Plaque Vascular Smooth Muscle Cells Research Articles

Metabolic reprogramming of immune cells by mitochondrial division inhibitor-1 to prevent post-vascular injury neointimal hyperplasia

Background and aimsNew treatments are needed to prevent neointimal hyperplasia that contributes to post-angioplasty and stent restenosis in patients with coronary artery disease (CAD) and peripheral arterial disease (PAD). We investigated whether modulating mitochondrial function using mitochondrial division inhibitor-1 (Mdivi-1) could reduce post-vascular injury neointimal hyperplasia by metabolic reprogramming of macrophages from a pro-inflammatory to anti-inflammatory phenotype. Methods and ResultsIn vivo Mdivi-1 treatment of Apoe−/− mice fed a high-fat diet and subjected to carotid-wire injury decreased neointimal hyperplasia by 68%, reduced numbers of plaque vascular smooth muscle cells and pro-inflammatory M1-like macrophages, and decreased plaque inflammation, endothelial activation, and apoptosis, when compared to control. Mdivi-1 treatment of human THP-1 macrophages shifted polarization from a pro-inflammatory M1-like to an anti-inflammatory M2-like phenotype, reduced monocyte chemotaxis and migration to CCL2 and macrophage colony stimulating factor (M-CSF) and decreased secretion of pro-inflammatory mediators. Finally, treatment of pro-inflammatory M1-type-macrophages with Mdivi-1 metabolically reprogrammed them to an anti-inflammatory M2-like phenotype by inhibiting oxidative phosphorylation and attenuating the increase in succinate levels and correcting the decreased levels of arginine and citrulline. ConclusionsWe report that treatment with Mdivi-1 inhibits post-vascular injury neointimal hyperplasia by metabolic reprogramming macrophages towards an anti-inflammatory phenotype thereby highlighting the therapeutic potential of Mdivi-1 for preventing neointimal hyperplasia and restenosis following angioplasty and stenting in CAD and PAD patients.

Lipid accumulation and novel insight into vascular smooth muscle cells in atherosclerosis.

Atherosclerosis is a chronic and progressive process. It is the most important pathological basis of cardiovascular disease and stroke. Vascular smooth muscle cells (VSMCs) are an essential cell type in atherosclerosis. Previous studies have revealed that VSMCs undergo phenotypic transformation in atherosclerosis to participate in the retention of atherogenic lipoproteins as well as the formation of the fibrous cap and the underlying necrotic core in plaques. The emergence of lineage-tracing studies indicates that the function and number of VSMCs in plaques have been greatly underestimated. In addition, recent studies have revealed that VSMCs make up at least 50% of the foam cell population in human and mouse atherosclerotic lesions. Therefore, understanding the formation of lipid-loaded VSMCs and their regulatory mechanisms is critical to elucidate the pathogenesis of atherosclerosis and to explore potential therapeutic targets. Moreover, combination of many complementary technologies such as lineage tracing, single-cell RNA sequencing (scRNA-seq), flow cytometry, and mass cytometry (CyTOF) with immunostaining has been performed to further understand the complex VSMC function. Correct identification of detrimental and beneficial processes may reveal successful therapeutic treatments targeting VSMCs and their derivatives during atherosclerosis. The purpose of this review is to summarize the process of lipid-loaded VSMC formation in atherosclerosis and to describe novel insight into VSMCs gained by using multiple advanced methods.

Telomere damage promotes vascular smooth muscle cell senescence and immune cell recruitment after vessel injury

Background and Aims: Vascular smooth muscle cells (VSMCs) accumulate in injury-induced neointimal lesions and atherosclerotic plaques in an oligoclonal fashion, yet plaque VSMCs show reduced proliferation and cell senescence. DNA damage leads to VSMC senescence and inflammation and VSMC senescence promotes atherosclerosis; however, the exact mechanism by which VSMC senescence promotes lesion formation is not known. Here, we investigated telomere damage-induced VSMC senescence, the contribution of senescence-induced inflammation and the mechanisms involved, the consequences of VSMC senescence in vivo after injury, and whether it promotes clonality.

SIRT6 Protects Smooth Muscle Cells From Senescence and Reduces Atherosclerosis.

