Synthetic Vascular Smooth Muscle Cells Research Articles

AMP-Activated Protein Kinase Alpha 2 Deletion Induces VSMC Phenotypic Switching and Reduces Features of Atherosclerotic Plaque Stability.

AMP-activated protein kinase (AMPK) has been reported to play a protective role in atherosclerosis. However, whether AMPKα2 controls atherosclerotic plaque stability remains unknown. The aim of this study was to evaluate the impact of AMPKα2 deletion on atherosclerotic plaque stability in advanced atherosclerosis at the brachiocephalic arteries and to elucidate the underlying mechanisms. Features of atherosclerotic plaque stability and the markers for contractile or synthetic vascular smooth muscle cell (VSMC) phenotypes were monitored in the brachiocephalic arteries from Apoe(-/-)AMPKα2(-/-) mice or VSMC-specific AMPKα2(-/-) mice in an Apoe(-/-) background (Apoe(-/-)AMPKα2(sm-/-)) fed Western diet for 10 weeks. We identified that Apoe(-/-)AMPKα2(-/-) mice and Apoe(-/-)AMPKα2(sm-/-) mice exhibited similar unstable plaque features, aggravated VSMC phenotypic switching, and significant upregulation of Kruppel-like factor 4 (KLF4) in the plaques located in the brachiocephalic arteries compared with those found in Apoe(-/-) and Apoe(-/-)AMPKα2(sm+/+) control mice. Pravastatin, an AMPK activator, suppressed VSMC phenotypic switching and alleviated features of atherosclerotic plaque instability in Apoe(-/-)AMPKα2(sm+/+) mice, but not in Apoe(-/-)AMPKα2(sm-/-) mice. VSMC isolated from AMPKα2(-/-) mice displayed a significant reduction of contractile proteins(smooth muscle actin-α, calponin, and SM-MHC [smooth muscle-mysion heavy chain]) in parallel with increased detection of synthetic proteins (vimentin and osteopontin) and KLF4, as observed in vivo. KLF4-specific siRNA abolished AMPKα2 deletion-induced VSMC phenotypic switching. Furthermore, pharmacological or genetic inhibition of nuclear factor-κB significantly decreased KLF4 upregulation in VSMC from AMPKα2(-/-) mice. Finally, we found that AMPKα2 deletion markedly promoted the binding of nuclear factor-κBp65 to KLF4 promoter. This study demonstrated that AMPKα2 deletion induces VSMC phenotypic switching and promotes features of atherosclerotic plaque instability in nuclear factor-κB-KLF4-dependent manner.

Intrinsic Deregulation of Vascular Smooth Muscle and Myofibroblast Differentiation in Mesenchymal Stromal Cells from Patients with Systemic Sclerosis.

IntroductionObliterative vasculopathy and fibrosis are hallmarks of systemic sclerosis (SSc), a severe systemic autoimmune disease. Bone marrow-derived mesenchymal stromal cells (MSCs) from SSc patients may harbor disease-specific abnormalities. We hypothesized disturbed vascular smooth muscle cell (VSMC) differentiation with increased propensity towards myofibroblast differentiation in response to SSc-microenvironment defining growth factors and determined responsible mechanisms.MethodsWe studied responses of multipotent MSCs from SSc-patients (SSc-MSCs) and healthy controls (H-MSCs) to long-term exposure to CTGF, b-FGF, PDGF-BB or TGF-β1. Differentiation towards VSMC and myofibroblast lineages was analyzed on phenotypic, biochemical, and functional levels. Intracellular signaling studies included analysis of TGF-β receptor regulation, SMAD, AKT, ERK1/2 and autocrine loops.ResultsVSMC differentiation towards both, contractile and synthetic VSMC phenotypes in response to CTGF and b-FGF was disturbed in SSc-MSCs. H-MSCs and SSc-MSCs responded equally to PDGF-BB with prototypic fibroblastic differentiation. TGF-β1 initiated myofibroblast differentiation in both cell types, yet with striking phenotypic and functional differences: In relation to H-MSC-derived myofibroblasts induced by TGF-β1, those obtained from SSc-MSCs expressed more contractile proteins, migrated towards TGF-β1, had low proliferative capacity, and secreted higher amounts of collagen paralleled by reduced MMP expression. Higher levels of TGF-β receptor 1 and enhanced canonical and noncanonical TGF-β signaling in SSc-MSCs accompanied aberrant differentiation response of SSc-MSCs in comparison to H-MSCs.ConclusionsDeregulated VSMC differentiation with a shift towards myofibroblast differentiation expands the concept of disturbed endogenous regenerative capacity of MSCs from SSc patients. Disease related intrinsic hyperresponsiveness to TGF-β1 with increased collagen production may represent one responsible mechanism. Better understanding of repair barriers and harnessing beneficial differentiation processes in MSCs could widen options of autologous MSC application in SSc patients.

Vascular Calcification in Uremia: New-Age Concepts about an Old-Age Problem.

