Human Coronary Artery Smooth Muscle Cells Research Articles

Exploring Bone Morphogenetic Protein-2 and -4 mRNA Expression and Their Receptor Assessment in a Dynamic In Vitro Model of Vascular Calcification

Background: Vascular calcification (VC) is a dynamic, tightly regulated process driven by cellular activity and resembling the mechanisms of bone formation, with specific molecules playing pivotal roles in its progression. We aimed to investigate the involvement of the bone morphogenic proteins (BMP-2, BMP-4, BMPR-1a/1b, and BMPR-2) system in this process. Our study used an advanced in vitro model that simulates the biological environment of the vascular wall, assessing the ability of a phosphate mixture to induce the osteoblastic switch in human coronary artery smooth muscle cells (HCASMCs). Methods: HCASMCs were grown in mono- and co-culture with human coronary artery endothelial cells (HCAECs) in a double-flow bioreactor (LiveBox2 and IVTech), allowing static and dynamic conditions through a peristaltic pump. The VC was stimulated by incubation in a calcifying medium for 7 days. A BMP system Real-Time PCR was performed at the end of each experiment. Results: In monocultures, BMP-2 expression increased in calcified HCASMCs in static (p = 0.01) and dynamic conditions. BMP-4 and the biological receptors were expressed in all the experimental settings, increasing mainly in dynamic flow conditions. In co-cultures, we observed a marked increase in BMP-2 and BMP-4, BMPR-1a (p = 0.04 and p = 0.01, respectively), and BMPR-2 (p = 0.001) in the calcifying setting mostly in dynamic conditions. Conclusions: The increase in BMP-2/4 in co-culture suggests that these genes might promote the switch towards an osteogenic-like phenotype, data also supported by the rise of both BMPR-1a and BMPR-2. Thus, our findings provide insights into the mechanisms by which dynamic co-culture modulates the BMP system activation in an environment mimicking in vivo VC’s cellular and mechanical characteristics.

Abstract 4116603: Interleukin-33 from Necrotic Tunica Media Plays a Key Role in the Exacerbations of Coronary Arteritis in Kawasaki Disease

Objective: Alarmins resulting from cell death or oxidative stress have been shown to be involved in the development of Kawasaki disease (KD) vasculitis. In our previous study, we demonstrated the potential role of (IL)-33 as an alarmin in the development of KD vasculitis. Although edematous dissociation (necrotic change) of the tunica media is thought to be a major source of IL-33 in KD vasculitis, it has not yet been elucidated. Methods: We investigated the impact of IL-33 released from necrotic human coronary artery smooth muscle cells (HCASMCs) on human coronary artery endothelial cells (HCAECs) by a co-culture assay using the Transwell ® cell culture insert system. Subsequently, we evaluated the anti-inflammatory effects of anti-IL-33 and anti-ST2 antibodies compared to the conventional therapies of KD, such as high-dose IgG or anti-tumor necrosis factor (TNF)-α antibody. Results: Primary necrosis of HCASMCs induced significant release of IL-33. In co-cultures of necrotic HCASMCs with HCAECs, necrotic HCASMCs significantly induced the production of various proinflammatory cytokines in HCAECs. Anti-IL-33 and anti-ST2 antibodies exhibited unique inhibitory effects on the production of platelet-derived growth factor-BB or IL-12(p70) in HCAECs. Conclusion: The results of the present study suggest the potential involvement of edematous dissociation of the tunica media in the development of KD vasculitis. Furthermore, the distinctive anti-inflammatory effects of the anti-IL-33/ST2 axis drugs suggest novel therapeutic options for patients with refractory KD.

Abstract 4137846: Smooth muscle expression of RNA editing enzyme ADAR1 controls vascular integrity and progression of atherosclerosis

Mapping the genomic architecture of complex disease has been predicated on the understanding that genetic variants influence disease risk through modifying gene expression. However, recent discoveries have revealed that a significant burden of disease heritability in common autoinflammatory disorders and coronary artery disease is mediated through genetic variation modifying post-transcriptional modification of RNA through adenosine-to-inosine (A-to-I) RNA editing. This common RNA modification is catalyzed by ADAR enzymes, where ADAR1 edits specific immunogenic double stranded RNA (dsRNA) to prevent activation of the double strand RNA (dsRNA) sensor MDA5 ( IFIH1 ) and stimulation of an interferon stimulated gene (ISG) response. Multiple lines of human genetic data indicate impaired RNA editing and increased dsRNA sensing to be an important mechanism of coronary artery disease (CAD) risk. Here, we provide a crucial link between observations in human genetics and mechanistic cell biology leading to progression of CAD. Through analysis of human atherosclerotic plaque, we implicate the vascular smooth muscle cell (SMC) to have a unique requirement for RNA editing, and that ISG induction occurs in SMC phenotypic modulation, implicating MDA5 activation. Through culture of human coronary artery SMCs, generation of a conditional SMC specific Adar1 deletion mouse model on a pro-atherosclerosis background, and with incorporation of single cell RNA sequencing cellular profiling, we further show that Adar1 controls SMC phenotypic state, is required to maintain vascular integrity, and controls progression of atherosclerosis and vascular calcification. Our data implicates MDA5 activation to occur in SMC phenotypic modulation and accelerates formation of chondromyocytes in a distinct cellular lineage trajectory — indicating a cell type and context specific requirement of RNA editing. Through this work, we describe a fundamental new mechanism of CAD, where RNA editing and sensing of dsRNA in a cell type and context specific mechanism mediates disease progression, bridging our understanding of human genetics and disease causality.