Vascular smooth muscle cell (VSMC) senescence promotes atherosclerosis and features of plaque instability, in part, through lipid-mediated oxidative DNA damage and telomere dysfunction. SIRT6 (Sirtuin 6) is a nuclear deacetylase involved in DNA damage response signaling, inflammation, and metabolism; however, its role in regulating VSMC senescence and atherosclerosis is unclear. We examined SIRT6 expression in human VSMCs, the role, regulation, and downstream pathways activated by SIRT6, and how VSMC SIRT6 regulates atherogenesis. SIRT6 protein, but not mRNA, expression was markedly reduced in VSMCs in human and mouse atherosclerotic plaques, and in human VSMCs derived from plaques or undergoing replicative or palmitate-induced senescence versus healthy aortic VSMCs. The ubiquitin ligase CHIP (C terminus of HSC70-interacting protein) promoted SIRT6 stability, but CHIP expression was reduced in human and mouse plaque VSMCs and by palmitate in a p38- and c-Jun N-terminal kinase-dependent manner. SIRT6 bound to telomeres, while SIRT6 inhibition using shRNA or a deacetylase-inactive mutant (SIRT6H133Y) shortened human VSMC lifespan and induced senescence, associated with telomeric H3K9 (histone H3 lysine 9) hyperacetylation and 53BP1 (p53 binding protein 1) binding, indicative of telomere damage. In contrast, SIRT6 overexpression preserved telomere integrity, delayed cellular senescence, and reduced inflammatory cytokine expression and changes in VSMC metabolism associated with senescence. SIRT6, but not SIRT6H133Y, promoted proliferation and lifespan of mouse VSMCs, and prevented senescence-associated metabolic changes. ApoE-/- (apolipoprotein E) mice were generated that overexpress SIRT6 or SIRT6H133Y in VSMCs only. SM22α-hSIRT6/ApoE-/- mice had reduced atherosclerosis, markers of senescence and inflammation compared with littermate controls, while plaques of SM22α-hSIRT6H133Y/ApoE-/- mice showed increased features of plaque instability. SIRT6 protein expression is reduced in human and mouse plaque VSMCs and is positively regulated by CHIP. SIRT6 regulates telomere maintenance and VSMC lifespan and inhibits atherogenesis, all dependent on its deacetylase activity. Our data show that endogenous SIRT6 deacetylase is an important and unrecognized inhibitor of VSMC senescence and atherosclerosis.

Vascular smooth muscle cells in atherosclerosis.

Vascular smooth muscle cells (VSMCs) are a major cell type present at all stages of an atherosclerotic plaque. According to the 'response to injury' and 'vulnerable plaque' hypotheses, contractile VSMCs recruited from the media undergo phenotypic conversion to proliferative synthetic cells that generate extracellular matrix to form the fibrous cap and hence stabilize plaques. However, lineage-tracing studies have highlighted flaws in the interpretation of former studies, revealing that these studies had underestimated both the content and functions of VSMCs in plaques and have thus challenged our view on the role of VSMCs in atherosclerosis. VSMCs are more plastic than previously recognized and can adopt alternative phenotypes, including phenotypes resembling foam cells, macrophages, mesenchymal stem cells and osteochondrogenic cells, which could contribute both positively and negatively to disease progression. In this Review, we present the evidence for VSMC plasticity and summarize the roles of VSMCs and VSMC-derived cells in atherosclerotic plaque development and progression. Correct attribution and spatiotemporal resolution of clinically beneficial and detrimental processes will underpin the success of any therapeutic intervention aimed at VSMCs and their derivatives.

Defective Base Excision Repair of Oxidative DNA Damage in Vascular Smooth Muscle Cells Promotes Atherosclerosis

Atherosclerotic plaques demonstrate extensive accumulation of oxidative DNA damage, predominantly as 8-oxoguanine (8oxoG) lesions. 8oxoG is repaired by base excision repair enzymes; however, the mechanisms regulating 8oxoG accumulation in vascular smooth muscle cells (VSMCs) and its effects on their function and in atherosclerosis are unknown. We studied levels of 8oxoG and its regulatory enzymes in human atherosclerosis, the mechanisms regulating 8oxoG repair and the base excision repair enzyme 8oxoG DNA glycosylase I (OGG1) in VSMCs in vitro, and the effects of reducing 8oxoG in VSMCs in atherosclerosis in ApoE-/- mice. Human plaque VSMCs showed defective nuclear 8oxoG repair, associated with reduced acetylation of OGG1. OGG1 was a key regulatory enzyme of 8oxoG repair in VSMCs, and its acetylation was crucial to its repair function through regulation of protein stability and expression. p300 and sirtuin 1 were identified as the OGG1 acetyltransferase and deacetylase regulators, respectively, and both proteins interacted with OGG1 and regulated OGG1 acetylation at endogenous levels. However, p300 levels were decreased in human plaque VSMCs and in response to oxidative stress, suggesting that reactive oxygen species-induced regulation of OGG1 acetylation could be caused by reactive oxygen species-induced decrease in p300 expression. We generated mice that express VSMC-restricted OGG1 or an acetylation defective version (SM22α-OGG1 and SM22α-OGG1K-R mice) and crossed them with ApoE-/- mice. We also studied ApoE-/- mice deficient in OGG1 (OGG1-/-). OGG1-/- mice showed increased 8oxoG in vivo and increased atherosclerosis, whereas mice expressing VSMC-specific OGG1 but not the acetylation mutant OGG1K-R showed markedly reduced intracellular 8oxoG and reduced atherosclerosis. VSMC OGG1 reduced telomere 8oxoG accumulation, DNA strand breaks, cell death and senescence after oxidant stress, and activation of proinflammatory pathways. We identify defective 8oxoG base excision repair in human atherosclerotic plaque VSMCs, OGG1 as a major 8oxoG repair enzyme in VSMCs, and p300/sirtuin 1 as major regulators of OGG1 through acetylation/deacetylation. Reducing oxidative damage by rescuing OGG1 activity reduces plaque development, indicating the detrimental effects of 8oxoG on VSMC function.

Characterization of Carotid Smooth Muscle Cells during Phenotypic Transition.