A hallmark of aging, and major contributor to the increased prevalence of cardiovascular disease in patients with chronic kidney disease (CKD), is the progressive structural and functional deterioration of the arteries and concomitant accrual of mineral. Vascular calcification (VC) was long viewed as a degenerative age-related pathology that resulted from the passive deposition of mineral in the extracellular matrix; however, since the discovery of "bone-related" protein expression in calcified atherosclerotic plaques over 20 years ago, a plethora of studies have evoked the now widely accepted view that VC is a highly regulated and principally cell-mediated phenomenon that recapitulates many features of physiologic ossification. Central to this theory are changes in vascular smooth muscle cell (VSMC) phenotype and viability, thought to be driven by chronic exposure to a number of dystrophic stimuli characteristics of the uremic state. Here, dedifferentiated synthetic VSMCs are seen to spawn calcifying matrix vesicles that actively seed mineralization of the arterial matrix. This review provides an overview of the major epidemiological, histological, and molecular aspects of VC in the context of CKD, and a counterpoint to the prevailing paradigm that emphasizes the primacy of VSMC-mediated mechanisms. Particular focus is given to the import of protein and small molecule inhibitors in regulating physiologic and pathological mineralization and the emerging role of mineral nanoparticles and their interplay with proinflammatory processes.

Cyclic nucleotide phosphodiesterase 1 and vascular aging.

VSMCs (vascular smooth muscle cells) play critical roles in arterial remodelling with aging, hypertension and atherosclerosis. VSMCs exist in diverse phenotypes and exhibit phenotypic plasticity, e.g. changing from a quiescent/contractile phenotype to an active myofibroblast-like, often called ‘synthetic’, phenotype. Synthetic VSMCs are able to proliferate, migrate and secrete ECM (extracellular matrix) proteinases and ECM proteins. In addition, they produce pro-inflammatory molecules, providing an inflammatory microenvironment for leucocyte penetration, accumulation and activation. The aging VSMCs have also shown changes in cellular phenotype, responsiveness to contracting and relaxing mediators, replicating potential, matrix synthesis, inflammatory mediators and intracellular signalling. VSMC dysfunction plays a key role in age-associated vascular remodelling. Cyclic nucleotide PDEs (phosphodiesterases), by catalysing cyclic nucleotide hydrolysis, play a critical role in regulating the amplitude, duration and compartmentalization of cyclic nucleotide signalling. Abnormal alterations of PDEs and subsequent changes in cyclic nucleotide homoeostasis have been implicated in a number of different diseases. In the study published in the latest issue of Clinical Science, Bautista Niño and colleagues have shown that, in cultured senescent human VSMCs, PDE1A and PDE1C mRNA levels are significantly up-regulated and inhibition of PDE1 activity with vinpocetine reduced cellular senescent makers in senescent VSMCs. Moreover, in the premature aging mice with genomic instability (Ercc1d/−), impaired aortic ring relaxation in response to SNP (sodium nitroprusside), an NO (nitric oxide) donor, was also largely improved by vinpocetine. More interestingly, using data from human GWAS (genome-wide association studies), it has been found that PDE1A single nucleotide polymorphisms is significantly associated with diastolic blood pressure and carotid intima–media thickening, two hallmarks of human vascular dysfunction in aging. These findings establish a strong relationship between PDE1 expression regulation and vascular abnormalities in aging.

Inhibitory role of reactive oxygen species in the differentiation of multipotent vascular stem cells into vascular smooth muscle cells in rats: a novel aspect of traditional culture of rat aortic smooth muscle cells.

Proliferative or synthetic vascular smooth muscle cells (VSMCs) are widely accepted to be mainly derived from the dedifferentiation or phenotypic modulation of mature contractile VSMCs, i.e., a phenotype switch from a normally quiescent and contractile type into a proliferative or synthetic form. However, this theory has been challenged by recent evidence that synthetic VSMCs predominantly originate instead from media-derived multipotent vascular stem cells (MVSCs). To test these hypotheses further, we re-examine whether the conventional rat aortic SMC (RASMC) culture involves the VSMC differentiation of MVSCs or the dedifferentiation of mature VSMCs and the potential mechanism for controlling the synthetic phenotype of RASMCs. We enzymatically isolated RASMCs and cultured the cells in both a regular growth medium (RGM) and a stem cell growth medium (SCGM). Regardless of culture conditions, only a small portion of freshly isolated RASMCs attaches, survives and grows slowly during the first 7days of primary culture, while expressing both SMC- and MVSC-specific markers. RGM-cultured cells undergo a process of synthetic SMC differentiation, whereas SCGM-cultured cells can be differentiated into not only synthetic SMCs but also other somatic cells. Notably, compared with the RGM-cultured differentiated RASMCs, the SCGM-cultured undifferentiated cells exhibit the phenotype of MVSCs and generate greater amounts of reactive oxygen species (ROS) that act as a negative regulator of differentiation into synthetic VSMCs. Knockdown of phospholipase A2, group 7 (Pla2g7) suppresses ROS formation in the MVSCs while enhancing SMC differentiation of MVSCs. These results suggest that cultured synthetic VSMCs can be derived from the SMC differentiation of MVSCs with ROS as a negative regulator.

