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Background: Atherosclerosis is driven by the infiltration of the arterial intima by diverse immune cells and smooth muscle cells (SMCs). CD8 + T cells promote lesion growth during atherosclerotic lesion development, but their role in advanced atherosclerosis is less clear. Here, we studied the role of CD8 + T cells and their effects on SMCs in established atherosclerosis., Methods: CD8 + T cells were depleted in (SMC reporter) low-density lipoprotein receptor-deficient ( Ldlr -/- ) mice with established atherosclerotic lesions. Atherosclerotic lesion formation was examined, and single-cell RNA sequencing of aortic SMCs and their progeny was performed. Additionally, coculture experiments with primary aortic SMCs and CD8 + T cells were conducted., Results: Although we could not detect differences in atherosclerotic lesion size, an increased plaque SMC content was noted in mice after CD8 + T-cell depletion. Single-cell RNA sequencing of aortic lineage-traced SMCs revealed contractile SMCs and a modulated SMC cluster, expressing macrophage- and osteoblast-related genes. CD8 + T-cell depletion was associated with an increased contractile but decreased macrophage and osteoblast-like gene signature in this modulated aortic SMC cluster. Conversely, exposure of isolated aortic SMCs to activated CD8 + T cells decreased the expression of genes indicative of a contractile SMC phenotype and induced a macrophage and osteoblast-like cell state. Notably, CD8 + T cells triggered calcium deposits in SMCs under osteogenic conditions. Mechanistically, we identified transcription factors highly expressed in modulated SMCs, including Runx1 , to be induced by CD8 + T cells in cultured SMCs in an IFNγ (interferon-γ)-dependent manner., Conclusions: We here uncovered CD8 + T cells to control the SMC phenotype in atherosclerosis. CD8 + T cells promote SMC dedifferentiation and drive SMCs to adopt features of macrophage-like and osteoblast-like, procalcifying cell phenotypes. Given the critical role of SMCs in atherosclerotic plaque stability, CD8 + T cells could thus be explored as therapeutic target cells during lesion progression., Competing Interests: None.

Hexarelin, a synthetic growth hormone-releasing peptide, is shown to be protective in cardiovascular diseases such as myocardial infraction and atherosclerosis. However, the functional role of hexarelin in abdominal aortic aneurysm (AAA) remains undefined. The present study determined the effect of hexarelin administration (200 μg/kg twice per day) in a mouse model of elastase-induced abdominal aortic aneurysm. Echocardiography and in situ pictures showed hexarelin decreased infrarenal aorta diameter. Histology staining showed elastin degradation was improved in hexarelin-treated group. Hexarelin rescued smooth muscle cell contractile phenotype with increased α-SMA and decreased MMP2. Furthermore, hexarelin inhibited inflammatory cell infiltration, NLRP3 inflammasome activation and IL-18 production. Particularly, hexarelin suppressed NF-κB signaling pathway which is a key initiator of inflammatory response. These results demonstrated that hexarelin attenuated AAA development by inhibiting SMC phenotype switch and NF-κB signaling mediated inflammatory response., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Accumulation of vascular smooth muscle cells (VSMCs) is a hallmark of multiple vascular pathologies, including following neointimal formation after injury and atherosclerosis. However, human VSMCs in advanced atherosclerotic lesions show reduced cell proliferation, extensive and persistent DNA damage, and features of premature cell senescence. Here, we report that stress-induced premature senescence (SIPS) and stable expression of a telomeric repeat-binding factor 2 protein mutant (TRF2 T188A ) induce senescence of human VSMCs, associated with persistent telomeric DNA damage. VSMC senescence is associated with formation of micronuclei, activation of cGAS-STING cytoplasmic sensing, and induction of multiple pro-inflammatory cytokines. VSMC-specific TRF2 T188A expression in a multicolor clonal VSMC-tracking mouse model shows no change in VSMC clonal patches after injury, but an increase in neointima formation, outward remodeling, senescence and immune/inflammatory cell infiltration or retention. We suggest that persistent telomere damage in VSMCs inducing cell senescence has a major role in driving persistent inflammation in vascular disease.

Familial hypercholesterolemia (FH) is increasingly associated with inflammation, a phenotype that persists despite treatment with lipid lowering therapies. The alternative C3 complement system (C3), as a key inflammatory mediator, seems to be involved in the atherosclerotic process; however, the relationship between C3 and lipids during plaque progression remains unknown. The aim of the study was to investigate by a systems biology approach the role of C3 in relation to lipoprotein levels during atherosclerosis (AT) progression and to gain a better understanding on the effects of C3 products on the phenotype and function of human lipid-loaded vascular smooth muscle cells (VSMCs). By mass spectrometry and differential proteomics, we found the extracellular matrix (ECM) of human aortas to be enriched in active components of the C3 complement system, with a significantly different proteomic signature in AT segments. Thus, C3 products were more abundant in AT-ECM than in macroscopically normal segments. Furthermore, circulating C3 levels were significantly elevated in FH patients with subclinical coronary AT, evidenced by computed tomographic angiography. However, no correlation was identified between circulating C3 levels and the increase in plaque burden, indicating a local regulation of the C3 in AT arteries. In cell culture studies of human VSMCs, we evidenced the expression of C3, C3aR (anaphylatoxin receptor) and the integrin α M β 2 receptor for C3b/iC3b (RT-PCR and Western blot). C3mRNA was up-regulated in lipid-loaded human VSMCs, and C3 protein significantly increased in cell culture supernatants, indicating that the C3 products in the AT-ECM have a local vessel-wall niche. Interestingly, C3a and iC3b (C3 active fragments) have functional effects on VSMCs, significantly reversing the inhibition of VSMC migration induced by aggregated LDL and stimulating cell spreading, organization of F-actin stress fibers and attachment during the adhesion of lipid-loaded human VSMCs. This study, by using a systems biology approach, identified molecular processes involving the C3 complement system in vascular remodeling and in the progression of advanced human atherosclerotic lesions.

Aims: Uromodulin is produced exclusively in the kidney and secreted into both urine and blood. Serum levels of uromodulin are correlated with kidney function and reduced in chronic kidney disease (CKD) patients, but physiological functions of serum uromodulin are still elusive. This study investigated the role of uromodulin in medial vascular calcification, a key factor associated with cardiovascular events and mortality in CKD patients., Methods and Results: Experiments were performed in primary human (HAoSMCs) and mouse (MOVAS) aortic smooth muscle cells, cholecalciferol overload and subtotal nephrectomy mouse models and serum from CKD patients. In three independent cohorts of CKD patients, serum uromodulin concentrations were inversely correlated with serum calcification propensity. Uromodulin supplementation reduced phosphate-induced osteo-/chondrogenic transdifferentiation and calcification of HAoSMCs. In human serum, pro-inflammatory cytokines tumour necrosis factor α (TNFα) and interleukin-1β (IL-1β) co-immunoprecipitated with uromodulin. Uromodulin inhibited TNFα and IL-1β-induced osteo-/chondrogenic signalling and activation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated β cells (NF-kB) as well as phosphate-induced NF-kB-dependent transcriptional activity in HAoSMCs. In vivo, adeno-associated virus (AAV)-mediated overexpression of uromodulin ameliorated vascular calcification in mice with cholecalciferol overload. Conversely, cholecalciferol overload-induced vascular calcification was aggravated in uromodulin-deficient mice. In contrast, uromodulin overexpression failed to reduce vascular calcification during renal failure in mice. Carbamylated uromodulin was detected in serum of CKD patients and uromodulin carbamylation inhibited its anti-calcific properties in vitro., Conclusions: Uromodulin counteracts vascular osteo-/chondrogenic transdifferentiation and calcification, at least in part, through interference with cytokine-dependent pro-calcific signalling. In CKD, reduction and carbamylation of uromodulin may contribute to vascular pathology., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

The pathobiology of atherosclerotic disease requires further elucidation to discover new approaches to address its high morbidity and mortality. To date, over 17 million cardiovascular-related deaths have been reported annually, despite a multitude of surgical and nonsurgical interventions and advances in medical therapy. Existing strategies to prevent disease progression mainly focus on management of risk factors, such as hypercholesterolemia. Even with optimum current medical therapy, recurrent cardiovascular events are not uncommon in patients with atherosclerosis, and their incidence can reach 10-15% per year. Although treatments targeting inflammation are under investigation and continue to evolve, clinical breakthroughs are possible only if we deepen our understanding of vessel wall pathobiology. Vascular smooth muscle cells (VSMCs) are one of the most abundant cells in vessel walls and have emerged as key players in disease progression. New technologies, including in situ hybridization proximity ligation assays, in vivo cell fate tracing with the CreER T2 -loxP system and single-cell sequencing technology with spatial resolution, broaden our understanding of the complex biology of these intriguing cells. Our knowledge of contractile and synthetic VSMC phenotype switching has expanded to include macrophage-like and even osteoblast-like VSMC phenotypes. An increasing body of data suggests that VSMCs have remarkable plasticity and play a key role in cell-to-cell crosstalk with endothelial cells and immune cells during the complex process of inflammation. These are cells that sense, interact with and influence the behavior of other cellular components of the vessel wall. It is now more obvious that VSMC plasticity and the ability to perform nonprofessional phagocytic functions are key phenomena maintaining the inflammatory state and senescent condition and actively interacting with different immune competent cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Sorokin, Vickneson, Kofidis, Woo, Lin, Foo and Shanahan.)