Vascular smooth muscle cells (VSMCs) are central players in carotid atherosclerosis plaque development. Although the precise mechanisms involved in plaque destabilization are not completely understood, it is known that VSMC proliferation and migration participate in plaque stabilization. In this study, we analyzed expression patterns of genes involved in carotid atherosclerosis development (e.g., transcription factors of regulation of SMC genes) of VSMCs located inside or outside the plaque lesion that may give clues about changes in phenotypic plasticity during atherosclerosis. VSMCs were isolated from 39 carotid plaques extracted from symptomatic and asymptomatic patients by endarterectomy. Specific biomarker expression, related with VSMC phenotype, was analyzed by qPCR, western immunoblot, and confocal microscopy. MYH11, CNN1, SRF, MKL2, and CALD1 were significantly underexpressed in VSMCs from plaques compared with VSMCs from a macroscopically intact (MIT) region, while SPP1, KLF4, MAPLC3B, CD68, and LGALS3 were found significantly upregulated in plaque VSMCs versus MIT VSMCs. The gene expression pattern of arterial VSMCs from a healthy donor treated with 7-ketocholesterol showed high similarity with the expression pattern of carotid plaque VSMCs. Our results indicate that VSMCs isolated from plaque show a typical SMC dedifferentiated phenotype with macrophage-like features compared with VSMCs isolated from a MIT region of the carotid artery. Additionally, MYH11, KLF5, and SPP1 expression patterns were found to be associated with symptomatology of human carotid atherosclerosis.

Impaired mitochondrial respiration in human carotid plaque atherosclerosis: A potential role for Pink1 in vascular smooth muscle cell energetics

Background and aimsDNA damage and mitochondrial dysfunction are thought to play an essential role in ageing and the energetic decline of vascular smooth muscle cells (VSMCs) essential for maintaining plaque integrity. We aimed to better understand VSMCs and identify potentially useful compensatory pathways that could extend their lifespan. Moreover, we wanted to assess if defects in mitochondrial respiration exist in human atherosclerotic plaques and to identify the appropriate markers that may reflect a switch in VSMC energy metabolism. MethodsHuman plaque tissue and cells were assessed for composition and evidence of DNA damage, repair capacity and mitochondrial dysfunction. Fresh plaque tissue was evaluated using high resolution oxygen respirometry to assess oxidative metabolism. Recruitment and processing of the mitochondrial regulator of autophagy Pink1 kinase was investigated in combination with transcriptional and protein markers associated with a potential switch to a more glycolytic metabolism. ResultsHuman VSMC have increased nuclear (nDNA) and mitochondrial (mtDNA) damage and reduced repair capacity. A subset of VSMCs within plaque cap had decreased oxidative phosphorylation and expression of Pink1 kinase. Plaque cells demonstrated increased glycolytic activity in response to loss of mitochondrial function. A potential compensatory glycolytic program may act as energetic switch via AMP kinase (AMPK) and hexokinase 2 (Hex2). ConclusionsWe have identified a subset of plaque VSMCs required for plaque stability that have increased mitochondrial dysfunction and decreased oxidative phosphorylation. Pink1 kinase may initiate a cellular response to promote a compensatory glycolytic program associated with upregulation of AMPK and Hex2.

165 Human atherosclerosis is characterised by oxidative dna damage due to defective base excision repair

RationaleHuman atherosclerotic plaques show extensive oxidative DNA damage in vascular smooth muscle cells (VSMCs) and macrophages, including accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG), the most abundant DNA base lesion on oxidative exposure....

P2Y12 Promotes Migration of Vascular Smooth Muscle Cells Through Cofilin Dephosphorylation During Atherogenesis.

P2Y12 is a well-recognized receptor expressed on platelets and the target of thienopyridine-type antiplatelet drugs. However, recent evidence suggests that P2Y12 expressed in vessel wall plays a role in atherogenesis, but the mechanisms remain elusive. In this study, we examined the molecular mechanisms of how vessel wall P2Y12 mediates vascular smooth muscle cells (VSMCs) migration and promotes the progression of atherosclerosis. Using a high-fat diet-fed apolipoprotein E-deficient mice model, we found that the expression of P2Y12 in VSMCs increased in a time-dependent manner and had a linear relationship with the plaque area. Moreover, administration of P2Y12 receptor antagonist for 12 weeks caused significant reduction in atheroma and decreased the abundance of VSMCs in plaque. In cultured VSMCs, we found that activation of P2Y12 receptor inhibited cAMP/protein kinase A signaling pathway, which induced cofilin dephosphorylation and filamentous actin disassembly, thereby enhancing VSMCs motility and migration. In addition, the number of P2Y12-positive VSMCs was decreased in the carotid artery plaque from patients receiving clopidogrel. Vessel wall P2Y12 receptor, which promotes VSMCs migration through cofilin dephosphorylation, plays a critical role in the development of atherosclerotic lesion and may be used as a therapeutic target for atherosclerosis.