Abstract 250: Oxidative Stress Mediated Increase of HMGB-1 and RAGE cCntributes to Ascending Aortic Aneurysm Development in vivo.

Background: Ascending-Thoracic Aortic Aneurysm (TAA) is a life-threatening condition for which the only treatment available is the surgical replacement/repair of the dilated aorta. It is known that oxidative stress (ROS) plays an important role in aortic wall remodeling involved in TAA: ROS induces pathological changes in vascular smooth muscle cells (VSMC) and triggers a disorganized and excessive production of matrix components and mediators of elastolysis. We demonstrated that chronic infusion of Angiotensin II (AngII) induces TAA formation in mice with concomitant accumulation of both ROS and synthetic VSMC, similar to that seen in human aneurysmal tissues. We also demonstrated that two ROS triggered molecules, the Receptor for Advanced Glycation end product (RAGE) and its ligand HMGB-1/amphoterin, are highly expressed in dysfunctional human aortic tissue. Recently a few microRNAs (miRs) have been proposed to play a role in aneurysm formation. We found that miR-143 is significantly down-regulated in human TAA tissues. We hypothesized that miR-143 may play a role in TAA development by regulating RAGE/HMGB-1 expression. Methods: RAGE and HMGB-1 expression was tested in control and aneurysmal human ascending aorta. C57BL6/J mice were fed a hypercholesterolemic diet and infused with saline or Ang II (1000 ng/kg/min) for 28 days. MiR-143, HMGB-1 and RAGE expression was evaluated by Western blotting and qPCR in murine ascending aorta samples and in human aortic VSMC treated with AngII (100nM) for 4 and 24 hrs. Results: Human aneurysmal aorta shows down-regulation of miR-143 concomitant with up-regulation HMGB-1 and RAGE expression. AngII infusion induces TAA in mice together with up-regulation of HMGB-1 and RAGE and miR-143 down-regulation. AngII treatment of VSMC induces RAGE and HMGB-1 expression and down-regulation of miR-143. A miR target analysis, using bioinformatic miR-mRNA algorithm tools, identified HMGB-1 as a direct target of miR-143. Conclusion: Our data suggest that miR-143, RAGE and HMGB-1 may cooperate in the process of aortic remodeling behind TAA formation. Further studies, investigating the interplay between miR-143 and HMGB-1/RAGE activation, may lead to potential new pharamacological targets for the treatment of TAA.

New endoplasmic reticulum stress regulator, Gipie, regulates the survival of vascular smooth muscle cells and the neointima formation after vascular injury.

The accumulation of unfolded protein in the endoplasmic reticulum (ER) initiates an adaptive stress response, termed the unfolded protein response. Previous studies suggested that ER stress might be involved in the formation of neointima after vascular injury. We recently discovered a novel regulator of ER stress, 78-kDa glucose-regulated protein-interacting protein induced by ER stress (Gipie). The objective of this study was to elucidate the role of Gipie using models of vascular disease. We investigated the functions of Gipie in cultured vascular smooth muscle cells (VSMCs) and in a vascular injury model of a rat carotid artery. The expression of Gipie was predominantly detected in synthetic VSMCs and to a much lesser extent in contractile VSMCs, which was augmented by treatment with thapsigargin. Gipie knockdown increased the phosphorylation levels of c-Jun N-terminal kinase and the number of apoptotic cells under ER stress. Moreover, Gipie knockdown decreased the mature form of collagen I in synthetic VSMCs. The expression of Gipie was rarely detected in the medial VSMCs of the intact carotid artery, whereas it was detected in most of the neointimal cells and some of the medial VSMCs after balloon injury. Depletion of Gipie in the rat carotid artery attenuated the neointimal thickening, which was accompanied by increased cell death in the neointima. Conversely, overexpression of Gipie augmented the neointimal thickening. Gipie participates in the ER stress response in VSMCs and plays an important role in neointima formation after vascular injury.