Aims: Defects in efficient endothelial healing have been associated with complication of atherosclerosis such as post-angioplasty neoatherosclerosis and plaque erosion leading to thrombus formation. However, current preventive strategies do not consider re-endothelialization in their design. Here, we investigate mechanisms linking immune processes and defect in re-endothelialization. We especially evaluate if targeting phosphoinositide 3-kinase γ immune processes could restore endothelial healing and identify immune mediators responsible for these defects., Methods and Results: Using in vivo model of endovascular injury, we showed that both ubiquitous genetic inactivation of PI3Kγ and hematopoietic cell-specific PI3Kγ deletion improved re-endothelialization and that CD4+ T-cell population drives this effect. Accordingly, absence of PI3Kγ activity correlates with a decrease in local IFNγ secretion and its downstream interferon-inducible chemokine CXCL10. CXCL10 neutralization promoted re-endothelialization in vivo as the same level than those observed in absence of PI3Kγ suggesting a role of CXCL10 in re-endothelialization defect. Using a new established ex vivo model of carotid re-endothelialization, we showed that blocking CXCL10 restore the IFNγ-induced inhibition of endothelial healing and identify smooth muscle cells as the source of CXCL10 secretion in response to Th1 cytokine., Conclusion: Altogether, these findings expose an unforeseen cellular cross-talk within the arterial wall whereby a PI3Kγ-dependent T-cell response leads to CXCL10 production by smooth muscle cells which in turn inhibits endothelial healing. Therefore, both PI3Kγ and the IFNγ/CXCL10 axis provide novel strategies to promote endothelial healing., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Context: Atherosclerosis is a chronic inflammatory disease in which the plaques were built up inside of the artery. Interleukin-8 (IL-8, CXCL8) is an inflammatory factor, known to play an important role in the development of atherosclerosis. G31P is an antagonist of the IL-8 receptor, which plays roles in vascular smooth muscle cell (VSMC) proliferation and migration. Objective: This study is to investigate the therapeutic effect of G31P on atherosclerosis through a mouse model. Materials and methods: A mouse model of atherosclerosis was generated through feeding the ApoE -/- mice with high fat diet for 12 weeks. G31P was injected subcutaneously into the mice. The levels of keratinocyte chemoattractant (KC), CXCR2, TNF-α, and IFN-γ were analyzed through ELISA. The expressions of MMP-2, MMP-9, PCNA, and Mef2a in aortic tissues were detected through RT-qPCR. In A7r5 cells, the levels of p-ERK, ROCK1, and ROCK2 were analyzed by western blot. Intracellular calcium levels were measured through Fluo-3 AM assay. Results and disccussion: G31P suppressed the abnormal lipid profile and decreased the levels of KC, MMP-2, MMP-9, PCNA, and Mef2a in a mouse model of atherosclerosis. In addition, G31P also inhibited the expressions of p-ERK, ROCK1, ROCK2, and decreased the calcium concentrations in A7r5 cells. Conclusions: These findings indicate the potential therapeutic effects of G31P in suppressing the development of atherosclerosis by antagonizing the IL-8 receptor. G31P inhibits the proliferation and migration of VSMCs through regulating the Rho-kinase, ERK, and calcium-dependent pathways.

Objective: This study aims to explore whether the inhibitory role of metformin could inhibit LPS-induced inflammatory response in vascular smooth muscle cells (VSMCs) and its underlying mechanism., Materials and Methods: VSMCs were extracted from aorta of Sprague Dawley rats. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed to detect VSMCs viability after treatment with different concentrations of metformin. Levels of monocyte chemoattractant protein-1 (MCP-1), interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in VSMCs were detected by ELISA (enzyme-linked immunosorbent assay) and qRT-PCR (quantitative Real time-polymerase chain reaction). Protein and mRNA levels of toll like receptor 4 (TLR4) and peroxisome proliferators activated receptor γ (PPAR-γ) in VSMCs were detected by Western blot and qRT-PCR, respectively. Finally, VSMCs were treated with the PPAR-γ antagonist GW9662 and inflammatory indicators in cells were detected., Results: No significant difference in VSMCs viability was found after 0-2 mM metformin treatment or 500 μg/L LPS induction for 24 h. After 500 μg/L LPS induction in VSMCs for 24 h, levels of MCP-1, TNF-α and IL-6 were remarkably elevated. Both mRNA and protein levels of TLR4 in VSMCs were upregulated after 500 μg/L LPS induction for 24 h, which were remarkably reversed by the treatment of different concentrations of metformin. Knockdown of TLR4 remarkably inhibited LPS-induced inflammatory response in VSMCs, manifesting as decreased levels of MCP1, TNF-α and IL-6, which were further downregulated after combination treatment of TLR4 knockdown and 20 mM metformin. Furthermore, both mRNA and protein levels of PPAR-γ in VSMCs were downregulated after 500 μg/L LPS induction for 24 h, which were remarkably reversed by the treatment of different concentrations of metformin. GW9662 treatment resulted in elevated expressions of MCP-1, TNF-α and IL-6, which were reversed by metformin treatment., Conclusions: Metformin can effectively inhibit the mRNA and protein expressions of IL-6, MCP-1, and TNF-α in LPS-induced VSMCs. The anti-inflammatory effects of metformin inhibit the inflammatory response through downregulating rely on the downregulation of TLR4 expression and upregulation ofng PPAR-γ activity.

Vascular smooth muscle cells (VSMCs) constitute the major cells in the media layer of arteries, and are critical to maintain the integrity of the arterial wall. They participate in arterial wall remodeling, and play important roles in atherosclerosis throughout all stages of the disease. Studies demonstrate that VSMCs can adopt numerous phenotypes depending on inputs from endothelial cells (ECs) of the intima, resident cells of the adventitia, circulating immune cells, hormones, and plasma lipoproteins. This plasticity allows them to perform multiple tasks in physiology and disease. In this minireview, we focus on a previously underappreciated activity of VSMCs, i.e., their impact on atherosclerosis immunity via formation of artery tertiary lymphoid organs (ATLOs).

Atherosclerosis is widely known to be a chronic inflammatory disease. C-reactive protein (CRP), an important inflammatory factor, plays an essential role in the pathogenesis of atherosclerosis. Nicotine, the main addictive component of cigarette, has been shown to induce the production of CRP. The aim of this study was to investigate the effect of rosmarinic acid (RA), a polyphenol with antiinflammatory activity, on nicotine-induced elevation of CRP in vascular smooth muscle cells (VSMCs). We found that pretreatment of VSMCs with RA attenuated nicotine-induced expression of CRP in a time- and dose-dependant manner. In addition, RA also inhibited the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome and reactive oxygen species (ROS) production resulting from nicotine treatment in VSMCs. To confirm these findings in vivo, we constructed a nicotine-induced atherosclerosis rat model. RA did not significantly reduce the serum nicotine level of the rats, whereas it significantly decreased the levels of serum lipids, including concentrations of cholesterol, triglycerides, and low-density lipoprotein cholesterol, and the serum level of CRP. RA also led to diminished nicotine-induced activation of NLRP3 inflammasome and elevation in the CRP level in the aortic tissue of the model rats. The results of this study suggested a protective role of RA in nicotine-induced atherosclerosis by inhibiting the ROS-NLRP3 inflammasome-CRP axial, and RA therefore represented a potential effective therapeutic approach to atherosclerosis, in particular for those who smoke., (© 2018 Wiley Periodicals, Inc.)

Objective: Damage and pathogen associated molecular patterns such as oxidized low-density lipoprotein (oxLDL) or bacillus Calmette-Guerin (BCG) vaccine can induce long term pro-inflammatory priming in monocytes and macrophages due to metabolic and epigenetic reprogramming-an emerging new concept called trained innate immunity. Vascular smooth muscle cells express pattern recognition receptors involved in trained innate immunity in monocytes. Here we investigated whether the mechanisms of trained innate immunity also control a proinflammatory phenotype in human coronary smooth muscle cells. Methods: Human coronary smooth muscle cells were primed with oxLDL or BCG for 24 h. After a resting time of 4 to 7 days, the cells were restimulated with either PAM3cys4, LPS or TNFα and cytokine production or mRNA expression were measured. Then, mechanisms of monocyte trained innate immunity were analyzed in smooth muscle cells, including receptors, intracellular pathways as well as metabolic and epigenetic reprogramming. Results: Priming with oxLDL or BCG lead to a significantly increased production of IL6, IL8 and MCP-1 following restimulation. OxLDL priming had little effect on the expression of macrophage or SMC marker genes. Proinflammatory priming of smooth muscle cells induced mTOR-HIF1α-signaling and could be blocked by mTOR-, TLR2-, and TLR4-inhibition. Finally, metabolic and epigenetic mechanisms of trained innate immunity in monocytes could be replicated in smooth muscle cells, including increased glucose consumption, lactate production, responsiveness to 6-fluoromevalonate and mevalonate treatment and inhibition of priming by the histone methyltransferase inhibitor methylthioadenosine (MTA). Conclusion: We demonstrate for the first time that mechanisms of the so called trained innate immunity control a proinflammatory phenotype in non-immune cells of the vascular wall. Our findings warrant further research into the specificity of trained innate immunity as an immune cell response as well as the mechanisms of vascular smooth muscle cells inflammation.

