Expression In Smooth Muscle Cells Research Articles

SDF-1/CXCR4 axis participants in the pathophysiology of adult patients with moyamoya disease

BackgroundMoyamoya disease (MMD) is characterized by an abundance of moyamoya vessels; however, the precise mechanism driving the spontaneous angiogenesis of these compensatory vessels remains unclear. Previous research has established a link between the stromal cell-derived factor-1 (SDF-1)/ CXC receptor 4 (CXCR4) axis and angiogenesis under hypoxic conditions. Nevertheless, the alterations in this axis within the cerebrospinal fluid, arachnoid membranes and vascular tissue of MMD patients have not been fully investigated. MethodsOur study enrolled 66 adult MMD patients and 61 patients with atherosclerotic vascular disease (ACVD). We investigated the SDF-1 concentration in cerebrospinal fluid (CSF) and CXCR4 expression level on the arachnoid membranes and vascular tissue. We utilized enzyme-linked immunosorbent assay and immunohistochemistr. Additionally, we cultured and stimulated human brain microvascular endothelial cells (HBMECs) and smooth muscle cells (SMCs) under oxygen and glucose deprivation (OGD) conditions followed by reoxygenation, to examine any changes in the SDF-1/CXCR4 axis. ResultsThe results demonstrated an elevation in the level of SDF-1 in CSF among MMD patients compared to those with ACVD. Moreover, the expression of CXCR4 in arachnoid membranes and vascular tissue showed a similar trend. Furthermore, the content of CXCR4 in HBMECs and SMCs increased with the duration of ischemia and hypoxia. However, it was observed that the expression of CXCR4 decreased at OGD/R 24h compared to OGD 24h. The temporal pattern of SDF-1 expression in HBMECs and SMCs mirrored that of CXCR4 expression. ConclusionThese findings indicate a critical role for the SDF-1/CXCR4 axis in the angiogenesis of moyamoya disease.

Smooth muscle cell-specific matrix metalloproteinase 3 deletion reduces osteogenic transformation and medial artery calcification.

Vascular calcification is highly prevalent in atherosclerosis, diabetes, and chronic kidney disease. It is associated with increased morbidity and mortality in patients with cardiovascular disease. Matrix metalloproteinase 3 (MMP-3), also known as stromelysin-1, is part of the large matrix metalloproteinase family. It can degrade extracellular matrix components of the arterial wall including elastin, which plays a central role in medial calcification. In this study, we sought to determine the role of MMP-3 in medial calcification. We found that MMP-3 was increased in rodent models of medial calcification as well as in vascular smooth muscle cells (SMCs) cultured in a phosphate calcification medium. It was also highly expressed in calcified tibial arteries in patients with peripheral arterial disease (PAD). Knockdown and inhibition of MMP-3 suppressed phosphate-induced SMC osteogenic transformation and calcification, whereas the addition of a recombinant MMP-3 protein facilitated SMC calcification. In an ex vivo organ culture model and a rodent model of medial calcification induced by vitamin D3, we found that MMP-3 deficiency significantly suppressed medial calcification in the aorta. We further found that medial calcification and osteogenic transformation were significantly reduced in SMC-specific MMP-3-deficient mice, suggesting that MMP-3 in SMCs is an important factor in this process. These findings suggest that MMP-3 expression in vascular SMCs is an important regulator of medial calcification and that targeting MMP-3 could provide a therapeutic strategy to reduce it and address its consequences in patients with PAD.

Bone-marrow-derived nerve growth factor receptor-positive dendritic cells contribute to the suppression of arterial remodelling via apoptosis

Abstract Background Bone marrow (BM) contributes to the pathogenesis of arteriosclerosis. Nerve growth factor receptor (NGFR) is expressed in BM stroma and is present in peripheral blood and the ischemic coronary artery. NGFR transduces signals towards cell survival or apoptosis in different cell types according to the coupling co-receptors. The previous clinical study demonstrated that low gene expression of NGFR in peripheral leucocytes in patients with acute coronary syndrome predicted 5-year repetitive coronary interventions at a de novo lesion. Purpose To investigate the hypothesis that BM-derived NGFR-positive (NGFR+) cells are associated with arterial remodelling. Methods Adult C57BL/6J male NGFR-wild-type (WT) or -knockout (KO) mice were subjected to unilateral carotid artery ligation after BM transplantation (BMT) from green fluorescent protein-positive (GFP+) NGFR-WT or -KO mice (N = 4-6). The ligated and the opposite-sided, sham-operated arteries were assessed by immunohistology and gene expression analysis using a PCR on day 28. Also, human peripheral blood NGFR+ mononuclear cells (MNCs) from a healthy volunteer were sorted using FACS Aria II and co-cultured with smooth muscle cells (SMCs) to examine early apoptosis (AnnexinV+7AAD-) in response to the NGFR ligands; proNGF and NGF. Also, human NGFR+MNCs from three healthy volunteers underwent RNA sequencing analysis and were immunophenotypically characterised by flow cytometry. Results In WT mice, NGFR+ cells accumulated in the neointima after ligation. The neointimal area was significantly larger in the mice with BM-specific depletion of NGFR than that in WT mice. After BMT from GFP+NGFR-WT mice, NGFR+GFP+ cells accumulated in the neointima, exhibited apoptosis (TUNEL+ and cleaved caspase-3+), and promoted F4/80+GFP+ macrophage accumulation. By contrast, in the BM-specific NGFR-KO model, medial SMCs (αSMA+GFP-) occupied the neointima with augmented proliferation, and GFP+ cells were rare. Notably, apoptotic NGFR+ cells were accompanied by the accumulation of macrophages (F4/80+NGFR-) and expressed IL-10, which were abolished by BM-specific depletion of NGFR. Furthermore, human NGFR+, but not NGFR- cells, co-cultured with SMCs were susceptible to apoptosis in response to proNGF. Additionally, PDGF stimulated NGF expression in SMCs in vitro, and proNGF was expressed in a ligated artery, implying that local proNGF might cause NGFR+ cell apoptosis. Meanwhile, RNA sequencing and flow cytometric analysis demonstrated that human NGFR+MNCs resemble dendritic cells (DCs) and predominantly express the plasmacytoid DCs markers. Consistently, NGFR+ cells in the neointima were positive for HLA-DR and CD11c. Conclusions BM-derived NGFR+ cells expressed dendritic cell markers and contributed to the suppression of arterial remodelling mediated by apoptosis. Apoptotic NGFR+ cells promoted macrophage chemotaxis and coordinately inhibited medial SMC migration to, and proliferation in, the neointima.