Akt1 isoform modulates phenotypic conversion of vascular smooth muscle cells

In this study, we investigated the role of Akt1 isoform in phenotypic change of vascular smooth muscle cells (VSMCs) and neointima formation. Laminin-induced conversion of synthetic VSMCs into contractile VSMCs was measured by expression of marker proteins for contractile VSMCs and collagen gel contraction assay. Culture of synthetic VSMCs on laminin-coated plates induced expression of marker proteins for contractile VSMCs and showed contraction in response to angiotensin II (AngII) stimulation. Silencing integrin-linked kinase attenuated activation of Akt and blocked phenotypic conversion of VSMCs resulting in the loss of AngII-dependent contraction. Laminin-induced phenotypic conversion of VSMCs was abrogated by phosphatidylinositol 3-kinase inhibitor or in cells silencing Akt1 but not Akt2. Proliferation of contractile VSMCs on laminin-coated plate was enhanced in cells silencing Akt1 whereas silencing Akt2 did not affect. Promoter activity of myocardin and SM22α was enhanced in contractile phenotype and overexpression of myocardin stimulated promoter activity of SM22α in synthetic phenotype. Promoter activity of myocardin and SM22α was reduced in cells silencing Akt1 and promoter activity of SM22α was restored by overexpression of myocardin in cells silencing Akt1. However, silencing of Akt2 affected neither promoter activity of myocardin nor SM22α. Finally, neointima formation in carotid artery ligation and high fat-diet-induced atherosclerosis was facilitated in mice lacking Akt1. This study demonstrates that Akt1 isoform stimulates laminin-induced phenotypic conversion of synthetic VSMCs by regulating the expression of myocardin. VSMCs become susceptible to shifting from contractile to synthetic phenotype by the loss of Akt1 in pathological conditions.

Expression of sarco (endo) plasmic reticulum calcium ATPase (SERCA) system in normal mouse cardiovascular tissues, heart failure and atherosclerosis

The sarco(endo)plasmic reticulum Ca2+ATPases (SERCA) system, a key regulator of calcium cycling and signaling, is composed of several isoforms. We aimed to characterize the expression of SERCA isoforms in mouse cardiovascular tissues and their modulation in cardiovascular pathologies (heart failure and/or atherosclerosis).Five isoforms (SERCA2a, 2b, 3a, 3b and 3c) were detected in the mouse heart and thoracic aorta. Absolute mRNA quantification revealed SERCA2a as the dominant isoform in the heart (~99%). Both SERCA2 isoforms co-localized in cardiomyocytes (CM) longitudinal sarcoplasmic reticulum (SR), SERCA3b was located at the junctional SR. In the aorta, SERCA2a accounted for ~91% of total SERCA and SERCA2b for ~5%. Among SERCA3, SERCA3b was the most expressed (~3.3%), mainly found in vascular smooth muscle cells (VSMC), along with SERCA2a and 2b.In failing CM, SERCA2a was down-regulated by 2-fold and re-localized from longitudinal to junctional SR. A strong down-regulation of SERCA2a was also observed in atherosclerotic vessels containing mainly synthetic VSMCs. The proportion of both SERCA2b and SERCA3b increased to 9.5% and 8.3%, respectively.In conclusion: 1) SERCA2a is the major isoform in both cardiac and vascular myocytes; 2) the expression of SERCA2a mRNA is ~30 fold higher in the heart compared to vascular tissues; and 3) nearly half the amount of SERCA2a mRNA is measured in both failing cardiomyocytes and synthetic VSMCs compared to healthy tissues, with a relocation of SERCA2a in failing cardiomyocytes. Thus, SERCA2a is the principal regulator of excitation–contraction coupling in both CMs and contractile VSMCs.

MiR‐21 normalizes vascular smooth muscle proliferation and improves coronary collateral growth in metabolic syndrome

Inadequate cell proliferation is considered a major causative factor for impaired coronary collateral growth (CCG). Proangiogenic growth factors (GFs) stimulate cell proliferation, but their administration does not promote CCG in patients. These GFs are increased in patients with metabolic syndrome and in animal models, where CCG is impaired. Here, we investigated whether excessive cell proliferation underlies impaired CCG in metabolic syndrome. Normal [Sprague-Dawley (SD)] and metabolic syndrome [James C. Russell (JCR)] rats underwent repetitive ischemia (RI; transient, repetitive coronary artery occlusion and myocardial ischemia). We have shown that CCG was maximal at d 9 of RI in SD rats but did not occur in JCR rats. The increase in cell proliferation (PCNA, Ki-67, cyclin A, phospho- cdc2, p21Waf, p27Kip) was transient (∼4-fold, d 3 RI) in SD rats but greater and sustained in JCR rats (∼8- to 6-fold, d 3-9 RI). In JCR rats, this was associated with increased and sustained miR-21 expression and accumulation of proliferating synthetic vascular smooth muscle cells in the lumen of small arterioles, which failed to undergo outward expansion. Administration of anti-miR-21 blocked RI-induced cell proliferation and significantly improved CCG in JCR rats (∼60%). miR-21-dependent excessive cell proliferation in the later stages of collateral remodeling correlates with impaired CCG in metabolic syndrome.