Objective: Previous in vitro and animal studies have suggested that osteopontin (OPN), an inflammatory extracellular matrix protein, is involved in the formation and growth of abdominal aortic aneurysms (AAAs). However, the mechanism by which this occurs continues to be nebulous. The relationship between OPN and inflammation-suppressing lymphocytes present in the human AAA condition was investigated and presented herein., Methods: Serum OPN concentrations were measured in healthy, risk factor-matched non-AAA and AAA patients by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was used to determine the source of OPN secretion using aortic tissue collected from multiorgan donors and AAA patients undergoing open surgical repair. Vascular smooth muscle cells (VSMCs) were exposed to various inflammatory mediators, and OPN expression was evaluated by quantitative reverse transcriptase-polymerase chain reaction and ELISA. The inflammatory nature of OPN and the aortic wall was determined using a TR1 suppressor cell induction assay as a surrogate and characterized by ELISA and fluorescence-activated cell sorting., Results: OPN was found to be elevated in both the plasma and aortic homogenate of AAA patients compared with controls. On immunohistochemistry, OPN localized to the tunica media of the diseased aorta but was minimally expressed in healthy aorta. In vitro, cigarette smoke extract was the most potent stimulator of OPN secretion by VSMCs and increased both messenger RNA and supernatant concentrations. OPN demonstrated an ability to inhibit the induction of interleukin 10-secreting TR1 lymphocytes, a depleted population in the AAA patient, from naive precursors. Last, neutralizing receptor targets of OPN in the setting of AAA homogenate coincubation abrogated the inhibition of TR1 induction., Conclusions: OPN, secreted by the VSMCs of the tunica media, is elevated in the circulating plasma and aortic wall of patients with AAA. It can inhibit the induction of the TR1 suppressor cell, leading to an overall proinflammatory state contributing to progressive aortic wall breakdown and dilation., (Copyright © 2017 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Purpose of Review: Smooth muscle cells (SMCs) form the thickened intimal layer in atherosclerosis-prone arteries in early life, and provide the initial site for retention and uptake of atherogenic lipoproteins. Here we review current knowledge regarding the importance of SMCs in the deposition of cholesterol in atherosclerotic plaque., Recent Findings: SMCs were found to comprise at least 50% of total foam cells in human coronary artery atherosclerosis, and exhibit a selective loss of expression of the cholesterol efflux promoter ATP-binding cassette transporter A1. Cholesterol loading induced a loss of SMC gene expression and an increase in macrophage and proinflammatory marker expression by cultured mouse and human arterial SMCs, with reversal of these effects upon removal of the excess cholesterol. Mice engineered to track all cells of SMC lineage indicated that, at most, SMCs make up about one-third of total cells in atherosclerotic plaque in these animals., Summary: SMCs appear to be the origin of the majority of foam cells in human atherosclerotic plaque. Recent studies suggest a renaissance of research on the role of SMCs in atherosclerosis is needed to make the next leap forward in the prevention and treatment of this disease.

Statins have often been used in atherosclerosis treatment because of its pleiotropic effects on inflammation. However, some adverse effects of high doses of statin show reverse effects after withdrawal. Cyanidin-3-glucoside (C3G) is a powerful anti-inflammation and antioxidant that has been of interest for use in combination with low doses of statin, which may be alternative treatment for atherosclerosis. The objective is to investigate the synergistic effect of atorvastatin and C3G in angiotensin II (Ang II)-induced inflammation in vascular smooth muscle cells. Human aortic smooth muscle cells (HASMCs) were exposed to Ang II with or without atorvastatin and C3G alone, or in combination. The results revealed that the combination of atorvastatin and C3G produces synergism against inflammation and oxidative stress. The mechanism of the combination of atorvastatin and C3G suppressed the translocation of the p65 subunit of NF-κB from cytosol to nucleus, and attenuated the expression of proteins including inducible nitric oxide synthase, intracellular adhesion molecule 1(ICAM-1), and vascular cell adhesion molecule 1(VCAM-1), in addition to nitric oxide (NO) production. Moreover, C3G exerts the antioxidative properties of atorvastatin through down-regulating NOX1 and promoting the activity of the Nrf2(-)ARE signaling pathway and downstream proteins including heme oxygenase (HO-1), NAD(P)H:quinoneoxidoreductase 1 (NQO-1), and glutamate-cysteine ligase catalytic subunit (γ-GCLC), besides increasing the activity of superoxide dismutase (SOD) enzymes. Taken together, these results suggest that a combination of low dose statins and C3G might serve as a potential regulator of the atherosclerosis process which is mediated by attenuating oxidative stress, thereby inhibiting NF-κB and activating Nrf2 signaling pathways induced by Ang II., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Several previous reports suggested that many commercially available antibodies directed against G protein-coupled receptors (GPCR) lack sufficient selectivity. Accordingly, it has been proposed that receptor antibodies should be validated by at least one of several criteria, such as testing tissues or cells after knockout or silencing of the corresponding gene. Here, we tested whether 12 commercially available antibodies directed against α-adrenergic receptor (AR) subtypes (α1A/B/D, α2A/B/C), atypical chemokine receptor 3 (ACKR3), and vasopressin receptor 1A (AVPR1A) suffice these criteria. We detected in flow cytometry experiments with human vascular smooth muscle cells that the fluorescence signals from each of these antibodies were reduced by 46 ± 10 %-91 ± 2 % in cells treated with commercially available small interfering RNA (siRNA) specific for each receptor, as compared with cells that were incubated with non-targeting siRNA. The tested antibodies included anti-ACKR3 (R&D Systems, mab42273), for which specificity has previously been demonstrated. Staining with this antibody resulted in 72 ± 5 % reduction of the fluorescence signal after ACKR3 siRNA treatment. Furthermore, staining with anti-α1A-AR (Santa Cruz, sc1477) and anti-ACKR3 (Abcam, ab38089), which have previously been reported to be non-specific, resulted in 70 ± 19 % and 80 ± 4 % loss of the fluorescence signal after α1A-AR and ACKR3 siRNA treatment, respectively. Our findings demonstrate that the tested antibodies show reasonable selectivity for their receptor target under our experimental conditions. Furthermore, our observations suggest that the selectivity of GPCR antibodies depends on the method for which the antibody is employed, the species from which cells/tissues are obtained, and on the type of specimens (cell, tissue/cell homogenate, or section) tested.

The pathophysiological mechanisms of the immune activation of smooth muscle cells are not well understood. Increased expression of Axl, a receptor tyrosine kinase, was recently found in arteries from patients after coronary bypass grafts. In the present study, we hypothesized that Axl-dependent immune activation of smooth muscle cells regulates vein graft remodeling. We observed a twofold decrease in intimal thickening after vascular and systemic depletion of Axl in vein grafts. Local depletion of Axl had the greatest effect on immune activation, whereas systemic deletion of Axl reduced intima due to an increase in apoptosis in vein grafts. Primary smooth muscle cells isolated from Axl knockout mice had reduced proinflammatory responses by prevention of the STAT1 pathway. The absence of Axl increased suppressor of cytokine signaling (SOCS)1 expression in smooth muscle cells, a major inhibitory protein for STAT1. Ultrasound imaging suggested that vascular depletion of Axl reduced vein graft stiffness. Axl expression determined the STAT1-SOCS1 balance in vein graft intima and progression of the remodeling. The results of this investigation demonstrate that Axl promotes STAT1 signaling via inhibition of SOCS1 in activated smooth muscle cells in vein graft remodeling., (Copyright © 2015 the American Physiological Society.)

Objective: Atherosclerosis, the cause of 50% of deaths in westernized societies, is widely regarded as a chronic vascular inflammatory disease. Vascular smooth muscle cell (VSMC) inflammatory activation in response to local proinflammatory stimuli contributes to disease progression and is a pervasive feature in developing atherosclerotic plaques. Therefore, it is of considerable therapeutic importance to identify mechanisms that regulate the VSMC inflammatory response., Approach and Results: We report that myocardin, a powerful myogenic transcriptional coactivator, negatively regulates VSMC inflammatory activation and vascular disease. Myocardin levels are reduced during atherosclerosis, in association with phenotypic switching of smooth muscle cells. Myocardin deficiency accelerates atherogenesis in hypercholesterolemic apolipoprotein E(-/-) mice. Conversely, increased myocardin expression potently abrogates the induction of an array of inflammatory cytokines, chemokines, and adhesion molecules in VSMCs. Expression of myocardin in VSMCs reduces lipid uptake, macrophage interaction, chemotaxis, and macrophage-endothelial tethering in vitro, and attenuates monocyte accumulation within developing lesions in vivo. These results demonstrate that endogenous levels of myocardin are a critical regulator of vessel inflammation., Conclusions: We propose myocardin as a guardian of the contractile, noninflammatory VSMC phenotype, with loss of myocardin representing a critical permissive step in the process of phenotypic transition and inflammatory activation, at the onset of vascular disease., (© 2015 American Heart Association, Inc.)