Role of S100A4 in the crosstalk between smooth muscle and inflammatory cells in atherosclerosis

Abstract Background During atherosclerotic plaque formation, smooth muscle cells (SMCs) accumulate in the intima and undergo a transition from a contractile to a synthetic phenotype. We previously showed that S100A4 is a marker of the synthetic SMCs in vitro and intimal SMCs in pig, human and mouse. In vitro, S100A4-treated SMCs acquire a pro-inflammatory phenotype. The systemic neutralization of extracellular S100A4 in ApoE-/- mice induces atherosclerotic plaque stabilization. Purpose To characterize the role of S100A4-expressing SMCs in atherosclerosis. Methods and results We established a coculture model between S100A4-stimulated human aortic SMCs and human blood-derived monocytes. Flow cytometry analysis of monocytes exposed to S100A4-treated SMCs for 3 days showed an upregulation of markers typical of inflammatory M1 (HLA-DR) and anti-inflammatory M2 (CD206) macrophages (fig. 1). Macrophages also upregulated mRNA expression of NPC Intracellular cholesterol transporter 1 and 2 by qPCR, suggesting modifications in their cholesterol metabolism. In vivo, in an SMC-lineage-tracing ApoE-/-mouse model, we identified S100A4-expressing SMCs at the onset of atherosclerosis in aortic media of 11-week-old mice fed a high cholesterol diet (HCD) for 3 weeks. Medial SMCs were characterized by means of single-cell RNA sequencing. Interestingly, 10 out of 11 identified clusters expressed S100A4, demonstrating broad and early involvement of S100A4 in shaping medial SMC identity. We assumed that blood-derived monocytes might be a driving force of early S100A4 activation in SMCs. To mitigate the impact of monocytes, 8-week-old ApoE-/- mice were fed an HCD for 9 weeks and injected with either clodronate-encapsulated or empty liposomes during the first 3 weeks. No differences in S100A4 expression in the media were observed by qPCR. Unexpectedly, ApoE-/- mice treated with clodronate demonstrated larger atherosclerotic lesions compared to control animals as measured by OilRedO staining (fig. 2). Studies are in progress to define the plaque composition. Conclusion Our findings shed light on the role of S100A4 in the crosstalk between SMCs and inflammatory cells in atherosclerosis and the fine-tuning of the phenotype of medial SMCs prior to their accumulation in the intima. We suggest that temporary monocyte depletion at the onset of the disease alters the mechanisms underlying plaque formation, worsening the outcome of the disease, while not affecting S100A4 expression in medial SMCs.

Epigenetic Induction of Smooth Muscle Cell Phenotypic Alterations in Aortic Aneurysms and Dissections.

Smooth muscle cell (SMC) phenotypic switching has been increasingly detected in aortic aneurysm and dissection (AAD) tissues. However, the diverse SMC phenotypes in AAD tissues and the mechanisms driving SMC phenotypic alterations remain to be identified. We examined the transcriptomic and epigenomic dynamics of aortic SMC phenotypic changes in mice with angiotensin II-induced AAD by using single-cell RNA sequencing and single-cell sequencing assay for transposase-accessible chromatin. SMC phenotypic alteration in aortas from patients with ascending thoracic AAD was examined by using single-cell RNA sequencing analysis. Single-cell RNA sequencing analysis revealed that aortic stress induced the transition of SMCs from a primary contractile phenotype to proliferative, extracellular matrix-producing, and inflammatory phenotypes. Lineage tracing showed the complete transformation of SMCs to fibroblasts and macrophages. Single-cell sequencing assay for transposase-accessible chromatin analysis indicated that these phenotypic alterations were controlled by chromatin remodeling marked by the reduced chromatin accessibility of contractile genes and the induced chromatin accessibility of genes involved in proliferation, extracellular matrix, and inflammation. IRF3 (interferon regulatory factor 3), a proinflammatory transcription factor activated by cytosolic DNA, was identified as a key driver of the transition of aortic SMCs from a contractile phenotype to an inflammatory phenotype. In cultured SMCs, cytosolic DNA signaled through its sensor STING (stimulator of interferon genes)-TBK1 (tank-binding kinase 1) to activate IRF3, which bound and recruited EZH2 (enhancer of zeste homolog 2) to contractile genes to induce repressive H3K27me3 modification and gene suppression. In contrast, double-stranded DNA-STING-IRF3 signaling induced inflammatory gene expression in SMCs. In Sting-/- mice, the aortic stress-induced transition of SMCs into an inflammatory phenotype was prevented, and SMC populations were preserved. Finally, profound SMC phenotypic alterations toward diverse directions were detected in human ascending thoracic AAD tissues. Our study reveals the dynamic epigenetic induction of SMC phenotypic alterations in AAD. DNA damage and cytosolic leakage drive SMCs from a contractile phenotype to an inflammatory phenotype.