Abstract 12203: Fxr1 Regulates Inflammatory mRA Stability and Vascular Disease

Introduction: Vascular smooth muscle cells (VSMC) play a critical role in the etiology and progression of many vascular diseases, and reduction of inflammatory gene expression in VSMC is a rational approach to limit the severity of these diseases. HuR is an mRNA stability protein that recognizes AU-rich elements (ARE) present almost exclusively in the 3’UTR of pro-inflammatory gene mRNA. Proteins and pathways which limit HuR activity in vascular cells may reduce inflammatory mRNA stability, but are currently uncharacterized. Using un-biased LC-MS/MS to identify HuR-interacting proteins under different inflammatory conditions, we identified one protein termed Fragile X-related protein (FXR1), which interacts with HuR in inflammatory, but not basal conditions. Results and Conclusions: Using immunohistochemical and molecular biological approaches, preliminary data shows that FXR1 expression is increased in VSMC in plaque from atherosclerotic mouse and human arteries. FXR1 translocates from the nucleus into the cytoplasm and co-localizes with HuR in cytoplasm. FXR1 expression is also increased by TNFα and oxLDL stimulation of cultured human VSMC. Importantly, siRNA knock down of FXR1 in VSMC increases inflammatory mRNA stability, increases abundance of ARE-containing inflammatory proteins, and increases cholesterol uptake. Surprisingly, FXR1 expression is also increased by anti-inflammatory interleukins. Since FXR1 is induced by both pro-inflammatory stimuli as well as IL-19, and yet deletion of FXR1 leads to enhanced expression of pro-inflammatory proteins, our overall hypothesis is that FXR1 expression is a negative, compensatory, counter-regulatory mechanism used by VSMC to respond to and dampen inflammation. Our specific hypothesis is that FXR1 antagonizes HuR function by numerous mechanisms leading to a reduction in stability of pro-inflammatory, ARE-containing mRNA. Currently, very little is understood concerning negative regulation of inflammatory mRNA stability, particularly so in vascular disease. Nothing at all is known about FXR1 expression and function in VSMC and vascular disease. This work may identify FXR1 as part of an anti-inflammatory gene expression program and uncover a novel role for FXR1 in vascular disease.