The transient receptor potential vanilloid type 1 (TRPV1) channel, a ligand-gated cation channel of the TRP subfamily, can be activated by multiple stimuli, including capsaicin. Currently, cumulative studies have demonstrated an interesting link between TRPV1 and cardiovascular diseases, including hypertension. Additionally, the protective effect of TRPV1 against hypertension and its related disorders has been proved to be partly involved with the improved action of vascular smooth muscle cells (VSMCs). This review focuses on the current knowledge of TRPV1 in improving VSMC function and attenuating hypertension., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Our previous study proved that CD137-CD137L interaction can regulate the expression of NFATc1. Here, we investigated whether CD137 signaling regulates the expression of NFATc1 in mice VSMCs through TRAF6/NF-κB p65 pathway. Data shows that the CD137 expression can be stimulated by TNF-α in a time-dependent manner in mouse VSMCs. Knockdown of TRAF6 by siTRAF6 significantly attenuated agonist-CD137mAb induced increase of NF-κB p65 and NFATc1 in VSMCs. Pretreatment with a NF-κB inhibitor PDTC for 30 min inhibited the expression of p-p65 in both cytoplasm and nucleus in VSMCs. Thus, the protein level of NFATc1 can be suppressed through inhibition of p-p65. Finally, we also show that the levels of IL-2 and IL-6 can be increased by agonist-CD137 stimulation and decreased when NFATc1 was suppressed. Our data suggest that activated CD137 signaling regulates the expression of NFATc1 and its downstream factors through TRAF6/NF-κB p65 pathways in VSMCs. These findings provide a novel target for treatment of atherosclerosis.

Objective: 17β-Estradiol (E2) offers cardiovascular protection in young female animals and postmenopausal women. In contrast, randomized trials of menopausal hormones performed in older women have shown harm or no cardiovascular benefit. We hypothesize that E2 effects on vascular inflammation are age dependent., Approach and Results: Young (10 weeks) and aged (52 weeks) female C57BL/6 mice were used as source for primary cultures of bone marrow-derived macrophages (BMMs) and vascular smooth muscle cells (VSMCs). E2 pretreatment of cells derived from young mice attenuated C-reactive protein (CRP)-induced expression of inflammatory mediators. In contrast, E2 pretreatment of cells from aged mice did not alter (BMMs) or paradoxically exaggerated (VSMCs) inflammatory mediator response to CRP. Using E2 receptor (ER) knockout mice, we demonstrated that E2 regulates inflammatory response to CRP in BMMs via ERα and in VSMCs via ERβ. BMMs derived from aged (versus young) mice expressed significantly less ERα mRNA and protein. A selective ligand of the novel ER GPR30 reproduced the E2 effects in BMMs and VSMCs. Unlike in young mice, E2 did not reduce neointima formation in ligated carotid arteries of aged CRP transgenic mice., Conclusions: E2 attenuates inflammatory response to CRP in BMMs and VSMCs derived from young but not aged mice and reduces neointima formation in injured carotid arteries of young but not aged CRP transgenic mice. ERα expression in BMMs is greatly diminished with aging. These data suggest that vasoprotective effects of E2 are age dependent and may explain the vasotoxic effects of E2 seen in clinical trials of postmenopausal women., (© 2014 American Heart Association, Inc.)

Purpose: Restenosis caused by intimal hyperplasia (IH) remains a significant drawback for vascular interventions. It is crucial to understand the molecular mechanisms that control activation of smooth muscle cells (SMCs) after the injury in order to develop strategies to prevent IH. The purpose of the present study was to investigate the early alterations in arterial-wall gene expression after balloon injury in the rat carotid artery with focus on the induction of an inflammatory response., Materials and Methods: Twenty-four male Sprague-Dawley rats were subjected to injury of the left common carotid artery by using a 2-F Fogarty catheter. The arteries were harvested 5, 10, and 20 hours after injury. Uninjured arteries from an additional eight rats were used as controls. RNA was isolated and used for genome-wide microarray expression analysis, followed by validation of selected genes with quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemistry was performed on the cross-sectioned vessels., Results: Analysis of gene expression by microarrays showed that the most differentially expressed genes were primarily associated with inflammation, cell proliferation, migration, and adhesion. As confirmed by qRT-PCR, microarray data showed a significant (P < .005) upregulation of cytokines and chemokines (IL-6, CCL2, CXCL1, AIMP1, and CD44) just 5 hours after injury. Immunohistochemistry demonstrated that CCL2 and the adhesion receptor CD44 were expressed by SMCs in the early response to injury and in the absence of leukocyte infiltration., Conclusions: Arterial injury is followed by an early induction of inflammatory genes in the vessel wall that appears to be confined to SMCs., (Copyright © 2014 SIR. Published by Elsevier Inc. All rights reserved.)

Oxidized low-density lipoprotein (oxLDL)-regulated secretion of inflammatory cytokines in smooth muscle cells (SMCs) is regarded as an important step in the progression of atherosclerosis; however, its underlying mechanism remains unclear. This study investigated the role of toll-like receptor 4 (TLR4) in oxLDL-induced expression of inflammatory cytokines in SMCs both in vivo and in vitro. We found that the levels of TLR4, interleukin 1-β (IL1-β), tumor necrosis factor-α (TNFα), monocyte chemoattractant protein 1 (MCP-1) and matrix metalloproteinase-2 (MMP-2) expression were increased in the SMCs of atherosclerotic plaques in patients with femoral artery stenosis. In cultured primary arterial SMCs from wild type mice, oxLDL caused dose- and time-dependent increase in the expression levels of TLR4 and cytokines. These effects were significantly weakened in arterial SMCs derived from TLR4 knockout mice (TLR4-/-). Moreover, the secretion of inflammatory cytokines was blocked by TLR4-specific antibodies in primary SMCs. Ox-LDL induced activation of p38 and NFκB was also inhibited in TLR4-/- primary SMCs or when treated with TLR4-specific antibodies. These results demonstrated that TLR4 is a crucial mediator in oxLDL-induced inflammatory cytokine expression and secretion, and p38 and NFκB activation.

Macrophages and vascular smooth muscle cells (VSMCs) are the major cell types involved in foam cell formation associated with atherosclerosis. The aim of this experiment was to clarify cell-specific regulation of LDL receptor in THP-1 macrophages and human VSMCs under physiological and inflammatory conditions and its potential mechanisms. Inflammatory stress was induced by adding lipopolysaccharide (LPS) to human THP-1 macrophages and human VSMCs. Intracellular total cholesterol, free cholesterol, and cholesterol ester were measured by an enzymic assay. Oil Red O staining was used to visualize lipid droplet accumulation in cells. Total cellular RNA was isolated from cells for detecting LDL receptor, sterol regulatory element binding protein (SREBP)-2 and SREBP cleavage-activating protein (SCAP) mRNA levels using real-time quantitative polymerase chain reaction. LDL receptor, SREBP-2 and SCAP protein expression were examined by Western blotting. The translocation of SCAP from ER to Golgi was detected by confocal microscopy. LDL loading increased intracellular cholesterol level, reducing LDL receptor mRNA level in both THP-1 macrophages and VSMCs under physiological conditions. The IC50 in VSMCs was 11.25 μg/ml, which is much lower than 18.125 μg/ml in THP-1 macrophages. With the increase in concentration of LPS (0-400 ng/ml), the LDL receptor mRNA levels were upregulated in both cells, but the curve of LDL receptor mRNA in VSMCs exhibited a flatter profile than that of THP-1 macrophages. Under the treatment of 200 ng/ml of LPS, the upregulation fold of the LDL receptor mRNA in THP-1 macrophages was much higher than that of VSMCs (0.33 vs 0.04). LDL receptor blocking agent heparin decreased lipid droplets induced by LPS significantly in THP-1 macrophages and VSMCs. LDL loading reduced the SREBP2 and SCAP protein expression under physiological conditions. Exposure to LPS caused overexpression of SREBP2 and SCAP despite a high concentration of LDL in the culture medium, and increased translocation of SCAP from the ER to the Golgi in the presence of 25 μg/ml of LDL. Inflammatory stress disrupts LDL receptor negative feedback regulation induced by intracellular cholesterol in both cell types, to a greater degree in THP-1 macrophages, which could be one reason why THP-1 macrophages are more prone to become foam cells under inflammatory stress.