Aryl-hydrocarbon receptor in smooth muscle cells protect against dioxin induced adverse remodeling of atherosclerosis.

Environmental exposure to dioxin has been linked to increased myocardial infarction. Smooth muscle cells (SMC) in the coronary vasculature play a critical role in atherosclerotic plaque remodeling due to their phenotypic plasticity, however, the detailed mechanism linking dioxin exposure to adverse SMC modulation is not well understood. Single-cell RNA and ATAC sequencing and histological analyses were performed on the aorta from mouse models of atherosclerosis exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) or control. Primary human coronary artery SMC (HCASMC) treated in culture with TCDD were used to perform RNA-Seq, ATAC-Seq, and functional phenotypic assays. ChIP-Seq was performed with antibodies against Aryl-hydrocarbon receptor (AHR) and TCF21, two of known SMC modulating transcription factors. Modulated SMC were the most transcriptionally responsive cell type to dioxin in the atherosclerotic aorta. Dioxin accelerated disease phenotype by promoting a modulated SMC phenotype early, resulting in increased lesion size, migration of SMC, and macrophage recruitment to the lesion. We found C3 expressing modulated SMCs to be likely contributing to the increased macrophage recruitment and inflammation. Analysis of the RNA-Seq data from HCASMC treated with TCDD showed differential enrichment of biological pathways related to cell migration, localization, and inflammation. Furthermore, ATAC-Seq data showed a significant activation for pathways regulating vascular development, cell migration, inflammation, and apoptosis. With TCDD treatment, there was also enrichment of AHR ChIP-Seq peaks, while the TCF21 enrichment decreased significantly. The SMC-specific Ahr knockout resulted in increased oxidative stress in SMC, increased lesion size and macrophage content, and loss of SMC lineage cells in the lesion cap when exposed to TCDD, consistent with a more vulnerable plaque phenotype. Dioxin adversely remodels atherosclerotic plaque by accelerating the SMC- phenotypic modulation, and increasing inflammation and oxidative stress resulting in increased macrophage recruitment and lesion size. Dioxin may adversely affect the SMC phenotype and disease state by affecting the TCF21 occupancy in the open chromatin regions. Furthermore, we observed that SMC-specific deletion of Ahr in mice resulted in worsening of dioxin mediated SMC modulation and atherosclerosis, suggesting that Ahr in SMC confers protection against dioxin by promoting a stable plaque phenotype and reducing dioxin induced oxidative stress. Exposure to dioxin, an environmental pollutant present in tobacco smoke and air pollution, accelerates smooth muscle cell modulation, and atherosclerosis.Dioxin exposure leads to inflammatory smooth muscle cell phenotype characterized by complement pathway activation and increased macrophage recruitment to plaqueAryl-hydrocarbon receptor in SMC protects against oxidative stress, and promotes a stable plaque phenotype.

Gut microbiota-derived Metabolite, Shikimic Acid, inhibits vascular smooth muscle cell proliferation and migration

Gut microbiota dysbiosis is linked to vascular wall disease, but the mechanisms by which gut microbiota cross-talk with the host vascular cells remain largely unknown. Shikimic acid (SA) is a biochemical intermediate synthesized in plants and microorganisms, but not mammals. Surprisingly, recent metabolomic profiling data demonstrate that SA is detectable in human and murine blood. In this study, analyzing data from germ-free rats, we provide evidence in support of SA as a bona fide gut microbiota-derived metabolite, emphasizing its biological relevance. Since vascular cells are the first cells exposed to circulating metabolites, in this study, we examined, for the first time, the effects and potential underlying molecular mechanisms of SA on vascular smooth muscle cell (VSMC) proliferation and migration, which play a key role in occlusive vascular diseases, such as post-angioplasty restenosis and atherosclerosis. We found that SA inhibits the proliferation and migration of human coronary artery SMCs. At the molecular level, unexpectedly, we found that SA activates, rather than inhibits, multiple pro-mitogenic signaling pathways in VSMCs, such as ERK1/2, AKT, and mTOR/p70S6K. Conversely, we found that SA activates the anti-proliferative AMP-activated protein kinase (AMPK) in VSMCs, a key cellular energy sensor and regulator. However, loss-of-function experiments demonstrate that AMPK does not mediate the inhibitory effects of SA on VSMC proliferation. In conclusion, these studies demonstrate that a microbiota-derived metabolite, SA, inhibits VSMC proliferation and migration in vitro and prompt further evaluation of the possible underlying molecular mechanisms and the potential protective role in VSMC-related vascular wall disease in vivo.