Circulation : Clinical Summaries

HomeCirculationVol. 132, No. 20Circulation: Clinical Summaries Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBCirculation: Clinical SummariesOriginal Research Put Into Perspective for the Practicing Clinician Originally published17 Nov 2015https://doi.org/10.1161/CIR.0000000000000323Circulation. 2015;132:1851–1852Adjusting for Risk Associated With Pediatric and Congenital Cardiac Catheterization: A Report From the NCDR IMPACT RegistryFor hospitals to be able to fairly compare their outcomes with those from other centers performing catheterization for congenital heart disease, validated methods for risk-standardizing outcomes must be developed. Risk standardization allows adjustment based on case-mix complexity and allows hospitals to compare their performance with that of other centers while accounting for the types of patients they treat and procedures they perform. The most widely accepted risk-standardization methodology for congenital cardiac catheterization to date is the Congenital Heart Disease Adjustment for Risk Method (CHARM). Using CHARM as a foundation, we sought to begin the development of a risk-standardization model for the National Cardiovascular Data Registry (NCDR) IMPACT (Improving Pediatric and Adult Congenital Treatment) Registry. Within IMPACT, we included 19 608 diagnostic and interventional cardiac catheterization procedures. We identified 8 patient and procedural characteristics predictive of experiencing a major adverse event, including patient age, renal insufficiency, and single-ventricle physiology, as well as procedure-type risk group and hemodynamic vulnerability (low systemic saturation, low mixed venous saturation, elevated systemic ventricular end-diastolic pressure, and elevated main pulmonary artery pressure) as defined by CHARM. A risk-standardization tool for the IMPACT Registry will fill an important gap in the care of pediatric and adult patients with congenital heart disease undergoing a diagnostic or interventional cardiac catheterization by identifying best practices at hospitals with the lowest risk-adjusted adverse event rates and disseminating these practices to all centers caring for this patient population. See p 1863.Efficacy and Safety of Vorapaxar With and Without a Thienopyridine for Secondary Prevention in Patients With Previous Myocardial Infarction and No History of Stroke or Transient Ischemic Attack: Results from TRA 2°P-TIMI 50We have previously reported the efficacy of vorapaxar, a first-in-class protease-activated receptor 1 antagonist. Vorapaxar has since been approved for clinical use in the United States for patients with a history of myocardial infarction (MI) without a history of stroke or transient ischemic attack. Given the prevalent use of thienopyridines in addition to aspirin in this post-MI population, we sought to understand the potential risks and benefits of adding vorapaxar to a thienopyridine as a second or third antiplatelet agent following MI. Evaluation of this large (n=16 897), well-characterized population revealed that vorapaxar significantly reduced the composite of cardiovascular death, myocardial infarction, and stroke in comparison with placebo regardless of thienopyridine use. Although bleeding was increased with vorapaxar in comparison with placebo, the risk was not significantly altered by thienopyridine use. These findings indicate that in patients with a previous MI, secondary prevention with vorapaxar may be beneficial when initiated early after stabilization from MI in conjunction with ongoing dual-antiplatelet therapy with aspirin and clopidogrel. See p 1871.Targeting Interleukin-1β Reduces Leukocyte Production After Acute Myocardial InfarctionThe acutely ischemic myocardium recruits millions of inflammatory blood leukocytes. To provide these cells, the bone marrow increases neutrophil and monocyte production, cells that are involved in reperfusion injury and infarct healing. Signals that communicate the increased demand of leukocytes after ischemia to the bone marrow are incompletely understood. Here, we provide data that support the role of circulating danger signals in alerting the bone marrow after myocardial infarction. Specifically, interleukin-1β levels increase systemically and result in accelerated hematopoietic stem and progenitor cell proliferation, myeloid cell production, and cell supply to the infarct. These actions occur directly via interleukin-1β signaling to hematopoietic cells and indirectly via bone marrow niche cells. Neutralization of interleukin-1β signaling with an antibody dampened bone marrow output of inflammatory leukocytes and supported inflammation resolution in the injured heart. This treatment also led to reduced post–myocardial infarction heart failure. Of note, a similar drug is clinically approved for cryopyrin-associated periodic syndromes and is currently being investigated for anti-inflammatory treatment in patients with atherosclerosis in the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) trial. See p 1880.Sulodexide for the Prevention of Recurrent Venous Thromboembolism: The Sulodexide in Secondary Prevention of Recurrent Deep Vein Thrombosis (SURVET) Study: A Multicenter, Randomized, Double-Blind, Placebo-Controlled TrialPatients with unprovoked venous thromboembolism are at high risk for recurrence after discontinuation of treatment with vitamin K antagonists (VKAs). Extending treatment with VKAs reduces the recurrence risk but increases the bleeding risk. In clinical practice, VKAs are generally discontinued when the perceived risk of bleeding outweighs the risk of recurrence. Drugs with low or no bleeding risk and less aggressive antithrombotic activity may represent adequate alternatives to continue anticoagulation with VKAs, or patients should be left to only physical management (elastic stockings) in cases of doubt. Rates of bleeding in general inferior to VKAs and efficacy not inferior to VKAs have been shown by the newer non-VKAs. However, compared with placebo, the extended anticoagulation with dabigatran, rivaroxaban, or apixaban, although reducing the risk of venous thromboembolism recurrence, carried a higher risk of major or clinically relevant nonmajor bleeding. The pooled data of the Warfarin and Aspirin (WARFASA) and Aspirin to Prevent Recurrent Venous Thromboembolism (ASPIRE) trials showed a significant risk reduction of venous thromboembolism recurrence, although at a lower extent than with the new non-VKAs, but still a worse result than placebo in terms of the occurrence of clinically relevant bleeding. In the 2 years of treatment in the Sulodexide in Secondary Prevention of Recurrent Deep Vein Thrombosis (SURVET) study, venous thromboembolism recurred in 15 of 307 patients on sulodexide and 30 of 308 on placebo (hazard ratio, 0.49; 95% confidence interval, 0.27–0.92; P=0.02). There were no differences in major or clinically relevant nonmajor bleeding between the sulodexide and placebo groups. Sulodexide appears to be an important treatment option when extended anticoagulation is potentially useful but associated with unwanted bleeding risk. See p 1891.Red Blood Cell Dysfunction Induced by High-Fat Diet: Potential Implications for Obesity-Related AtherosclerosisHigh-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. Here, we examined the impact of HFD on red blood cells (RBCs), which may play an important modulatory role in atherosclerosis by binding inflammatory chemokines and interacting with macrophages and endothelium within atherosclerotic plaques. We detected a marked increase in the level of chemokines bound to the RBCs of mice fed a 60% HFD for 12 weeks in comparison with mice fed a normal chow diet. Further investigations demonstrated that these chemokines were bound to RBCs via the Duffy antigen receptor for chemokines. Exposure of RBCs from HFD-fed mice to an endothelial monolayer in vitro significantly enhanced macrophage transendothelial migration, confirming the functional importance of RBC-bound chemokines in the setting of HFD. In addition to increasing the level of chemokines bound to RBCs, HFD increased RBC membrane cholesterol content and phosphatidylserine externalization (a marker or RBC damage or senescence), fostering RBC-macrophage inflammatory interactions and promoting the uptake of RBC by macrophages in vitro and by the spleen in vivo. Finally, RBCs from HFD-fed mice augmented macrophage adhesion to the endothelium when incubated with isolated aortic segments, indicating endothelial activation. We propose that RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic. See p 1898.Vascular Smooth Muscle Cell Senescence Promotes Atherosclerosis and Features of Plaque VulnerabilityProliferation of vascular smooth muscle cells (VSMCs) is a central axiom of both historical and current models of atherosclerosis, either as a response to injury or inflammation. The detrimental effect of VSMC proliferation has been endorsed by the success of antiproliferatives on drug-eluting stents, which have virtually eliminated in-stent stenosis. However, atherosclerosis is a disease of ageing, where proliferation of normal cells usually slows down. Indeed, human plaque VSMCs show premature senescence both in culture and in vivo, associated with telomere shortening and extensive DNA damage. Using human and mouse arteries, plaques and cells, and 2 novel transgenic mouse models, we show that human plaque VSMCs show reduced expression and telomere association of the key telomere protein TRF2. TRF2 reduces DNA damage, accelerates DNA repair, and protects VSMCs against senescence. In contrast, a TRF2 mutant (TRF2T188A) increases DNA damage, impairs DNA repair, and promotes VSMC senescence. TRF2T188A increased atherosclerosis and necrotic core formation in apolipoprotein E–/– mice. In contrast, transgenic mice expressing TRF2 only in VSMCs show increased relative fibrous cap and reduced necrotic areas, promoting features of stable plaques. Our work indicates that VSMC senescence promotes plaque formation and has marked effects on the fibrous cap and necrotic core, directly promoting features of plaque vulnerability. In contrast to earlier views, our work indicates that VSMC proliferation may protect against atherogenesis, and senescence, not proliferation, might promote atherosclerosis and vulnerable plaques. The study also explains for the first time how specific progeroid diseases might be associated with atherosclerosis and premature heart attack. See p 1909. Previous Back to top Next FiguresReferencesRelatedDetails November 17, 2015Vol 132, Issue 20 Advertisement Article InformationMetrics © 2015 American Heart Association, Inc.https://doi.org/10.1161/CIR.0000000000000323 Originally publishedNovember 17, 2015 PDF download Advertisement