Background: Absent in melanoma (AIM2) was recently identified to act as a cytosolic DNA sensor in innate immunity. Considering the role of chronic inflammation in atherosclerosis, we hypothesized that AIM2 may act as a damage signal that is activated in response to cellular stress likewise in vascular cells of larger arteries. We thus addressed AIM2 expression in healthy arterial wall and in different vascular lesions. In addition, AIM2 expression was characterized in cultured human aortic endothelial cells (HAoECs), smooth muscle cells (HAoSMCs), and T/G-HA-vascular smooth muscle cells (VSMCs) in response to different stimuli., Methods: Carotid and aortic lesions from patients who underwent surgery and normal arterial specimens were analyzed by immunohistochemistry for AIM2 expression. Cultured HAoECs, HAoSMCs, and T/G-HA-VSMCs were stimulated in vitro with proinflammatory cytokines (tumor necrosis factor-α, interferon-γ) or poly(dA:dT) and analyzed for AIM2 transcript and protein expression., Results: AIM2 was detected in ECs of the intima and vasa vasorum of normal carotid artery and aorta. Moreover, AIM2 was moderately expressed in VSMCs of normal media and intima layers, as well as in VSMCs of atherosclerotic lesions. Increased AIM2 expression was detected around the necrotic core of atherosclerotic carotid lesions and in the vasa vasorum neovasculature of aortic aneurysms. Subsequent in vitro analysis identified an endogenous AIM2 expression in cultured HAoECs, HAoSMCs, and T/G-HA-VSMCs that was markedly increased upon treatment of the cells with tumor necrosis factor-α, interferon-γ, or cytosolic DNA., Conclusions: ECs and VSMC are able to respond to inflammatory signals by upregulation of AIM2 expression, indicating a role of AIM2 in vascular pathogenesis., (Copyright © 2014 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

Reactive oxygen species (ROS) mediate cell-signaling processes in response to various ligands and play important roles in the pathogenesis of cardiovascular diseases. The present study reports that interleukin-22 (IL-22) elicits signal transduction in vascular smooth muscle cells (SMCs) through a ROS-dependent mechanism. We find that pulmonary artery SMCs express IL-22 receptor alpha 1 and that IL-22 activates STAT3 through this receptor. IL-22-induced signaling is found to be mediated by NADPH oxidase, as indicated by the observations that the inhibition and siRNA knock-down of this enzyme inhibit IL-22 signaling. IL-22 triggers the oxidative modifications of proteins through protein carbonylation and protein glutathionylation. Mass spectrometry identified some proteins that are carbonylated in response to IL-22 stimulation, including α-enolase, heat shock cognate 71kDa protein, mitochondrial 60kDa heat shock protein, and cytoplasmic 2 actin and determined that α-tubulin is glutathionylated. Protein glutathionylation and STAT3 phosphorylation are enhanced by the siRNA knock-down of glutaredoxin, while IL-22-mediated STAT3 phosphorylation is suppressed by knocking down thioredoxin interacting protein, an inhibitor of thioredoxin. IL-22 is also found to promote the growth of SMCs via NADPH oxidase. In rats, pulmonary hypertension is found to be associated with increased smooth muscle IL-22 expression. These results show that IL-22 promotes the growth of pulmonary vascular SMCs via a signaling mechanism that involves NADPH oxidase-dependent oxidation., (© 2013.)

Objective: Elevated levels of oxidative stress have been reported in abdominal aortic aneurysms (AAA), but which reactive oxygen species promotes the development of AAA remains unclear. Here, we investigate the effect of hydrogen peroxide (H2O2)-degrading enzyme catalase on the formation of AAA., Approach and Results: AAA were induced with the application of calcium chloride (CaCl2) on mouse infrarenal aortas. The administration of PEG-catalase, but not saline, attenuated the loss of tunica media and protected against AAA formation (0.91 ± 0.1 versus 0.76 ± 0.09 mm). Similarly, in a transgenic mouse model, catalase overexpression in the vascular smooth muscle cells preserved the thickness of tunica media and inhibited aortic dilatation by 50% (0.85 ± 0.14 versus 0.57 ± 0.08 mm). Further studies showed that injury with CaCl2 decreased catalase expression and activity in the aortic wall. Pharmacological administration or genetic overexpression of catalase restored catalase activity and subsequently decreased matrix metalloproteinase activity. In addition, a profound reduction in inflammatory markers and vascular smooth muscle cell apoptosis was evident in aortas of catalase-overexpressing mice. Interestingly, as opposed to infusion of PEG-catalase, chronic overexpression of catalase in vascular smooth muscle cells did not alter the total aortic H2O2 levels., Conclusions: The data suggest that a reduction in aortic wall catalase activity can predispose to AAA formation. Restoration of catalase activity in the vascular wall enhances aortic vascular smooth muscle cell survival and prevents AAA formation primarily through modulation of matrix metalloproteinase activity.

Phenotypes of lung smooth muscle cells in health and disease are poorly characterized. This is due, in part, to a lack of methodologies that allow for the independent and direct isolation of bronchial smooth muscle cells (BSMCs) and vascular smooth muscle cells (VSMCs) from the lung. In this paper, we describe the development of a bi-fluorescent mouse that permits purification of these two cell populations by cell sorting. By subjecting this mouse to an acute allergen based-model of airway inflammation that exhibits many features of asthma, we utilized this tool to characterize the phenotype of so-called asthmatic BSMCs. First, we examined the biophysical properties of single BSMCs from allergen sensitized mice and found increases in basal tone and cell size that were sustained ex vivo. We then generated for the first time, a comprehensive characterization of the global gene expression changes in BSMCs isolated from the bi-fluorescent mice with allergic airway inflammation. Using statistical methods and pathway analysis, we identified a number of differentially expressed mRNAs in BSMCs from allergen sensitized mice that code for key candidate proteins underlying changes in matrix formation, contractility, and immune responses. Ultimately, this tool will provide direction and guidance for the logical development of new markers and approaches for studying human lung smooth muscle.

A number of cardiovascular diseases, such as restenosis, aneurysm, and atherosclerosis, lead to vascular remodeling associated with complex adaptive reactions of different cell populations. These reactions include growth of smooth muscle cells, proliferation of endothelial cells, and the inflammatory response of macrophages. MicroRNAs (miRNAs), a class of short RNAs, play key roles in various biological processes and in the development of human disease by post-transcriptional regulation of gene expression. Here, we review the molecular mechanisms of a subset of miRNAs involved in vascular remodeling, including miR-143/145, miR-221/222, miR-126, miR-21, and miR-155. Some of these miRNAs, such as miR-143/145 and miR-126, have been shown to be protective during vascular remodeling, whereas others, such as miR-21, may promote the cellular response that leads to neointima formation. The increasing knowledge regarding the roles of miRNAs in vascular remodeling opens novel avenues for the treatment of various cardiovascular diseases. However, more in vivo studies on the functional roles of these miRNAs are required in the future.

Objective: Vascular smooth muscle cell (VSMC) migration is critically important for neointimal formation after vascular injury and atherosclerosis lesion formation. Copper (Cu) chelator inhibits neointimal formation, and we previously demonstrated that Cu transport protein antioxidant-1 (Atox1) is involved in Cu-induced cell growth. However, role of Atox1 in VSMC migration and neointimal formation after vascular injury is unknown., Approach and Results: Here, we show that Atox1 expression is upregulated in injured vessel, and it is colocalized with the Cu transporter ATP7A, one of the downstream targets of Atox1, mainly in neointimal VSMCs at day 14 after wire injury. Atox1(-/-) mice show inhibition of neointimal formation and extracellular matrix expansion, which is associated with a decreased VSMCs accumulation within neointima and lysyl oxidase activity. Mechanistically, in cultured VSMC, Atox1 depletion with siRNA inhibits platelet-derived growth factor-induced Cu-dependent VSMC migration by preventing translocation of ATP7A and small G protein Rac1 to the leading edge, as well as Cu- and Rac1-dependent lamellipodia formation. Furthermore, Atox1(-/-) mice show decreased perivascular macrophage infiltration in wire-injured vessels, as well as thioglycollate-induced peritoneal macrophage recruitment., Conclusions: Atox1 is involved in neointimal formation after vascular injury through promoting VSMC migration and inflammatory cell recruitment in injured vessels. Thus, Atox1 is a potential therapeutic target for VSMC migration and inflammation-related vascular diseases.

Rationale: Human genetics have implicated the 5-lipoxygenase enzyme in the pathogenesis of cardiovascular disease, and an inhibitor of the 5-lipoxygenase activating protein (FLAP) is in clinical development for asthma., Objective: Here we determined whether FLAP deletion modifies the response to vascular injury., Methods and Results: Vascular remodeling was characterized 4 weeks after femoral arterial injury in FLAP knockout mice and wild-type controls. Both neointimal hyperplasia and the intima/media ratio of the injured artery were significantly reduced in the FLAP knockouts, whereas endothelial integrity was preserved. Lesional myeloid cells were depleted and vascular smooth muscle cell (VSMC) proliferation, as reflected by bromodeoxyuridine incorporation, was markedly attenuated by FLAP deletion. Inflammatory cytokine release from FLAP knockout macrophages was depressed, and their restricted ability to induce VSMC migration ex vivo was rescued with leukotriene B(4). FLAP deletion restrained injury and attenuated upregulation of the extracellular matrix protein, tenascin C, which affords a scaffold for VSMC migration. Correspondingly, the phenotypic modulation of VSMC to a more synthetic phenotype, reflected by morphological change, loss of α-smooth muscle cell actin, and upregulation of vascular cell adhesion molecule-1 was also suppressed in FLAP knockout mice. Transplantation of FLAP-replete myeloid cells rescued the proliferative response to vascular injury., Conclusions: Expression of lesional FLAP in myeloid cells promotes leukotriene B(4)-dependent VSMC phenotypic modulation, intimal migration, and proliferation.