Exploring the expression and potential function of follicle stimulating hormone receptor in extragonadal cells related to abdominal aortic aneurysm.

Follicle stimulating hormone (FSH) is identified to play a role in postmenopausal disease and hypothesized to affect abdominal aortic aneurysm (AAA) onset/progression in postmenopausal women. We aimed to detect FSHR gene expression in AAA tissue and cell types involved in AAA formation. FSH stimulation of human umbilical cord endothelial cells (HUVECs), smooth muscle cells (HUCs) and PMA-differentiated macrophages to assess gene expression of FSHR and various markers. Human macrophages activated with various stimuli were assessed for FSHR gene expression. AAA dataset, AAA tissue samples and AAA-derived smooth muscle cells (SMC) obtained from elderly female donors were assessed for FSHR gene expression. AAA-SMCs were stimulated with FSH to assess its effect on gene expression. Lastly, oxidized low-density-lipoprotein (ox-LDL) uptake and abundance of cell surface protein markers were assessed by flow cytometry after FSH stimulation of human monocytes. FSH stimulation showed similar levels of gene expression in HUVECs and HUCs. Only ACTA2 was downregulated in HUCs. In PMA-differentiated macrophages, gene expression of inflammation markers was unchanged after FSH stimulation. FSHR gene expression was found to be low in the AAA datasets. Female AAA-SMCs show occasional FSHR gene expression at a very low level, yet stimulation with FSH did not affect gene expression of SMC- or inflammation markers. FSH stimulation did not impact ox-LDL uptake or alter cell surface protein expression in monocytes. While FSHR gene expression was detected in human testis tissue, it was below quantification level in all other investigated cell types, even upon activation of macrophages with various stimuli. Despite previous reports, we did not detect FSHR gene expression in various extragonadal cell types, except in occasional female AAA-SMCs. No clear effect on cell activation was observed upon FSH stimulation in any cell type. Our data suggest that a direct effect of FSH in AAA-related extragonadal cells is unlikely to influence AAA.

Abstract 663: Smooth Muscle Cell-specific MMP-3 Deletion Reduces Osteogenic Transformation And Medial Artery Calcification

Objective: Vascular calcification is highly prevalent in atherosclerosis, diabetes, and chronic kidney disease. It is associated with increased morbidity and mortality in patients with cardiovascular disease. Matrix metalloproteinase 3 (MMP-3), also known as stromelysin-1, is part of the large matrix metalloproteinase family. It can degrade extracellular matrix components of the arterial wall including elastin, which plays a central role in medial calcification. In this study, we sought to determine the role of MMP-3 in medial calcification. Methods and Results: We found that MMP-3 expression was increased in vascular smooth muscle cells (SMCs) cultured in a phosphate calcification medium. It was also highly expressed in the calcified tibial arteries in patients with peripheral arterial disease (PAD). Knockdown and inhibition of MMP-3 suppressed phosphate-induced SMC osteogenic transformation and calcification, whereas the addition of a recombinant MMP-3 protein facilitated SMC calcification. In an ex vivo organ culture model and a rodent model of medial calcification induced by vitamin D 3 , we found that MMP-3 deficiency significantly suppressed aortic medial calcification. We further found that medial calcification and osteogenic transformation were significantly reduced in SMC-specific MMP-3-deficient mice, suggesting that MMP-3 in SMCs is crucial for medial calcification. Conclusion: These findings suggest that MMP-3 expression in vascular SMCs is an important regulator of medial calcification and that targeting MMP-3 could provide a therapeutic strategy to reduce it and address its consequences in patients with PAD.

Genetic Regulation of SMC Gene Expression and Splicing Predict Causal CAD Genes.

Coronary artery disease (CAD) is the leading cause of death worldwide. Recent meta-analyses of genome-wide association studies have identified over 175 loci associated with CAD. The majority of these loci are in noncoding regions and are predicted to regulate gene expression. Given that vascular smooth muscle cells (SMCs) play critical roles in the development and progression of CAD, we aimed to identify the subset of the CAD loci associated with the regulation of transcription in distinct SMC phenotypes. We measured gene expression in SMCs isolated from the ascending aortas of 151 heart transplant donors of various genetic ancestries in quiescent or proliferative conditions and calculated the association of their expression and splicing with ~6.3 million imputed single-nucleotide polymorphism markers across the genome. We identified 4910 expression and 4412 splicing quantitative trait loci (sQTLs) representing regions of the genome associated with transcript abundance and splicing. A total of 3660 expression quantitative trait loci (eQTLs) had not been observed in the publicly available Genotype-Tissue Expression dataset. Further, 29 and 880 eQTLs were SMC-specific and sex-biased, respectively. We made these results available for public query on a user-friendly website. To identify the effector transcript(s) regulated by CAD loci, we used 4 distinct colocalization approaches. We identified 84 eQTL and 164 sQTL that colocalized with CAD loci, highlighting the importance of genetic regulation of mRNA splicing as a molecular mechanism for CAD genetic risk. Notably, 20% and 35% of the eQTLs were unique to quiescent or proliferative SMCs, respectively. One CAD locus colocalized with a sex-specific eQTL (TERF2IP), and another locus colocalized with SMC-specific eQTL (ALKBH8). The most significantly associated CAD locus, 9p21, was an sQTL for the long noncoding RNA CDKN2B-AS1, also known as ANRIL, in proliferative SMCs. Collectively, our results provide evidence for the molecular mechanisms of genetic susceptibility to CAD in distinct SMC phenotypes.