Abstract 165: cAMP-Phosphodiesterase 1C Regulates Neointimal Hyperplasia by Controlling Platelet-Derived Growth Factor Receptor ß Degradation via the Endosome-Lysosome Pathway

Neointimal hyperplasia is associated with the development of diverse vascular diseases such as atherosclerosis, vein bypass graft disease and restenosis after percutaneous coronary interventions. Vascular injury-mediated neointimal hyperplasia involves the phenotypic modulation, migration and proliferation of vascular smooth muscle cells (VSMCs). Cyclic nucleotide is vital in regulating VSMC migration and proliferation, which are controlled by cyclic nucleotide phosphodiesterase (PDE) isozymes. To identify the regulation and function of PDEs in VSMC pathogenesis of vascular diseases, we performed systematic screening for all 22 known PDE genes in normal contractile VSMCs and proliferating synthetic VSMCs. Interestingly, we observed that expression of PDE1C was very low in normal contractile VSMCs but drastically elevated in synthetic VSMCs in vitro as well as in vivo in various mouse models of vascular injuries, including carotid artery ligation, femoral artery wire injury and vein bypass graft. Consistently, PDE1C is also highly induced in VSMCs in neointimal lesion of human coronary artery. More importantly, injury-induced neointimal formation was significantly attenuated in pde1c-deficient mice or by perivascular administration of PDE1 inhibitor. Furthermore, PDE1 inhibition also reduced the spontaneous vascular remodeling of human saphenous vein explant in an ex vivo culture model. Mechanistic studies revealed that PDE1C plays a critical role in regulating the stability of PDGF receptor beta (PDGFR-β). We found that PDE1C knockdown or inhibition markedly decreased the levels of PDGFR-β protein but not mRNA, which was blocked by endosome and lysosome inhibitors. Furthermore, PDE1C inhibition is capable of promoting PDGFR-β internalization and attenuating PDGFR-β membrane accumulation. Finally, we found that PDE1C co-localized with PDGFR-β on cell membrane, which was able to regulate PDGFR-β protein degradation through modulating a transmembrane adenylyl cyclase-cAMP-PKA signaling. Taken together, our present data demonstrated that PDE1C plays a critical role in regulating VSMC proliferation and neointimal hyperplasia, in part, through promoting endosome/lysosome dependent PDGFR-β protein degradation.

Abstract 481: Downregulation of MicroRNA-143 Induces Proximal Aorta Dilatation in C57BL6/J Mice

Background: Thoracic Aortic Aneurysm (TAA) is a silent life-threatening condition which leads to death if not surgically treated. TAA development is secondary to the remodeling of the aortic wall structure due to pathological changes in both vascular smooth muscle cells (VSMC) and extracellular components. The phenotypic switch of VSMC from a contractile to a pathological synthetic state by inducing excessive production of matrix components is associated with the aberrant remodeling of the aorta. MicroRNA (miR)-143 and -145 are critical in controlling VSMC phenotype and are highly expressed in the aorta. In investigating the molecular mechanisms controlling VSMC phenotype we found that accumulation of reactive oxygen species (ROS) linearly correlates with synthetic VSMC accumulation as well as dilatation of the ascending aorta in humans. Furthermore ROS contribute to the onset of TAA in a murine model based on Angiotensin II (AngII) infusion. Interestingly we found that miR-143 is down regulated in patients with aneurysmal aorta. We hypothesized that miR-143 may be regulated by ROS and its down regulation could contribute to TAA development. Methods: MiR expression, MMP-2, MMP-9, and VSMC markers of phenotype (Vimentin, Elk-1, Myocardin) were tested in human isolated aortic VSMC treated with ROS. C57BL6/J mice were subjected to AngII for 28 days in the presence or absence of LNA-anti-miR-143. Two-dimensional M Mode echocardiography was performed to measure aortic diameter. Results: ROS treatment of human VSMC induces a dose dependent down regulation of miR-143 and -145 together with up regulation of MMP-2, MMP-9 and VSMC synthetic markers Vimentin and Elk-1. Both miR-143/145 are down regulated in the aneurysmal proximal aorta of C57BL6/J mice treated with AngII. Injection of LNA-anti-miR-143 alone induces dilatation of the proximal aorta but has no effect on AngII induced TAA development. Conclusion: ROS induced down regulation of miR-143 is involved in the pathological remodeling of the proximal aorta leading to TAA. Understanding the mechanism behind ROS-mediated regulation of miR-143 may unveil new targets for the development of therapeutic strategies against TAA.

Isolation, culture and identification of vascular smooth muscle cells with synthetic phenotype