Background: Modulation of inflammatory signaling has been elucidated in several disease models. Acrolein, an environmental pollutant, has been linked to diseases such as atherosclerosis and to the inflammatory process involving nuclear factor κB (NFκB). Serum response factor (SRF), a transcription factor, regulates cell development, differentiation and proliferation through signaling molecules such as extracellular signal-regulated kinase 1/2 (ERK1/2) and CD36. We hypothesized that acrolein toxicity involves SRF in the process of activating NFκB and may involve CD36/ERK1/2., Methods: Vascular smooth muscle cells (VSMCs) were exposed to acrolein (0.5 μg/mL) in the presence or absence of 10 nM QNZ (NFκB inhibitor), 300 nM CCG1423 (SRF inhibitor) and 50 μM PD98059 (ERK1/2 inhibitor). Protein and RNA were isolated. Changes in expression were determined by Western blot and polymerase chain reaction (PCR) array., Results: Subtoxic doses of acrolein increased ERK1/2, SRF and NFκB protein expression, whereas CD36 expression was unchanged. Increase in NFκB expression was accompanied by an increase in activity. ERK1/2 inhibition only blunted SRF expression. SRF inhibition blunted NFκB expression but not that of ERK1/2. CD36 expression was unchanged in the presence of either inhibitor. PCR array analysis indicated up-regulation of nine genes (>4- to 50-fold) and down-regulation of six genes (>4- to 12-fold) involved in inflammatory signaling., Conclusions: We propose that SRF is required in acrolein activation of NFκB and is ERK1/2 dependent.

Objective: Migration of vascular smooth muscle cells (VSMCs) from the media into intima contributes to the development of atherosclerosis. Gene deletion experiments implicate a role for toll-like receptor 2 (TLR2) in atherogenesis. However, the underlying mechanisms remain unclear. We postulate that TLR2 promotes VSMC migration by enhancing interleukin (IL)-6 production., Methods and Results: Migration assays revealed that TLR2 agonists promoted VSMC migration but not cell proliferation or viability. TLR2 deficiency or inhibition of TLR2 signaling with anti-TLR2 antibody suppressed TLR2 agonist-induced VSMC migration and IL-6 production, which was mediated via p38 mitogen-associated protein kinase and extracellular signal-regulated kinase 1/2 signaling pathways. Neutralizing anti-IL-6 antibodies impaired TLR2-mediated VSMC migration and formation of filamentous actin fiber and lamellipodia. Blockade of p38 mitogen-associated protein kinase or extracellular signal-regulated kinase 1/2 activation inhibited TLR2 agonist pam3CSK4-induced phosphorylation of cAMP response element-binding protein, which regulates IL-6 promoter activity through the cAMP response element site. Moreover, cAMP response element-binding protein small interfering RNA inhibited pam3CSK4-induced IL-6 production and VSMC migration. Additionally, Rac1 small interfering RNA inhibited pam3CSK4-induced VSMC migration but not IL-6 production., Conclusions: Our results suggest that on ligand binding, TLR2 activates p38 mitogen-associated protein kinase and extracellular signal-regulated kinase 1/2 signaling in VSMCs. These signaling pathways act in concert to activate cAMP response element-binding protein and subsequent IL-6 production, which in turn promotes VSMC migration via Rac1-mediated actin cytoskeletal reorganization.

Leukotrienes are pro-inflammatory mediators that are locally produced in coronary atherosclerotic plaques. The response induced by cysteinyl leukotrienes (CysLT) in human coronary arteries may be altered under pathological conditions, such as atherosclerosis. The aim of the present study was to elucidate cysteinyl leukotriene signaling in vascular smooth muscle cells (SMCs) and the effects of inflammation on this process. Immunohistochemical analysis of human carotid endarterectomy samples revealed that the CysLT(1) leukotriene receptor was expressed in areas that also stained positive for α-smooth muscle actin. In human coronary artery smooth muscle cells, lipopolysaccharide significantly upregulated the CysLT(1) receptor and significantly enhanced the changes in intracellular calcium induced by leukotriene C(4) (LTC(4)). In these cells, the CysLT(1) receptor exhibited a perinuclear expression, and LTC(4) stimulation predominantly enhanced nuclear calcium increase, which was significantly inhibited by the CysLT(1) receptor antagonist MK-571. Microarray analysis revealed, among a number of significantly upregulated genes after 24 h stimulation of human coronary artery smooth muscle cells with LTC(4), a 5-fold increase in mRNA levels for plasminogen activator inhibitor (PAI)-2. The LTC(4)-induced increase in PAI-2 expression was confirmed by real-time quantitative PCR and ELISA and was inhibited by the CysLT(1) receptor antagonist MK-571 and by calcium chelators. In summary, pro-inflammatory stimulation of vascular SMCs upregulated a perinuclear CysLT(1) receptor expression coupled to nuclear calcium signaling and changes in gene expression, such as upregulation of PAI-2. Taken together, these findings suggest a role of nuclear CysLT(1) receptor signaling in vascular SMCs inducing gene expression patterns associated with atherosclerosis.

Vascular calcification predicts an increased risk for cardiovascular events in atherosclerosis, diabetes, and end-stage kidney diseases. Matrix Gla protein (MGP), an inhibitor of calcification, limits calcium phosphate deposition in the vessel wall. There are many factors contributing to the progression of atherosclerosis, including hypertension, hyperlipidemia, the renin-angiotensin system, and inflammation. Angiotensin II (ANG II) plays a crucial role in the atherogenic process through not only its pressor responses but also its growth-promoting and inflammatory effects. In this study, we investigated the role of MGP in ANG II-induced exacerbation of vascular calcification in human vascular smooth muscle cells (VSMCs). The expression of MGP, calcification, and apoptosis in human VSMCs were examined by Western blot analysis, real-time PCR, in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, and enzyme-linked immunosorbent assay, respectively. Increase in VSMC calcification in human atherosclerotic plaques upregulates MGP expression and apoptosis in a negative feedback manner. ANG II inhibited MGP expression in VSMCs via and in vitro in a dose- and time-dependent manner through ANG II type 1 receptor and NF-κB signaling pathway. Meanwhile, MGP inhibited the calcification, caspase-3 activity, activation of runt-related transcription factor 2, and release of inflammatory cytokines by VSMCs induced by calcification medium (2.5 mM P(i)) and ANG II in vitro. These observations provide evidence that ANG II exacerbates vascular calcification through activation of the transcription factors, runt-related transcription factor 2 and NF-κB, and regulation of MGP, inflammatory cytokines expression in human VSMCs.

Vascular calcification is a major risk factor for the cardiovascular disease, yet its underlying molecular mechanisms remain to be elucidated. Recently, we identified that osteogenic signals via bone morphogenetic protein (BMP)-2 exerted by vascular smooth muscle cells (VSMCs) play a crucial role in the formation of atherosclerotic plaque calcification. Here we report a synergistic interaction between macrophages and VSMCs with respect to plaque calcification. Treatment with conditioned medium (CM) of macrophages dramatically enhanced BMP-2 expression in VSMCs, while it substantially reduced the expression of matrix Gla-protein (MGP) that inhibits the BMP-2 osteogenic signaling. As a result, macrophages significantly accelerated the osteoblastic differentiation of C2C12 cells induced by VSMC-CM. In contrast, macrophage-CM did not enhance the osteoblastic gene expressions in VSMCs, indicating that macrophages unlikely induced the osteoblastic trans-differentiation of VSMCs. We then examined the effect of recombinant TNF-α and IL-1β on the VSMC-derived osteogenic signals. Similar to the macrophage-CM, both cytokines enhanced BMP-2 expression and reduced MGP expression in VSMCs. Nevertheless, only the neutralization of TNF-α but not IL-1β attenuated the effect of macrophage-CM on the expression of these genes in VSMCs, due to the very low concentration of IL-1β in the macrophage-CM. On the other hand, VSMCs significantly enhanced IL-1β expression in macrophages, which might in turn accelerate the VSMC-mediated osteogenic signals. Together, we identified a unique role of macrophages in the formation of plaque calcification in coordination with VSMCs. This interaction between macrophages and VSMCs is a potential therapeutic target to treat and prevent the atherosclerotic plaque calcification., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Both paclitaxel and zotarolimus are currently employed in vascular interventional therapies, such as drug-eluting stents, and are under investigation for use in other novel drug-device combination products. Paclitaxel is a microtubule-stabilizing compound with potent antiproliferative properties and antimigration effects, whereas zotarolimus is a potent mammalian target of rapamycin inhibitor with antiproliferative and antiinflammatory properties. This study was intended to compare paclitaxel and zotarolimus for intravascular applications in which drug exposure time may be reduced, such as in drug-coated balloons. These applications are generally aimed at reducing neointimal hyperplasia by limiting smooth muscle cell (SMC) proliferation and inflammatory cell recruitment, while minimally interfering with vessel reendothelialization after balloon denudation. In the cellular models described in this study, transient exposure of zotarolimus resulted in the sustained inhibition of SMC proliferation and reduced endothelial cell (EC) proinflammatory cytokine expression, while not affecting EC migration and viability. Transient exposure of paclitaxel inhibited SMC proliferation, EC migration, and overall cell viability, with no effect on expression of the proinflammatory biomarkers studied.

Vascular calcification is a major risk factor of cardiovascular mortality, particularly for patients with end-stage renal disease and diabetes. Although chronic inflammation is one of the etiologic factors, the underlying mechanism is not fully understood. To clarify this, we studied how nuclear factor-kappa B (NF-κB) induction, a mediator of inflammation, might promote vascular calcification. Activation of NF-κB by tumor necrosis factor (TNF) promoted inorganic phosphate-induced calcification in human aortic smooth muscle cells. Pyrophosphate (an inhibitor of calcification) efflux to the extracellular matrix was suppressed along with the decreased expression of ankylosis protein homolog (ANKH), a transmembrane protein that controls pyrophosphate efflux of cells. The restoration of ANKH expression in these cells overcame the decreased pyrophosphate efflux and calcification. Tristetraprolin, a downstream product of NF-κB activation, may mediate destabilization of ANKH mRNA as its knockdown by shRNA increased ANKH expression and decreased calcification. Furthermore, a rat chronic renal failure model, with increased serum TNF levels, activated NF-κB and decreased ANKH levels. In contrast, the inhibition of NF-κB maintained ANKH expression and attenuated vascular calcification both in vivo and in vitro. Both human calcified atherosclerotic lesions and arteries from patients with chronic kidney disease had activated NF-κB and decreased ANKH expression. Thus, TNF-activated NF-κB promotes inflammation-accelerated vascular calcification by inhibiting ankylosis protein homolog expression and consequent pyrophosphate secretion.