Genetic regulation of circular RNA expression in human aortic smooth muscle cells and vascular traits.

Circular RNAs (circRNAs) are a class of non-coding RNAs that have cell-type-specific expression and are relevant in cardiovascular disease. Aortic smooth muscle cells (SMCs) play a crucial role in cardiovascular disease. In this study, we employed a systems genetics approach to identify SMC circRNA transcripts and their relevance in cardiovascular traits across the genome. We quantified circRNA expression across 151 quiescent and proliferative human aortic SMCs from donors of various genetic ancestries. We identified 1,589 expressed circRNAs. Between quiescent and proliferative SMCs, we identified 173 differentially expressed circRNAs. To characterize the genetic regulation of circRNA expression, we associated the genotypes of 6.3 million single nucleotide polymorphisms (SNPs) with circRNA abundance and found 96 circRNAs that were associated with genetic loci. Three SNPs were associated with circRNA expression in proliferative SMCs but not quiescent SMCs. We identified six SNPs that had distinct association directions with circRNA isoforms from the same gene. Lastly, to identify the relevance of circRNAs in cardiovascular disease, we overlapped genetic loci associated with circRNA expression with vascular disease-related genome-wide association studies loci. We identified 14 blood pressure, one myocardial infarction, and three coronary artery disease loci, which were associated with a circRNA transcript but not an mRNA transcript. Overall, our results provide insight into the genetic basis of vascular disease traits mediated by circRNA expression.

MicroRNA-322 inhibition of calcification of arterial smooth muscle cells by regulation of galactosyltransferase 1-associating protein UBE2Q1 and Runx2

Background and purpose: Vascular smooth muscle cell (SMC) calcification represents a prominent phenotypic alteration in atherosclerosis. MicroRNA-322 (miR-322) is crucially involved in myogenic stem cell growth and differentiation. The galactosyltransferase 1-associated protein (GTAP) is a ubiquitin-conjugating enzyme E2Q1 (UBE2Q1) that serves as a critical mediator of post-translational regulation of certain cellular enzymes and transcription factors. Runt-related transcription factor 2 (Runx2) plays a critical role in arterial calcification. However, the interplay between miR-322, UBE2Q1, and Runx2 during cardiovascular calcification remain largely unknown. Therefore, the purpose of this study is to delineate the molecular mechanisms by which miR-322 regulates vascular calcification. Methods: Here we examined miR-322 expression in murine SMC, and determined whether miR-322 regulates SMC calcification via modulating expression of UBE2Q1 and calcifying proteins. Murine SMC cultures or aortic segments were exposed to inorganic phosphate (Pi) for induction of calcification. Expressions of calcification-related genes in SMC with lentivirus-mediated knockdown of UBE2Q1 were determined with Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Luciferase reporter assay was performed to validate miR-322 target binding and SMC were transfected with anti-miR-322 oligonucleotides to inhibit miR-322 function. Results: Aortic rings derived from UBE2Q1−/− mice exhibited much higher calcium content compared to aortic rings from wildtype (WT) animals, following calcification induction. Knockdown of UBE2Q1 by lentiviral short hairpin RNA (shRNA) significantly enhanced the calcium deposition and expression of osteogenic gene Runx2 in SMC. Enhanced UBE2Q1 expression dramatically reduced calcification while promoting expression of contractile proteins SM22α and α-SMA. Treatment with anti-miR-322 diminished the luciferase activity in SMC transfected with the reporter gene driven by the 3′-untranslated region of UBE2Q1 mRNA. Anti-miR-322 treatment also inhibited calcification significantly. Conclusions: Our study identified miR-322 regulates vascular calcification by targeting UBE2Q1. The miR-322–dependent regulation of UBE2Q1 and calcification represents a novel regulatory mechanism that controls vascular SMC function during the pathogenesis of vascular calcification.

Divergent Roles of Ephrin-B2/EphB4 Guidance System in Pulmonary Hypertension.