Objective To investigate a method to obtain vascular smooth muscle cells (VSMCs),and isolate,culture and identify the VSMCs with highly synthetic phenotype.Methods The primary and subculture of VSMCs from thoracic aorta of SD rats was done by modified collage Ⅱ enzymatic dispersion and trypsin digestion,respectively.The third passage VSMCs were treated with platelet-derived growth factorBB (PDGF-BB).The cell viability and migration were detected by methyl thiazol tetrazolium (MTT) assay and wound-healing assay,respectively.The VSMCs were observed using inverted microscope and crystal violet staining for morphological observation.Immunofluorescence staining and immunocytochemical staining were used to examine contractile marker α-smooth muscle actin (SMA) and synthetic markers nonmuscle MHC isoform-B (SMemb),Tropomyosin-4 (TPM-4) for VSMCs identification and quantitative analysis.Results The post-confluent cells in the control group exhibited the hill and valley growth pattern typical of cultured VSMCs.More than 95％ of cells were positively stained by both stains for SMA.VSMCs under PDGF-BB treatment underwent a significant morphological alteration,from spindle to polygonal shape (P ＜0.05).The viability and mobility of VSMCs was increased by 11.48％ and 1.92 times,respectively (P ＜0.05).The expression of both SMemb and TPM-4 proteins was increased,and that of SMA protein decreased,significantly (P ＜ 0.05).Conclusion PDGF-BB treatment could lead to a significant phenotype modulation (highly synthetic VSMCs) in accompanied with enhancement of viability and mobility,highly increased synthetic markers while decreased contractile marker,significantly. Key words: Vascular smooth muscle cells; Cell phenotype; Platelet-derived growth factor; Cell culture

Abstract 18191: Mitochondrial Regulation of the Hyperproliferative Vascular Smooth Muscle Cell Phenotype

Background: During arterial restenosis and atherosclerosis, vascular smooth muscle cells (VSMCs) develop a highly proliferative and synthetic phenotype. Because such phenotypic changes are likely integrated with the energetic state of the cell, we hypothesized that changes in mitochondrial function regulate VSMC plasticity. Methods and Results: Exposure of VSMCs to platelet-derived growth factor (PDGF) promoted loss of the contractile proteins α-smooth muscle actin and calponin and was accompanied by remarkable changes in mitochondrial morphology. Confocal analysis showed that 97% of mitochondria in contractile VSMCs were filamentous; however, synthetic VSMCs showed a mitochondrial pool with >70% fragmented mitochondria (p<0.05). Mitochondrial membrane potential decreased by 86±1% with the attainment of the synthetic phenotype (p<0.01; n=3), but was not associated with mitophagy or decreases in the abundance of key proteins involved in oxidative phosphorylation. Compared with contractile VSMCs, synthetic VSMCs showed a 51±1.9% decrease in the abundance of mitofusin 2 (Mfn2; p<0.001; n=3) with no significant changes in Drp1, Fis1, or Opa1. Analysis of mitochondrial function in intact VSMCs showed a 20±3.7% decrease in glucose oxidation in synthetic cells, which was accompanied by a 17±2.6% increase in fatty acid oxidation (p<0.02; n=3). In permeabilized cells, complex II-mediated respiration was decreased by 20±2% in synthetic VSMCs (p<0.05; n=4). An inhibitor of mitochondrial fragmentation, mdivi1, decreased mitochondrial fragmentation by 50±6% (p<0.001; n=3), abolished the hyperproliferative response to PDGF (p<0.03; n=4), and reversed changes in mitochondrial respiration, yet it did not prevent PDGF-induced losses of contractile proteins. Conclusions: These results indicate that changes in mitochondrial morphology and bioenergetics contribute to the hyperproliferation of synthetic VSMCs, but do not affect the degradation of the contractile proteins. We propose that mitochondrial fragmentation that occurs during transition to the synthetic phenotype regulates mitochondrial substrate selection and bioenergetics and is a therapeutic target for vascular disorders characterized by VSMC hyperproliferation.

Oxidative stress modulates vascular smooth muscle cell phenotype via CTGF in thoracic aortic aneurysm.

Dissection and rupture of the ascending aorta are life-threatening conditions resulting in 80% mortality. Ascending aortic replacement in patients presenting with thoracic aortic aneurysm (TAA) is determined by metric measurement. However, a significant number of dissections occur outside of the parameters suggested by the current guidelines. We investigate the correlation among altered haemodynamic condition, oxidative stress, and vascular smooth muscle cell (VSMC) phenotype in controlling tissue homoeostasis. We demonstrate using finite element analysis (FEA) based on computed tomography geometries that TAA patients have higher wall stress in the ascending aorta than non-dilated patients. We also show that altered haemodynamic conditions are associated with increased levels of reactive oxygen species (ROS), direct regulators of the VSMC phenotype in the microregional area of the ascending aorta. Using in vitro and ex vivo studies on human tissues, we show that ROS accumulation correlates with media layer degeneration and increased connective tissue growth factor (CTGF) expression, which modulate the synthetic VSMC phenotype. Results were validated by a murine model of TAA (C57BL/6J) based on Angiotensin II infusion showing that medial thickening and luminal expansion of the proximal aorta is associated with the VSMC synthetic phenotype as seen in human specimens. Increased peak wall stress correlates with change in VSMC towards a synthetic phenotype mediated by ROS accumulation via CTGF. Understanding the molecular mechanisms that regulate VSMC towards a synthetic phenotype could unveil new regulatory pathways of aortic homoeostasis and impact the risk-stratification tool for patients at risk of aortic dissection and rupture.