Abstract 15762: Gapdh Interaction With Ape1 Endonuclease Protects Vascular Smooth Muscle Cells Against Apoptosis: Potential Role of These Enzymes in Prevention of Atherosclerotic Plaque Destabilization

We have shown that oxidized lipids (OxLDL) co-localized with apoptotic vascular smooth muscle cells (VSMC) in atherosclerotic plaque and that OxLDL downregulated the major glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cultured VSMC via H2O2-dependent mechanism. We hypothesized that maintenance of sufficient GAPDH levels is critical for cell survival under oxidative stress. H2O2 (220 uM, 12h) decreased GAPDH protein (55±5% decrease) and induced apoptosis (TUNEL assay) in human VSMC. Human VSMC transfection with pCMV-GAPDH increased GAPDH protein (3.5-fold) and prevented H2O2-induced apoptosis (74±4% decrease) vs. pCMV-GFP. We generated rat VSMC with constitutive 2-fold overexpression GAPDH (R3-VSMC). R3-VSMC had increased glycolysis (2.5 folds increase, pyruvate levels), ATP levels (38±2% increase) and reduced oxidative DNA damage (40±2% reduction in number of apurinic/apyrimidinic (AP) sites) and these effects correlated with suppressed H2O2-induced cell apoptosis (>90% reduction, TUNEL assay; 45.7±3.6% reduction, Cell Death ELISA). GAPDH-targeted siRNA reduced GAPDH protein (for 55%) and potentiated H2O2-induced apoptosis (9.4±0.3 folds increase, TUNEL; 8.6±0.4 folds increase, ELISA) in WT-VSMC and blocked anti-apoptotic effect seen in R3-VSMC. GAPDH known to bind apurinic/apyrimidinic (AP) endonuclease 1 (APE1), a major DNA repair enzyme. We found that GAPDH was co-immunoprecipitated and co-localized with APE1 in cytosolic/nuclear fraction and H2O2 induced further GAPDH-Ape1 co-localization and migration toward nuclei. APE1 specific activity was increased in H2O2-treated R3-VSMC vs. H2O2-treated WT VSMC, suggesting that GAPDH preserved APE1 activity under oxidative stress. APE1-targeted siRNA abrogated the protection of GAPDH overexpression against H2O2-induced apoptosis in VMSC. In summary, GAPDH downregulation mediates oxidant-induced VSMC apoptosis and forced expression of GAPDH protects VSMC from H2O2-induced cell death potentially via activation of APE1-dependent DNA repair. Our data suggest that preservation of GAPDH and APE1 activity in plaque VSMC could be a potential strategy to stabilize plaque and prevent acute coronary events.

Vascular Smooth Muscle Cell Senescence Promotes Atherosclerosis and Features of Plaque Vulnerability.

Although vascular smooth muscle cell (VSMC) proliferation is implicated in atherogenesis, VSMCs in advanced plaques and cultured from plaques show evidence of VSMC senescence and DNA damage. In particular, plaque VSMCs show shortening of telomeres, which can directly induce senescence. Senescence can have multiple effects on plaque development and morphology; however, the consequences of VSMC senescence or the mechanisms underlying VSMC senescence in atherosclerosis are mostly unknown. We examined the expression of proteins that protect telomeres in VSMCs derived from human plaques and normal vessels. Plaque VSMCs showed reduced expression and telomere binding of telomeric repeat-binding factor-2 (TRF2), associated with increased DNA damage. TRF2 expression was regulated by p53-dependent degradation of the TRF2 protein. To examine the functional consequences of loss of TRF2, we expressed TRF2 or a TRF2 functional mutant (T188A) as either gain- or loss-of-function studies in vitro and in apolipoprotein E(-/-) mice. TRF2 overexpression bypassed senescence, reduced DNA damage, and accelerated DNA repair, whereas TRF2(188A) showed opposite effects. Transgenic mice expressing VSMC-specific TRF2(T188A) showed increased atherosclerosis and necrotic core formation in vivo, whereas VSMC-specific TRF2 increased the relative fibrous cap and decreased necrotic core areas. TRF2 protected against atherosclerosis independent of secretion of senescence-associated cytokines. We conclude that plaque VSMC senescence in atherosclerosis is associated with loss of TRF2. VSMC senes cence promotes both atherosclerosis and features of plaque vulnerability, identifying prevention of senescence as a potential target for intervention.