Smooth muscle cell (SMC) expansion is one key morphological hallmark of pathologically altered vasculature and a characteristic feature of pulmonary vascular remodeling in pulmonary hypertension. Normal embryonal vessel maturation requires successful coverage of endothelial tubes with SMC, which is dependent on ephrin-B2 and EphB4 ligand-receptor guidance system. In this study, we investigated the potential role of ephrin-B2 and EphB4 on neomuscularization in adult pulmonary vascular disease. Ephrin-B2 and EphB4 expression is preserved in smooth muscle and endothelial cells of remodeled pulmonary arteries. Chronic hypoxia-induced pulmonary hypertension was not ameliorated in mice with SMC-specific conditional ephrin-B2 knockout. In mice with global inducible ephrin-B2 knockout, pulmonary vascular remodeling and right ventricular hypertrophy upon chronic hypoxia exposure were significantly diminished compared to hypoxic controls, while right ventricular systolic pressure was unaffected. In contrast, EphB4 receptor kinase activity inhibition reduced right ventricular systolic pressure in hypoxia-induced pulmonary hypertension without affecting pulmonary vascular remodeling. Genetic deletion of ephrin-B2 in murine pulmonary artery SMC, and pharmacological inhibition of EphB4 in human pulmonary artery smooth muscle cells, blunted mitogen-induced cell proliferation. Loss of EphB4 signaling additionally reduced RhoA expression and weakened the interaction between human pulmonary artery smooth muscle cells and endothelial cells in a three-dimensional coculture model. In sum, pulmonary vascular remodeling was dependent on ephrin-B2-induced Eph receptor (erythropoietin-producing hepatocellular carcinoma receptor) forward signaling in SMC, while EphB4 receptor activity was necessary for RhoA expression in SMC, interaction with endothelial cells and vasoconstrictive components of pulmonary hypertension.

442-P: Sorbs2 Deficiency–Mediated Vascular BK Channel Dysfunction in Type 2 Diabetes

Vascular BK channels, composed of the pore-forming α-subunits (BK-α) and the regulatory β1 subunits (BK-β1) , is a key determinant of vasoreactivity, and its function is impaired in diabetes. However, our understanding of the mechanisms causing BK channel dysfunction in diabetic vessels is incomplete. The Sorbin and SH3 domain-containing protein 2 (Sorbs2) is abundantly expressed in vascular smooth muscle cells (SMCs) . We hypothesize that Sorbs2 is critical for vascular BK channel physiology. In this study, we compared vascular BK channel function between Sorbs2 KO and WT mice at 4 months of age. There was no difference in body weight and blood glucose between WT and Sorbs2 KO mice (body weight: 25.9±1.3 g of WT vs. 23.3±1.5 g of KO, n=14, p=N.S.; blood glucose: 166.4±7.7 mg/dL of WT vs. 152.2±12.2 mg/dL of KO, n=14, p=N.S.) . However, Sorbs2 KO mice have markedly reduced cell surface and cytoplasmic expression of BK-α and BK-β1, as well as BK channel-mediated vasodilation in carotid arteries with a maximal dilatation of 64.70±1.92% for WT mice (n=4) and 20.74±4.66% for KO mice (n=5, p<0.05) . Patch clamp experiments revealed that BK channel sensitivity to Ca2+ activation was impaired in the coronary SMCs of Sorbs2 KO mice (EC50: 2.87±0.68 µM, n=13, p<0.05) with decreased Hill coefficient (nH: 1.26) , compared to those of WT mice (EC50: 0.48±0.µM, nH: 2.70, n=13) . In vivo studies showed that Sorbs2 interacted with BK-α and BK-β1 protein in vascular SMCs. Silencing of Sorbs2 by shRNA decreased BK-α and BK-β1 expression, while adenoviral expression of Sorbs2 increased BK-α and BK-β1 expression in vascular SMCs. Most importantly, Sorbs2 expression was significantly reduced in the arteries of db/db type 2 diabetic mice and high fat diet-induced diabetic mice. Our results indicate that Sorbs2 plays an important role in regulating BK channel function and expression in vascular SMCs. Sorbs2 deficiency is a risk factor of BK channelopathy and vasculopathy in diabetes, independent of obesity and glucotoxicity. Disclosure T.Lu: None.

101-OR: Cell-Specific Role of Insulin in a Model of Restenosis in Insulin-Sensitive and Insulin-Resistant Conditions

Angioplasty and stenting are widely used in the treatment of atherosclerosis. However, these procedures may fail due to post-angioplasty restenosis, which remains a problem for patients with diabetes. Currently, the vascular role of insulin is unclear. Its role on endothelial cells (EC) is vasculoprotective, whereas its effects on vascular smooth muscle cells (SMC) are mainly mitogenic according to in vitro data. We have previously shown a suppressive effect of insulin treatment on neointimal growth in rodent models of restenosis in insulin sensitive conditions that was abolished in insulin resistant conditions. The objective of this study was to determine the EC-and SMC-specific effects of insulin treatment on neointimal growth in a murine model of restenosis in insulin sensitive and insulin resistant conditions. Mice were generated by crossing insulin receptor (IR) floxed mice with tamoxifen inducible Cre recombinant mice under the control of Cdh5 (vascular endothelial cadherin) or SMMHC (smooth muscle heavy chain) promoters. Mice were fed with a low-fat (LFD) or a high-fat-high-sucrose (HFSD) diet and implanted with insulin pellet (0.U/day) or vehicle (control) prior to femoral artery wire injury. We found that compared to IR-floxed controls, tamoxifen-induced IRf/f-Cdh5-Cre+ or IRf/f-SMMHC-Cre+ mice showed a 94% and 80% decrease in IR expression in ECs or SMCs, respectively. In LFD-fed insulin sensitive conditions, insulin decreased neointimal area (NA) and intima-to-media ratio (I/M) in controls (Cdh5 and-SMMHC-floxed and wildtype-Cre+ mice) but not in endothelial and SMC IR deficient mice. In HFSD-fed insulin resistant conditions, insulin had no effect in either controls or endothelial and SMC IR deficient mice. These data demonstrate that insulin action in both ECs and SMCs is required for the anti-restenotic effect of insulin in insulin sensitive conditions, which is abolished but not reversed in insulin resistance even when endothelial IR is deficient. Disclosure J.Z. Wang: None. C.X. Zhang: None. A. Giacca: None.