Objective: The adventitia is increasingly recognized as an important player during the development of intimal hyperplasia. However, the mechanism of adventitial cell recruitment to the subintimal space remains largely undefined. We have shown previously that gene transfer of protein kinase C-delta (PKCδ) increases apoptosis of smooth muscle cells following balloon injury. In the current study, we investigated a potential role of PKCδ in regulating the recruitment of adventitial cells., Methods and Results: Conditioned media from PKCδ-overexpressing smooth muscle cells stimulated migration and CCR2 expression of adventitial fibroblasts through a MCP-1 dependent mechanism. Following balloon injury of rat carotid arteries, overexpression of PKCδ in smooth muscle cells significantly increased MCP-1 and CCR2 expression and the number of adventitia-originated cells detected in the neointima. Administration of an anti-MCP-1 antibody markedly diminished the recruitment of adventitial cells. Combined PKCδ overexpression and anti-MCP-1 inhibited intimal hyperplasia more effectively than either approach alone., Conclusions: Our data suggest that PKCδ regulates recruitment of adventitial cells to the neointima via a mechanism involving upregulation of the MCP-1/CCR2 signaling axis in injured arteries. Blockage of MCP-1 while enhancing apoptosis may serve as a potential therapeutic strategy to attenuate intimal hyperplasia.

Objective: Vascular smooth muscle cells (VSMC) from type 2 diabetic db/db mice exhibit enhanced proinflammatory responses implicated in accelerated vascular complications. We examined the role of microRNA(miR)-200 family members and their target Zeb1, an E-box binding transcriptional repressor, in these events., Methods and Results: The expression levels of miR-200b, miR-200c, and miR-429 were increased, although protein levels of Zeb1 were decreased in VSMC and aortas from db/db mice relative to control db/+ mice. Transfection of miR-200 mimics into VSMC downregulated Zeb1 by targeting its 3'-UTR, upregulated the inflammatory genes cyclooxygenase-2 and monocyte chemoattractant protein-1, and promoted monocyte binding in db/+VSMC. In contrast, miR-200 inhibitors reversed the enhanced monocyte binding of db/dbVSMC. Zeb1 gene silencing with siRNAs also increased these proinflammatory responses in db/+VSMC confirming negative regulatory role of Zeb1. Both miR-200 mimics and Zeb1 siRNAs increased cyclooxygenase-2 promoter transcriptional activity. Chromatin immunoprecipitation showed that Zeb1 occupancy at inflammatory gene promoters was reduced in VSMC from type 2 diabetic db/db mice. Furthermore, Zeb1 knockdown increased miR-200 levels demonstrating a feedback regulatory loop., Conclusion: Disruption of the reciprocal negative regulatory loop between miR-200 and Zeb1 under diabetic conditions enhances proinflammatory responses of VSMC implicated in vascular complications.

Heme oxygenase-1 (HO-1) has potent anti-inflammatory activity and recognized vascular protective effects. We have recently described the expression and vascular protective effects of an anti-inflammatory interleukin (IL-19), in vascular smooth muscle cells (VSMC) and injured arteries. The objective of this study was to link the anti-inflammatory effects of IL-19 with HO-1 expression in resident vascular cells. IL-19 induced HO-1 mRNA and protein in cultured human VSMC, as assayed by quantitative RT-PCR, immunoblot, and ELISA. IL-19 does not induce HO-1 mRNA or protein in human endothelial cells. IL-19 activates STAT3 in VSMC, and IL-19-induced HO-1 expression is significantly reduced by transfection of VSMC with STAT3 siRNA or mutation of the consensus STAT binding site in the HO-1 promoter. IL-19 treatment can significantly reduce ROS-induced apoptosis, as assayed by Annexin V flow cytometry. IL-19 significantly reduced ROS concentrations in cultured VSMC. The IL-19-induced reduction in ROS concentration is attenuated when HO-1 is reduced by siRNA, indicating that the IL-19-driven decrease in ROS is mediated by HO-1 expression. IL-19 reduces vascular ROS in vivo in mice treated with TNFα. This points to IL-19 as a potential therapeutic for vascular inflammatory diseases and a link for two previously unassociated protective processes: Th2 cytokine-induced anti-inflammation and ROS reduction.

Objective: To investigate the expression of NOD2 in human VSMCs, its role in the production of inflammatory cytokines in VSMC and the possible interaction of NOD2-mediated signaling pathway with those mediated by TLR2 and TLR4., Methods: Human coronary artery smooth muscle cells were stimulated with NOD2 agonist MDP alone or in combination with either TLR2 agonist PAM3 or TLR4 agonist LPSs. The mRNA expression of NOD2 and FGF-2 were measured by RT-PCR. The concentration of IL-8 and TNF-α in the culture supernatants was determined by ELISA. VSMC proliferation ability was analyzed by MTT assay., Results: MDP up regulated the expression of NOD2 mRNA in VSMC in a time-dependent manner, up regulated the expression of FGF-2 mRNA in VSMC, induced the production of IL-8 and TNF-α, and promoted the proliferation of VSMC. Additionally, MDP synergied with LPS and PAM3 to promote the proliferation of VSMC and induce the production of IL-8 and TNF-α., Conclusion: The activation of NOD2-mediated innate immune signaling pathway can increase the proliferation ability of VSMC and induce the production of inflammatory cytokines in VSMC. It is also shown a synergistic effect with TLR2- and TLR4-mediated signaling pathways in this process.

Objective: Stimuli activating vascular smooth muscle cell death can constrain the neointimal response to arterial damage and prevent vascular thickening. Conversely, endothelial cell death increases endothelial dysfunction and thrombosis risk. We investigated the combined effect of atorvastatin and TNF-α on vascular cell death., Methods and Results: Cell death was investigated in cultures of human aortic smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs). Atorvastatin downregulated NF-κB and enhanced JNK activity and cell death in VSMC cultured with TNF- α. In the absence of TNF-α, percentages (mean and StDev) of annexin V positive cells were 17.4 ± 6.6%, 19.3 ± 5.9%, 22.9 ± 9.4% and 35.0 ± 20.0 % with 0, 1, 3 and 10 µM atorvastatin, respectively. The cytotoxic effect of statin was significant at the highest dose of 10 μM (p=0.001). In the presence of TNF-α, percentages of annexin V positive cells were 27.1 ± 10.6%, 34.2 ± 8.5%, 37.4 ± 14.6, and 54.1 ± 20.0% with 0, 1, 3 and 10 µM atorvastatin, respectively. The cytotoxic effect of statin was significant at each dose used (p≤0.02), in the presence of TNF-α. The cell death sensitising effect of atorvastatin was apparently mediated by down modulation of PKCβ activity, because it was reproduced by the specific PKCβ inhibitor LY317615 and prevented by the PKC activator phorbol-12-myristate-13-acetate (PMA). This effect was cell context dependent because it was not observed in HUVECs. PKCβ was found to be constitutively active in VSMCs but not in HUVECs, thereby explaining the differential effect among the two cell types. Measurement of phosphoPKCβ protein levels in arterial specimens confirmed increased activation of this kinase in the smooth muscle layer, in comparison with endothelium. We show that PKCβ provides survival signals to vascular smooth muscle cells and not the endothelium., Conclusion: Our study suggests that atorvastatin enhances TNF-α-induced cell death in vascular smooth muscle- but not endothelial - cells; by a cell-context-dependent mechanism, involving PKCβ inhibition.

Objective: Despite pro-fibrotic effects, transforming growth factor (TGF)-β prevents arteriosclerosis by suppressing effector leukocytes and promoting smooth muscle differentiation. However, previous observations of increased TGF-β expression in arteriosclerotic plaques are not consistent with that of an effective protective factor. We investigated the expression, regulation, and responses of TGF-β in human arterial tissues and cells., Methods and Results: The expression of TGF-β by intrinsic vascular cells was lower in arteriosclerotic than non-diseased coronary arteries. Activation of resident and infiltrating leukocytes did not elicit TGF-β production from coronary artery segments in organ culture. Instead, the basal expression of TGF-β by coronary arteries decreased after vessel procurement and ex vivo culture. Activation of cultured smooth muscle cells and endothelial cells with phorbol ester and ionophore also decreased TGF-β expression. Isolated cell types representing those found in the artery wall were all capable of signaling in response to TGF-β, however production of the cytoprotective molecule, interleukin-11 was cell type-dependent and restricted to smooth muscle cells and fibroblasts. Interleukin-11 reduced smooth muscle cell apoptosis to T cell effectors., Conclusions: Inflammation and cellular activation diminish the basal expression of TGF-β by quiescent human vascular cells. Induction of interleukin-11 may contribute to the anti-arteriosclerotic actions of TGF-β., (Published by Elsevier Ireland Ltd.)