Effects of DNA Damage in Smooth Muscle Cells in Atherosclerosis

DNA damage and the DNA damage response have been identified in human atherosclerosis, including in vascular smooth muscle cells (VSMCs). However, although double-stranded breaks (DSBs) are hypothesized to promote plaque progression and instability, in part, by promoting cell senescence, apoptosis, and inflammation, the direct effects of DSBs in VSMCs seen in atherogenesis are unknown. To determine the presence and effect of endogenous levels of DSBs in VSMCs on atherosclerosis. Human atherosclerotic plaque VSMCs showed increased expression of multiple DNA damage response proteins in vitro and in vivo, particularly the MRE11/RAD50/NBS1 complex that senses DSB repair. Oxidative stress-induced DSBs were increased in plaque VSMCs, but DSB repair was maintained. To determine the effect of DSBs on atherosclerosis, we generated 2 novel transgenic mice lines expressing NBS1 or C-terminal deleted NBS1 only in VSMCs, and crossed them with apolipoprotein E(-/-) mice. SM22α-NBS1/apolipoprotein E(-/-) VSMCs showed enhanced DSB repair and decreased growth arrest and apoptosis, whereas SM22α-(ΔC)NBS1/apolipoprotein E(-/-) VSMCs showed reduced DSB repair and increased growth arrest and apoptosis. Accelerating or retarding DSB repair did not affect atherosclerosis extent or composition. However, VSMC DNA damage reduced relative fibrous cap areas, whereas accelerating DSB repair increased cap area and VSMC content. Human atherosclerotic plaque VSMCs show increased DNA damage, including DSBs and DNA damage response activation. VSMC DNA damage has minimal effects on atherogenesis, but alters plaque phenotype inhibiting fibrous cap areas in advanced lesions. Inhibiting DNA damage in atherosclerosis may be a novel target to promote plaque stability.

Identification of small proline-rich repeat protein 3 as a novel atheroprotective factor that promotes adaptive Akt signaling in vascular smooth muscle cells.

Atherosclerosis is the primary driver of cardiovascular disease, the leading cause of death worldwide. Identification of naturally occurring atheroprotective genes has become a major goal for the development of interventions that will limit atheroma progression and associated adverse events. To this end, we have identified small proline-rich repeat protein (SPRR3) as selectively upregulated in vascular smooth muscle cells (VSMCs) of atheroma-bearing arterial tissue versus healthy arterial tissue. In this study, we sought to determine the role of SPRR3 in atheroma pathophysiology. We found that atheroprone apolipoprotein E-null mice lacking SPRR3 developed significantly greater atheroma burden. To determine the cellular driver(s) of this increase, we evaluated SPRR3-dependent changes in bone marrow-derived cells, endothelial cells, and VSMCs. Bone marrow transplant of SPRR3-expressing cells into SPRR3(-/-)apolipoprotein E-deficient recipients failed to rescue atheroma burden. Similarly, endothelial cells did not exhibit a response to SPRR3 loss. However, atheromas from SPRR3-deficient mice exhibited increased TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive VSMCs compared with control. Cell death in SPRR3-deficient VSMCs was significantly increased in vitro. Conversely, SPRR3-overexpressing VSMCs exhibited reduced apoptosis compared with control. We also observed a PI3K (phosphatidylinositol 3-kinase)/Akt-dependent positive association between SPRR3 expression and levels of active Akt in VSMCs. The survival advantage seen in SPRR3-overexpressing VSMCs was abrogated after the addition of a PI3K/Akt pathway inhibitor. These results indicate that SPRR3 protects the lesion from VSMC loss by promoting survival signaling in plaque VSMCs, thereby significantly decreasing atherosclerosis progression. As the first identified atheroma-specific VSMC prosurvival factor, SPRR3 represents a potential target for lesion-specific modulation of VSMC survival.

Abstract TP433: Free Leptin And Soluble Leptin Receptor: Novel Circulating Markers Of Carotid Plaque - Related Symptomatology And Potential Role In Plaque Stability (from The Opal-lille Carotid Endarterectomy Study)