SREBP1 regulates Lgals3 activation in response to cholesterol loading

Aberrant smooth muscle cell (SMC) plasticity is etiological to vascular diseases. Cholesterol induces SMC phenotypic transition featuring high LGALS3 (galectin-3) expression. This proatherogenic process is poorly understood for its molecular underpinnings, in particular, the mechanistic role of sterol regulatory-element binding protein-1 (SREBP1), a master regulator of lipid metabolism. Herein we show that cholesterol loading stimulated SREBP1 expression in mouse, rat, and human SMCs. SREBP1 positively regulated LGALS3 expression (and vice versa), whereas Krüppel-like factor-15 (KLF15) acted as a negative regulator. Both bound to the Lgals3 promoter, yet at discrete sites, as revealed by chromatin immunoprecipitation-qPCR and electrophoretic mobility shift assays. SREBP1 and LGALS3 each abated KLF15 protein, and blocking the bromo/extraterminal domain-containing proteins (BETs) family of acetyl-histone readers abolished cholesterol-stimulated SREBP1/LGALS3 protein production. Furthermore, silencing bromodomain protein 2 (BRD2; but not other BETs) reduced SREBP1; endogenous BRD2 co-immunoprecipitated with SREBP1’s transcription-active domain, its own promoter DNA, and that of Lgals3. Thus, results identify a previously uncharacterized cholesterol-responsive dyad—SREBP1 and LGALS3, constituting a feedforward circuit that can be blocked by BETs inhibition. This study provides new insights into SMC phenotypic transition and potential interventional targets.

HIF1α and NFκB Regulate Mineralocorticoid Receptor Gene Expression in Aging Human Vascular Smooth Muscle Cells

BackgroundMineralocorticoid receptor (MR) activation drives aging‐associated cardiovascular diseases including hypertension, vascular stiffness and heart failure. Vascular MR expression increases with age and drives the aging phenotype but the mechanism of MR gene regulation is unknown. The MR has two mRNA isoforms (1α and 1β) that differ in only the 5’‐untranslated region (exon 1α or 1β) and in the promoter sequence (P1 or P2, respectively). Thus, we investigated the transcriptional mechanisms regulating MR gene expression in aging human smooth muscle cells (SMC).MethodsMR mRNA and protein expression were quantified in primary human aortic SMC (HASMC) and human aortic tissue comparing adult (average age 40s) and aged (average age 70s) male and female donors and comparing adult HASMC or Pac1 (rat SMC line) treated with H2O2 to induce aging. MR promoter activity was tested by luciferase reporter assays. In silico analysis of the MR promoters identified putative transcription factor (TF) binding sites and inhibitor studies and chromatin immunoprecipitations (ChIP) were performed to validate the predicted regulatory mechanisms.ResultsIncreased SMC donor age or H2O2 treatment were associated with significantly increased expression of both of the MR mRNA isoforms, MR protein level, and activity of both MR promoters in HASMCs. The hypoxia‐inducible TF, HIF1α, and the inflammatory TF, NFκB, were predicted in silico to bind to the MR promoters and found to increase significantly with age in HASMCs. HIF1α was induced by H2O2 in HASMCs and this was associated with significant enrichment of HIF1α binding specifically at a site in the 1a exon, near the MR P1 promoter. HIF1α inhibition attenuated expression specifically of the MR‐1α mRNA isoform resulting in decreased MR protein in HASMC. NFκB also increased with age in HASMCs and contributes to MR expression. In response to H2O2,NFκB binding is enriched at to two different binding sites, one each in the P1 and P2 promoters and NFκB inhibition decreased expression of both MR mRNA isoforms (1α and 1β) and MR protein in SMC. Finally, HIF1α and NFκB expression each positively correlated with donor age and with MR expression (p<0.0001) in human aortic tissue from males and females.ConclusionThe MR gene is regulated by the inflammatory transcription factor NFkB and by the hypoxia/oxidative stress‐induced TF HIF1α, to increase MR expression in SMC of the aging human vasculature. This molecular insight may explain enhanced MR activity in cardiovascular aging and could potentially contribute to other cardiovascular conditions driven by oxidative stress and inflammation.

Abstract 199: Conditional Inactivation Of Lysyl Oxidase In Smooth Muscle Cells Reduces Atherosclerosis Burden And Plaque Calcification In Hyperlipidemic Mice Through Suppression Of The β-catenin/tcf4 Signaling Axis.

