/serum response factor (SRF) activity (eg, ETS domain-containing protein-1 [Elk-1] and Kruppel-like factor [KLF] 4/5). Interestingly, myocardin activity seems to be regulated by an intrinsic loop because it stabilizes the expression of miR-145, whereas miR-143 seems to inhibit myocardin expression. miR-633 is a novel player with a dual effect on the VSMC phenotype not only by attenuating proliferation, migration, and proteolytic activity, but also by limiting their …

Regulation of Rho GTPases by N-Cadherin Cell-Cell Contact Formation in Vascular Smooth Muscle Cells

The directional migration of medial smooth muscle cells (SMC) into the vessel intima in vascular diseases such as atherosclerosis leads to stenosis and occlusion of the arterial lumen. An important initiating factor is the loss of direct SMC contacts with the overlying endothelial cells and with adjacent SMCs. We have shown that N-cadherin, a calcium-dependent cell-cell adhesion molecule, becomes asymmetrically distributed around the cell periphery when SMCs lose contact with endothelial cells, and that restoration of symmetric N-cadherin distribution may arrest migration.