Leptin, an adipocyte-derived cytokine with various effects on the cardiovascular system, circulates in a free biologically active form and a bound form to a soluble leptin receptor (sObR) involved in modulating leptin activity. The links between free leptin, sObR, human atherosclerotic carotid plaque phenotype remain unknown. In the present study, we investigated the potential links between serum/plaque free leptin, serum sObR, carotid plaque stability and related symptomatology in 146 consecutive patients (71 asymptomatic and 75 symptomatic) scheduled for carotid endarterectomy. Free leptin was assessed in serum (17.05±23.19 ng/mL, range 0.47-191) and carotid plaques (0.204±0.486 ng/mg protein) and sObR in serum (21.48±8.81 ng/ml). Serum free leptin concentrations were correlated negatively with serum sObR concentrations and positively with intra-plaque free leptin concentrations (p<0.0001, r=0.491, p<0.0001, r=0.726, respectively). Serum free leptin and sObR concentrations were independently associated with carotid artery symptomatology (ß=--0.048; 95%CI [-0.093;-0.002]; -0.102, 95% CI [-0.994;-0.026], respectively). Regarding plaque phenotype, histological examination of carotid endarterectomy specimens revealed plaque stability index (collagen to macrophages area ratio) and Vascular Smooth Muscle Cell (VSMC) content to be significantly higher in asymptomatic patients compared to symptomatic ones (2.1 (1.9-2.5) vs. 1.5 (1.1-1.8), p< 0.0001; 39.2 (35.9-55.0) vs. 20.4 (17.8-26.3), p= 0.0003, respectively). When serum or plaque leptin levels were above median value, plaque stability index, plaque VSMC content, plaque VSMC proliferation index and ERK activation were significantly higher (p< 0.02). Interestingly, via ERK pathway activation, at 20 ng/mL, leptin induced a migratory signal and higher collagen synthesis in human VSMC, followed by a proliferative response of VSMC at the concentration of 75 ng/mL. For the first time, we reported free leptin and sObR to be novel circulating markers of carotid plaque - related symptomatology. Free leptin could be an active player in carotid plaque stability via its effects on human VSMC.

13 Human plaque vascular smooth muscle cells show differential expression of genes associated with oxidative DNA damage

RationaleOxidative DNA damage is a prominent feature of human atherosclerotic plaques. DNA damage results in vascular smooth muscle cell (VSMC) premature senescence, cell cycle arrest and apoptosis, features observed in...

Bone marrow-derived smooth muscle-like cells are infrequent in advanced primary atherosclerotic plaques but promote atherosclerosis.

Although vascular smooth muscle cells (VSMCs) provide the major structural integrity of atherosclerotic plaques, their origin has been questioned. In particular, although some studies identified plaque VSMCs originating from bone marrow or peripheral blood, their frequency is controversial and their function unknown. We used genetic tracking of cell fate through smooth muscle cell (SMC)-specific LacZ reporter activity and VSMC-selective apoptosis to investigate the frequency, distribution, and role of marrow-derived VSMCs in atherogenesis. Cultured mouse bone marrow-derived smooth muscle-like cells expressed SMC markers and functional SMC promoter-driven transgenes over time. Transplantation of apolipoprotein E (ApoE)(-/-) mice with smooth muscle myosin heavy chain-Cre/ROSA26R/ApoE(-/-) marrow showed that 0.7±0.14% cells expressed LacZ in atherosclerotic plaques, located superficially in early plaques, and in necrotic cores but not fibrous caps of advanced lesions. Cells expressing both progenitor and SMC markers showed a similar distribution and frequency. Apoptosis of marrow-derived SMC-like cells transplanted from SM22α-human diphtheria toxin receptor/ApoE(-/-) mice retarded atherogenesis, with reduced plaque macrophage content. Cultured marrow-derived SMC-like cells secreted proinflammatory cytokines and promoted macrophage migration, VSMC proliferation, and collagen synthesis. Bone marrow-derived SMC-like cells are infrequent in advanced primary atherosclerotic plaques and absent in fibrous caps. However, these cells secrete proinflammatory cytokines and mitogens and promote atherosclerosis.

Neuropeptide Y receptors in carotid plaques of symptomatic and asymptomatic patients: Effect of inflammatory cytokines

Aims Cytokines released by the immune cells at the site of plaque milieu induce smooth muscle cell apoptosis to promote plaque instability. But, neuropeptide Y (NPY), a pleotropic factor, may modulate the effects of cytokines in atherosclerotic plaques of patients with carotid stenosis. Our aim was to investigate the relative expression of NPY-Y1, NPY-Y2 and NPY-Y5 receptors on carotid plaque vascular smooth muscle cells (pVSMCs) of symptomatic (S) and asymptomatic (AS) patients and examine the effect of inflammatory cytokines on the expression of NPY receptors, that may attenuate plaque rupture. Methods and results In healthy carotid artery, there were significantly increased immunopositivity and increased mRNA transcripts of NPY-Y1 and NPY-Y5 receptors in thin sections and isolated VSMCs, respectively, compared to S and AS plaques. However, the NPY-Y2 expression was higher in S and AS pVSMCs than controls. Stimulation of the cells with TNF-α, IL-12 or IFN-γ (50 ng/ml) decreased mRNA transcripts of NPY-Y1 and NPY-Y5 and increased NPY-Y2 mRNAs in VSMCs of healthy carotid artery. The effect of the cytokines on mRNA transcripts of NPY-Y5 and NPY-Y2 in pVSMCs of S and AS patients was similar to healthy VSMCs, but with variable effect on NPY-Y1. Conclusion Increased expression of NPY-Y2 receptors in symptomatic pVSMCs than in healthy and asymptomatic subjects suggests a potential role of NPY-Y2 in plaque instability. This is further supported by the pronounced effect of atheroma-associated cytokines to increase NPY-Y2 mRNA transcripts in pVSMCs of patients with carotid stenosis.