Elevated expression of connective tissue growth factor, osteopontin and increased collagen content in human ascending thoracic aortic aneurysms

Little is known about the molecular mechanisms of ascending thoracic aortic aneurysms (ATAAs). Abnormal extracellular matrix changes and variations of vascular smooth muscle cells (VSMCs) have been implicated in abdominal aortic aneurysm formation. Our objective was to investigate the alterations of collagen, stimulators of collagen synthesis and synthetic VSMCs in patients with ATAA. Surgical samples from ATAA were taken from 20 patients, and 18 control aortas were obtained during coronary artery bypass surgery. All aortic wall specimens were fixed for histology and immunohistochemistry for collagen, connective tissue growth factor (CTGF) and osteopontin. Realtime polymerase chain reaction was used to determine their mRNA expression. Histology and semi- quantitative analysis demonstrated that protein levels of collagen, CTGF and osteopontin significantly increased by 1.9-, 1.4- and 2.2-fold, respectively (P< 0.01 for all) in the ATAA group than in the control group. Similar results were shown in mRNA levels of type laland Illal collagen, CTGF and osteopontin. The protein levels of CTGF and osteopontin were positively correlated with aortic diameter (r= 0.67, r= 0.73; P< 0.01 for both). In conclusion, overexpression of aortic CTGF and synthetic VSMCs marker (osteopontin), which is likely to be responsible for elevated aortic collagen content, may provide a potential mechanism for aneurysmal enlargement.

Abstract 287: Oxidative Stress Modulates Vascular Smooth Muscle Cell Phenotype via Connective Tissue Growth Factor in Thoracic Aortic Aneurysm

Rationale. Thoracic Aortic Aneurysm (TAA) is characterized by a dysregulation of cellular and extracellular components of the aortic wall which contribute to the weakening of the ascending aorta. Oxidative stress has long been implicated in the pathogenesis of aneurysms for its role in inducing cellular apoptosis and matrix degradation. However, the link between oxidative stress and TAA formation remains to be firmly established. Connective Tissue Growth Factor (CTGF) regulates matrix biosynthesis as well as Vascular Smooth Muscle Cells (VSMC) proliferation and apoptosis. We hypothesized that Reactive Oxygen Species (ROS), modulated by altered hemodynamic conditions, could regulate VSMC phenotype and matrix composition via CTGF, affecting - in turn - the aortic tissue structure. Methods. Finite element analysis (FEA) based on computed tomography geometries was used to estimate wall stress in control and TAA patients. In vitro and ex vivo studies on human tissues and isolated VSMC were used to demonstrate the correlation between ROS, CTGF, and VSMC synthetic phenotype in TAA development. Results were validated in vivo by a murine model of TAA based on Angiotensin II (AngII) infusion. Results. TAA patients have higher wall stress in the ascending aorta than non-dilated patients. High aortic wall stress is associated with increased levels of ROS, media degeneration and accumulation of synthetic VSMCs in TAA tissues. ROS directly induce CTGF expression, which modulates synthetic VSMC phenotype in human isolated cells. ROS effect is reverted in vitro either using a neutralizing antibody against CTGF or by antioxidant administration. In AngII- induced TAA in mice, medial thickening and luminal expansion of the proximal aorta are associated with ROS accumulation, CTGF up-regulation and VSMC with a synthetic phenotype as seen in human specimens. Conclusions. Increased peak wall stress correlates with change in VSMCs towards a synthetic phenotype mediated by ROS accumulation via CTGF. Understanding the molecular mechanisms that regulate VSMCs towards a synthetic phenotype could unveil new regulatory pathways for aortic homeostasis and identify new therapeutic targets for patients at risk of aortic dissection and rupture.

192 MODULATED EXOSOME SECRETION BY VASCULAR SMOOTH MUSCLE CELLS IS A NOVEL REGULATORY MECHANISM OF VASCULAR CALCIFICATION

Vascular calcification is a regulated pathological process similar to bone formation which is mediated by vascular smooth muscle cells (VSMCs) undergoing osteogenic transdifferentiation. Initiation of vascular calcification occurs in small membrane-bound matrix vesicles (MVs), secreted by VSMCs into the extracellular matrix however the mechanisms regulating MV biogenesis and secretion are unclear. Fetuin-A, a circulating protein abundant in MVs was used to trace the origin of VSMC-derived MVs. Alexa488-labelled fetuin-A was rapidly uptaken by human VSMCs and appeared in the late endosomal system and multivesicular bodies (MVBs) indicating that MVs are secreted from the endosomal compartment similar to exosomes; extracellular vesicles originating from MVBs and secreted by the range of cells. Biochemical analysis of MVs showed that they were enriched with exosomal markers, CD9 and CD63. Furthermore, inhibition of sphingomyelin phosphodiesterase 3 (SMPD3), which in involved in exosome biogenesis abrogated MV secretion by VSMCs confirming that MVs represent exosomes. Calcifying conditions induced exosome secretion by VSMCs and this was accompanied by elevated SMPD3 expression. Importantly, an inhibition of SMPD3 prevented VSMC calcification. Phenotypic modulation of VSMCs and loss of the contractile phenotype resulted in elevated exosome secretion while contractile VSMCs secreted significantly less exosomes. Notably, elevated extracellular calcium rapidly induced calcification of synthetic VSMCs whilst contractile VSMCs did not calcify. In agreement with this data, abundant immunohistochemical staining for CD63 was observed only in atherosclerotic human aorta in close association with calcified areas with little staining detected in the healthy vessel wall. Taken together this study demonstrates that MVs originate from MVBs and are secreted via the exosomal pathway. Loss of the contractile phenotype and mineral imbalance promote VSMC calcification by enhanced exosome secretion. Targeting the mechanisms of VSMC exosome secretion and/or their loading with calcification inhibitors may provide novel therapeutic interventions aimed for the prevention of vascular calcification.