Chronic rejection manifests as transplant vasculopathy, which is characterized by intimal thickening of the vessels of the allograft. Intimal thickening is thought to result from the migration and proliferation of vascular smooth muscle cells (SMC) in the vessel media, followed by deposition of extracellular matrix proteins. The development of post-transplantation anti-human leukocyte antigen (HLA) antibodies (Ab) is strongly correlated with the development of transplant vasculopathy and graft loss. Here we demonstrate that cross-linking of HLA class I molecules on the surface of human SMC with anti-HLA class I Ab induced cell proliferation and migration. Class I ligation also increased phosphorylation of focal adhesion kinase (FAK), Akt, and ERK1/2 in SMC. Knockdown of FAK by siRNA attenuated class I-induced phosphorylation of Akt and ERK1/2, as well as cell proliferation and migration. These results indicate that ligation of HLA class I molecules induces SMC migration and proliferation in a FAK-dependent manner, which may be important in promoting transplant vasculopathy., (Copyright © 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Aims: Peroxisome proliferator-activated receptor γ (PPARγ) has been reported to attenuate intimal hyperplasia. This study aimed to test the hypothesis that PPARγ inhibits intimal hyperplasia through suppressing Toll-like receptor 4 (TLR4)-mediated inflammation in vascular smooth muscle cells., Methods and Results: TLR4(-/-) mice on a C57BL/6J background were used. Increased TLR4 and pro-inflammatory cytokines were observed in wire-injury-induced carotid neointima and in platelet-derived growth factor (PDGF)-activated vascular smooth muscle cells. The TLR4 deficiency protected the injured carotid from neointimal formation and impaired the cellular proliferation and migration in response to lipopolysaccharide and PDGF. Rosiglitazone attenuated intimal hyperplasia. Overexpression of PPARγ suppressed PDGF-induced proliferation and migration and inhibited TLR4-mediated inflammation in vascular smooth muscle cells, while PPARγ silencing exerted the opposite effect. Lipopolysaccharide counteracted the inhibitory effect of PPARγ on PDGF-induced proliferation and migration. Eritoran suppressed the proliferation and migration induced by PDGF and PPARγ silencing. Vascular smooth muscle cells derived from TLR4(-/-) mice showed impaired proliferation and migration upon PDGF activation and displayed no response to PPARγ manipulation., Conclusion: PPARγ inhibits vascular smooth muscle cell proliferation and migration by suppressing TLR4-mediated inflammation and ultimately attenuates intimal hyperplasia after carotid injury.

Objective: The arterial media, populated by vascular smooth muscle cells (VSMC), is an immunoprivileged compartment and, in contrast to the intima or adventitia containing endothelial cells, is generally spared by inflammatory processes, such as arteriosclerosis. To determine mechanisms of medial immunoprivilege, we investigated the ability of human VSMC versus endothelial cells to activate allogeneic T cells in vitro., Methods and Results: Unlike cultured endothelial cells, cultured VSMC do not activate allogeneic memory CD4 or CD8 T cells and fail to effectively support T-cell proliferation to the polyclonal activator, phytohemagglutinin, consistent with a defect in costimulation function. Although many costimulators are comparably expressed on both cell types, endothelial cells but not VSMC basally express OX40 ligand and upregulate inducible costimulator ligand in response to proinflammatory cytokines. OX40 ligand-transduced, but not control- or inducible costimulator ligand-transduced, VSMC acquire the capacity to stimulate allogeneic memory CD4 T cells to produce cytokines and to proliferate in the presence of supplemental l-tryptophan. OX40 ligand overexpression, although not essential, also enhances allogeneic memory CD8 T-cell responses to VSMC after l-tryptophan supplementation., Conclusions: The inability of cultured VSMC to activate memory T cells results from a lack of essential costimulators, particularly OX40 ligand, in addition to indoleamine 2,3-dioxygenase-mediated tryptophan depletion.

Rationale and Objective: Our previous studies have determined that the F11 receptor (F11R; aka JAM-A) exerts a critical force in the adhesion of human platelets to inflamed endothelial cells (ECs), and thus can play a significant role in the initiation of atherosclerotic plaque formation. In the present study, we focus on a subsequent event in plaque development--the migration of smooth muscle cells (SMCs) from the media to the intima of inflamed vessels. Here we report our investigation of the expression of F11R in atherosclerotic arteries of coronary artery disease (CAD) patients, and of the role of F11R in the migration of SMCs involved in atherogenesis., Methods and Results: Histological staining and specific-antibody immunofluorescence of excised, human atherosclerotic coronary arteries revealed the expression of F11R in the SMCs of the intima. RT-PCR and SDS-PAGE followed by immunoblotting procedures demonstrated that F11R mRNA and the F11R protein levels were enhanced by the stimulation of cultured human aortic SMCs with a combined treatment of proinflammatory cytokines (TNFalpha+INFgamma+IL-beta1). Neither the F11R message nor protein was expressed in non-stimulated SMCs. The functional role of F11R in SMCs' migration was studied in cytokine-stimulated SMCs by interference of F11R expression with siRNA. Silencing of the F11R gene of cytokines-treated SMCs inhibited the expression of both F11R mRNA and F11R protein. Functionally, silencing of the F11R gene blocked the proliferation and migration of these inflamed SMCs, both critical events in atherogenesis., Conclusions: The new findings of this study are that F11R mRNA and F11R protein are expressed in SMCs of the intima (but not in the media) of atherosclerotic arteries of CAD patients, and that F11R is required for the proliferation and migration of inflamed SMCs. Based on these findings, we conclude that in addition to the previously reported role of F11R in the initiation of plaque formation, F11R plays also an important role in the subsequent growth of atherosclerotic plaques. Identification of drugs that interfere with these pathophysiologic actions of F11R thus represents an effective new approach for the prevention and treatment of atherosclerosis, heart attacks and stroke., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

We used a direct-contact endothelial cell-smooth muscle cell (EC-SMC) co-culture to examine whether quiescent SMCs regulate the EC inflammatory response to tumor necrosis factor (TNF)-alpha. ECs were cultured under static and physiological flow conditions. Compared with TNF-alpha-treated ECs in monoculture, TNF-alpha-treated ECs in co-culture had less NF-kappaB nuclear translocation; less intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin surface protein expression; no change in TNF receptor expression, but greater Kruppel-like factor 2 (KLF2) gene expression. After flow preconditioning for 24 h at 15 dyne/cm(2), and exposure of ECs to flow and TNF-alpha for 4.5 h, ECs in co-culture had less ICAM-1, VCAM-1, and E-selectin surface protein expression. Exposure to flow greatly increased KLF2 gene expression levels in both EC cultures; as a result, ECs in co-culture and monoculture had similar levels of post-flow KLF2 gene expression. The reduced levels of TNF-alpha-induced adhesion molecule expression in co-culture required the presence of quiescent SMCs; adhesion to decellularized extracellular matrix (ECM) or co-culture with fibroblasts produced only a modest reduction in EC adhesion molecule expression. Furthermore, co-culture of quiescent SMCs and ECs on the opposite side of a 10-microm-thick porous membrane did not alter the TNF-alpha-mediated ICAM-1 surface protein expression. Although the ECM produced by SMCs plays some role in reducing TNF-alpha-mediated inflammation, these results suggest that the direct contact between ECs and quiescent SMCs is required to inhibit TNF-alpha-mediated activation.

Aim: This immunohistochemical investigation was to analyze the relationship between platelet activation/aggregation, inflammatory cell infiltration, the differentiation state of neointimal smooth muscle cells (SMCs), expression of platelet-derived growth factor (PDGF), and endothelial cell regeneration at sites of bare-metal stents (BMS) in patients with acute coronary syndrome (ACS)., Methods: Sixteen coronary arteries after stenting were obtained at autopsy from ACS patients. Serial frozen sections were stained with antibodies against SMCs (1A4, HHF-35, CGA-7), macrophages, neutrophils, endothelial cells, GP IIb/IIIa, P-selectin, PDGF-B, and PDGF-beta receptor., Results: Up to 12 days after BMS, the stent sites contained P-selectin-positive activated platelets with neutrophil infiltration. From 24 to 55 days after BMS, parts of the platelet thrombi were still positive for P-selectin, and infiltration of neutrophils and macrophages was also found. Neointimal SMCs at these stages stained positive with 1A4 but negative with CGA-7, and PDGF-B and PDGF-beta receptor were expressed in macrophages and SMCs. At sites from 3 months onward, platelet thrombi and neutrophil infiltration were not detected, and the neointima contained increased numbers of highly differentiated SMCs with CGA-7 positivity. The P-selectin-positive area was positively correlated with the neutrophil count and macrophage-positive area (neutrophils, r=0.86, p<0.0005; macrophage, r=0.66, p<0.05). In contrast, the P-selectin-positive area was negatively correlated with the HHF-35-positive area and CGA-7-positive area (HHF-35, r=-0.90, p<0.0001; CGA-7, r=-0.82, p<0.005)., Conclusion: These observations suggest that P-selectin-positive platelet thrombi in the neointima are positively associated with the inflammatory cell infiltration and reversely associated with the phenotypic redifferentiation of neointimal SMCs after BMS in ACS patients.