Introduction: In atherosclerosis development and complications, the importance of lysyl oxidase (LOX), a copper-dependent amino-oxidase that crosslinks collagen and elastin and controls gene expression, is not well established. Hypothesis: Inactivation of LOX in smooth muscle cells (SMC) decreases atherosclerosis burden and plaque calcification in hyperlipidemic mice. Methods: qRT-PCR, Western blot, and scRNAseq demonstrated the loss of Lox gene expression in SMC of ApoE conditional mice (Lox f/f Myh11-CreERT2 ApoE-/-) but not in littermate control animals after tamoxifen injections. Surprisingly, vascular loss of LOX did not increase the risk of aneurysms nor affected the aorta's stiffness. However, it reduced atherosclerosis burden with respect to control mice (13±2 versus 23±1%, p < 0.01) after 16 weeks of high fat diet (HFD). There was also a significant reduction in calcium deposition (5±0.4 versus11.8±3, p < 0.05) within the plaque of LOX knockout mice versus control. Aortic whole genome transcriptomic analysis revealed a positive correlation between Lox gene expression and Frizzled-related protein 3 ( Frzb ), a biphasic modulator of Wnt signaling that facilitates the association of β-catenin with TCF4 in the nucleus. scRNAseq analysis confirmed the downregulation of Bmp2 and Myc , both β-catenin/TCF4 regulated genes, in SMC following gene deletion of Lox and 8 weeks of HFD. LOX knockout SMCs also had lower expression of Frzb and were more resistant to osteoblastic transformation in vitro. Conclusions: Vascular inactivation of LOX protects the vasculature from the proatherogenic effects of FRZB in SMCs.

β‐catenin C‐terminal Domain/Sphingosine‐1‐Phosphate Receptor 1 Axis is a Potential Therapeutic Target in Vascular Remodeling

Vascular smooth muscle cells (SMCs), in part via activation of the Wnt/β‐catenin signaling pathway, contribute importantly to vascular remodeling. While the C‐terminal domain of β‐catenin links the pathway to gene expression, how this domain and related molecular mechanisms are involved in vascular remodeling is unknown. Inhibitors targeting specifically the C‐terminal domain of β‐catenin have been developed, but their use in obstructive vascular disease is not yet justified because of this gap of knowledge. Our group has previously shown that β‐catenin C‐terminal domain in SMCs is essential for arterial wall assembly during embryogenesis, but its role in vascular remodeling in adulthood is unknown. Therefore, the objective of this study was to define the importance of β‐catenin C‐terminal domain in SMCs during vascular remodeling and the underlying molecular mechanisms.To test the hypothesis that β‐catenin C‐terminal domain in SMCs is required for vascular remodeling, mice bearing a mutant β‐catenin allele (ΔC) that prevents the transcription of the C‐terminal domain were crossed with the SMC‐specific SMA‐CreERT2 line to generate mice bearing one β‐catenin flox allele and one knockin mutant (Ctnnb1ΔC/flox) or wt (Ctnnb1wt/flox) allele. In this system, tamoxifen administration removes the flox allele, resulting in mice bearing the mutant allele (SMβCΔC mice) or the wt allele (SMβCwt/‐ mice) as the only sources of β‐catenin in SMCs. To induce vascular remodeling, we performed carotid artery ligation one week after tamoxifen injection in male and female adult mice. Carotid arteries from both male and female SMβCΔC mice showed reduced neointima formation after injury induced by ligation compared to SMβCwt/‐ mice. Arteries from SMβCΔC mice also showed reduced SMC proliferation and increased SMC apoptosis and differentiation markers. We also isolated mouse aortic SMCs (MASMCs) from Ctnnb1ΔC/flox mice, and transduced these cells with GFP‐ or Cre‐expressing adenovirus to obtain control (βCControl) or cells expressing β‐cateninΔC(βCΔC) and found that βCΔC MASMCs show reduced growth compared to βCControl. To gain further insight on the mechanisms underlying the effects of β‐catenin C‐terminus, we performed whole‐transcriptome analysis by RNA‐seq of βCΔC and βCControl MASMCs and found a downregulation of the sphingosine‐1‐phosphate receptor 1 (S1pr1) gene in βCΔC MASMCs. This receptor is known to be important in vascular remodeling, but its regulation is not fully elucidated. Interestingly, we found decreased S1PR1 expression in SMCs of injured arteries from SMβC∆C mice. Consistent with this observation, βCΔC MASMCs showed reduced S1PR1 mRNA and protein levels and decreased S1pr1 promoter activity, which were all rescued by the transfection of a constitutively active form of β‐catenin. By using a S1PR1lox/stop/lox conditional gain‐of‐function mouse line, we found that overexpression of S1PR1 in SMCs rescues the injury‐induced neointima formation in SMβC∆C mice. Consistently, overexpression of S1PR1 in SMCs restored βCΔC MASMC growth towards βCControl levels. Taken together, our results define an essential SMC β‐catenin C‐terminal domain–S1PR1 axis that drives neointima formation after arterial injury, and identify multiple points for potential therapeutic intervention to control vascular remodeling in disease.