Mitochondrial fission induced by platelet-derived growth factor regulates vascular smooth muscle cell bioenergetics and cell proliferation

Vascular smooth muscle cells (VSMCs) develop a highly proliferative and synthetic phenotype in arterial diseases. Because such phenotypic changes are likely integrated with the energetic state of the cell, we hypothesized that changes in cellular metabolism regulate VSMC plasticity. VSMCs were exposed to platelet-derived growth factor-BB (PDGF) and changes in mitochondrial morphology, proliferation, contractile protein expression, and mitochondrial metabolism were examined. Exposure of VSMCs to PDGF resulted in mitochondrial fragmentation and a 50% decrease in the abundance of mitofusin 2. Synthetic VSMCs demonstrated a 20% decrease in glucose oxidation, which was accompanied by an increase in fatty acid oxidation. Results of mitochondrial function assays in permeabilized cells showed few changes due to PDGF treatment in mitochondrial respiratory chain capacity and coupling. Treatment of VSMCs with Mdivi-1—an inhibitor of mitochondrial fission—inhibited PDGF-induced mitochondrial fragmentation by 50% and abolished increases in cell proliferation; however, it failed to prevent PDGF-mediated activation of autophagy and removal of contractile proteins. In addition, treatment with Mdivi-1 reversed changes in fatty acid and glucose oxidation associated with the synthetic phenotype. These results suggest that changes in mitochondrial morphology and bioenergetics underlie the hyperproliferative features of the synthetic VSMC phenotype, but do not affect the degradation of contractile proteins. Mitochondrial fragmentation occurring during the transition to the synthetic phenotype could be a therapeutic target for hyperproliferative vascular disorders.

Abstract 18739: Autophagy Regulates Vascular Smooth Muscle Cell Synthetic Phenotype Transition and Resistance to Oxidative Stress

Arterial lesions due to atherosclerosis and restenosis are characterized by heightened oxidative stress and the transformation of vascular smooth muscle cells (VSMCs) to a synthetic phenotype. However, it is not known how synthetic VSMCs (sVSMCs) respond to the stress encountered in advanced vascular lesions. Our previous studies show that electrophilic stress caused by lipid peroxidation products promotes autophagy and endoplasmic reticulum stress. In this study, we tested the hypothesis that conversion to the synthetic phenotype primes VSMCs to survive under conditions of high oxidative stress. Synthetic VSMCs, elicited by exposure to platelet-derived growth factor-BB (PDGF-BB), maintained lower levels of endogenous protein-aldehyde adducts and demonstrated a heightened ability (2.5-fold; p&lt;0.05) to remove proteins modified by the lipid peroxidation product 4-hydroxy- trans -2-nonenal (HNE) than contractile VSMC (cVSMC). Moreover, HNE detoxification in sVSMCs was enhanced; the primary pathway of HNE detoxification—aldehyde dehydrogenase-mediated oxidation to 4-hydroxynonenoic acid—was increased by 30% and glutathione conjugation was increased by 50% (p&lt;0.05). The sVSMCs showed a 20% increase in viability compared with cVSMCs when challenged with HNE (p&lt;0.05). This resistance to stress in sVSMCs was found to be due primarily to robust induction of the autophagic program. PDGF-BB exposure increased autophagic flux (&gt;2-fold vs. untreated cells; p&lt;0.05), as assessed by quantifying GFP-microtubule-associated protein 1 light chain 3 (LC3) puncta as well as electron microscopy and LC3II abundance. Blockade of autophagy by spautin-1 or 3-methyladenine prevented the PDGF-BB-induced decreases in expression and abundance of calponin and α-SM-actin, suggesting that autophagy is required for VSMC phenotype conversion. Inhibiting autophagy also prevented removal of HNE-damaged proteins and prevented PDGF-induced increases in NAD + - and glutathione-linked HNE detoxification (p&lt;0.05). Collectively, these results show that PDGF-BB induces autophagy in VSMCs, which is required for conversion of VSMCs to the synthetic phenotype and for resistance to the oxidative stress characteristic of vascular lesions.