Abstract 466: Fhl5 Enhances Vascular Calcification By Regulating Smooth Muscle Cell Transcriptional Networks

Coronary artery calcium (CAC) is a hallmark of coronary artery disease (CAD) with increased CAC levels associated with increased risk for CAD related morbidity and mortality. The development of CAC is a pathological process driven by the modulation of smooth muscle cells (SMCs) and maladaptive extracellular matrix remodeling. In the most recent multi-ancestry meta-analysis of more than 35,000 individuals, FHL5 (Four-and-a-half LIM domains 5) was prioritized as a novel CAC gene through Bayesian colocalization and epigenomic analyses. scRNA-seq studies of human coronary and carotid arteries revealed high enrichment of FHL5 gene expression in SMC and pericyte populations. These preliminary studies motivate our hypothesis that FHL5 functions as a transcriptional regulator of vascular remodeling pathways associated with increased CAC risk. Following treatment of SMCs with a calcifying cocktail, stable FHL5 overexpression promoted the osteogenic SMC phenotypic transition, characterized by increased levels of osteogenic activators and reduced expression of SMC contractile markers. This osteogenic gene signature correlated with increased SMC calcification, consistent with the direction of effect of the CAC risk allele. To provide insights into the transcriptional program regulated by FHL5 that contributes to the vascular calcification phenotype, we mapped FHL5 binding sites in SMCs using the CUT&RUN method. We identified 17,201 FHL5 binding sites that overlapped the active enhancer mark H3K27ac. FHL5 target genes were enriched in pathways linked to vascular remodeling, such as cell adhesion and extracellular matrix organization. Moreover, risk variants for pulse pressure, a metric of arterial stiffness that is highly correlated with CAC, was enriched in FHL5 binding sites. Lastly, integration with FHL5 trans-eQTL target genes in STARNET arterial tissues revealed a network of CAD-associated candidate genes regulated by FHL5 in vitro and in vivo that impact SMC proliferation, migration, and calcification. Taken together, we provide evidence that FHL5 regulates a network of genes linked to arterial stiffness, which may explain the underlying mechanism of its genetic association with CAC in individuals from the general population.

Inducible Smooth Muscle‐Specific Knockout of Acid Sensing Ion Channel 1 Protects Against Pulmonary Hypertension

Acid‐sensing ion channel 1 (ASIC1) is a proton‐gated cation channel that conducts both Na+ and Ca2+. ASIC1 is expressed in multiple cell types including vascular smooth muscle cells (SMCs) and endothelial cells (ECs). Our laboratory has previously shown that ASIC1 contributes to pulmonary arterial constriction and the development of chronic hypoxia (CH)‐induced pulmonary hypertension. However, the contribution of SMC versus EC ASIC1 to CH‐induced pulmonary hypertension is unknown. We tested the hypothesis that SMC expression of ASIC1 contributes to pulmonary hypertension and vascular remodeling by examining the development of CH‐induced pulmonary hypertension (barometric pressure of 380 mmHg for 6 weeks) in inducible SMC‐specific ASIC1 knockout (SMC‐Asic1‐/‐) and EC‐specific ASIC1 knockout (EC‐Asic1‐/‐) mice. The SMC‐Asic1‐/‐animals were used to examine the prevention and reversal of pulmonary hypertension by either inducing Asic1 knockout before CH exposure or following the establishment of pulmonary hypertension (3 weeks post‐CH). Pulmonary hypertension was assessed by direct cardiac puncture in anesthetized mice to measure right ventricular systolic pressure. Similar to global ASIC1 knockout (Asic1‐/‐) mice, SMC‐Asic1‐/‐ mice were protected from the development of CH‐induced pulmonary hypertension. In contrast, EC‐Asic1‐/‐ mice developed pulmonary hypertension comparable to wildtype Asic1+/+ mice (Figure 1). Furthermore, CH‐induced pulmonary hypertension was reversed by induction of SMC‐specific ASIC1 knockout following established pulmonary hypertension. CH‐induced vascular remodeling was assessed using smooth muscle α‐actin immunofluorescence to determine the medial thickness of small pulmonary arteries. The degree of arterial muscularization was significantly attenuated and/or reversed in SMC‐Asic1‐/‐ arteries (P<0.05). These findings were additionally supported by decreased CH‐induced proliferation (P<0.05) and migration (P<0.05) of pulmonary arterial SMC from Asic1‐/‐ mice, assessed by flow cytometry of BrdU positive cells and transwell migration assay, respectively. Together these data demonstrate that SMC, but not EC, ASIC1 contributes to CH‐induced pulmonary hypertension and vascular remodeling. Furthermore, these studies provide evidence for the therapeutic potential of ASIC1 inhibition to reverse pulmonary hypertension.

Fibroblast-Secreted Phosphoprotein 1 Mediates Extracellular Matrix Deposition and Inhibits Smooth Muscle Cell Contractility in Marfan Syndrome Aortic Aneurysm.

Fibrillin 1 (Fbn1) mutation causes Marfan syndrome (MFS) with thoracic aortic aneurysm (TAA) as the main complication. The mechanisms for extracellular matrix (ECM) homeostasis disruption in MFS TAA are unclear. Here, we found ECM-related gene secreted phosphoprotein 1 (Spp1) increased in Fbn1C1041G/+ mice using transcriptome sequencing and a distinct fibroblast subcluster with Spp1 as the strongest marker was identified with analysis of the MFS mouse aortic single-cell sequencing dataset. Immunostaining confirmed elevated Spp1 in adventitial fibroblasts, and Spp1 might regulate fibroblast and smooth muscle cell (SMC) communication primarily through Itga8/Itgb1. Then, we observed Spp1 reduced contractile genes Acta2 and Tagln expression in SMCs and increased collagen expression in fibroblasts, which might contribute to TAA development. Finally, we also found elevated SPP1 plasma level was associated with an increased risk of TAA in patients. Therefore, SPP1 may serve as a biomarker and therapeutic target for TAA.