Fast and reliable quantification of aldosterone, cortisol and cortisone via LC-MS/MS to study 11β-hydroxysteroid dehydrogenase activities in primary cell cultures

Cell culture experiments can support characterization of enzymatic activities in healthy and tumorous human tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) enables simultaneous measurement of several steroids from a single sample, facilitating analysis of molecular pathways involved in steroid biosynthesis. We developed a reliable but fast method for quantification of cortisol, cortisone and aldosterone in cell culture supernatant. Validation, including investigation of matrix-matched calibration, was performed for two different cell types. Utility of the method was demonstrated in the study of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity under conditions of glucocorticoid and mineralocorticoid excess in different cell types. Aldosterone, cortisol and cortisone were extracted by liquid-liquid extraction (LLE) with methyl tert-butyl ether from 1 mL of cell culture supernatant. Steroids were separated on a Kinetex biphenyl column (50 ×2.1 mm, 2.6 µm) with gradient elution of water and methanol containing 2 mM ammonium format and analysed in multiple reaction monitoring mode after positive electrospray ionization. Application of the method included cell culture experiments with two different primary cell types, human coronary artery smooth muscle cells (HCSMC) and human coronary artery endothelial cells (EC). Cells were treated with different concentrations of cortisol, aldosterone and mifepristone, a glucocorticoid receptor antagonist and quantitative PCR was performed. The method exhibits high precision (CV ≤ 6 %) and accuracy (deviation from nominal concentration ≤ 6 %) for concentrations above the limit of quantification (LoQ) which is 0.11, 0.56 and 0.69 nmol/L for aldosterone, cortisone and cortisol, respectively. Calibration curves did not differ when prepared in media or solvent. The method enabled us to confirm activity of HSD11B2 and concentration dependent conversion of cortisol to cortisone in HCSMC (median conversion ratio at 140 nM cortisol = 1.46 %). In contrast we did not observe any HSD11B2 activity in EC. Neither addition of high aldosterone, nor addition of 1 µM mifepristone had impact on glucocorticoid concentrations. Quantitative PCR revealed expression of HSD11B1 and HSD11B2 in HCSMC but not in EC. We present a fast and reliable method for quantification of cortisol, cortisone and aldosterone in cell culture supernatants. The method enabled us to study HSD11B2 activity in two different cell types and will support future experiments investigating mechanisms of target organ damage in conditions of glucocorticoid and mineralocorticoid excess.

Necrotic Change of Tunica Media Plays a Key Role in the Development of Coronary Artery Lesions in Kawasaki Disease.

Alarmins resulting from cell death or oxidative stress are involved in the development of Kawasaki disease (KD) vasculitis. In a previous study, we demonstrated the potential role of interleukin (IL)-33 as an alarmin in the development of KD vasculitis. Although edematous dissociation (necrotic change) of the tunica media is thought to be a major source of IL-33 in KD vasculitis, it has not yet been elucidated. We investigated the impact of IL-33 released from necrotic human coronary artery smooth muscle cells (HCASMCs) on human coronary artery endothelial cells (HCAECs) using a coculture assay. Subsequently, we evaluated the anti-inflammatory effects of anti-IL-33 and anti-suppression of tumorigenicity 2 (ST2) antibodies compared with conventional therapies of KD, such as high-dose IgG or anti-tumor necrosis factor (TNF)-α antibody. Primary necrosis of HCASMCs induced significant release of IL-33. In cocultures of necrotic HCASMCs with HCAECs, the necrotic HCASMCs significantly induced the production of various proinflammatory cytokines in the HCAECs. Anti-IL-33 and anti-ST2 antibodies exhibited unique inhibitory effects on the production of platelet-derived growth factor-BB or IL-12(p70) in HCAECs. There is potential involvement of edematous dissociation of the tunica media in the development of KD vasculitis. Furthermore, the distinctive anti-inflammatory effects of the anti-IL-33/ST2 axis drugs suggest novel therapeutic options for patients with refractory KD.

Abstract Tu010: Matrix metallopeptidase 9 contributes to the beginning of plaque and is a potential biomarker for the early identification of atherosclerosis in asymptomatic patients with diabetes

Aims: To determine the roles of matrix metallopeptidase-9 (MMP9) on human coronary artery smooth muscle cells (HCASMCs) in vitro , early beginning of atherosclerosis in vivo in diabetic mice, and patients with diabetes. Methods: Active hMMP9 ( act-hMMP9) was added to HCASMCs and markers of inflammation were measured. Act-hMMP9 (n=16) or placebo (n=15) was administered to d i abetic KK.Cg- A y /J (KK) mice. Carotid artery inflammation and atherosclerosis measurements were made at 2 and 10 weeks after treatment. An observational study of newly diagnosed drug naïve patients with type 2 diabetes mellitus (T2DM n=234) and healthy matched controls (n=41) was performed and patients had ultrasound of carotid arteries and some had coronary computed tomography angiogram for the assessment of atherosclerosis. Serum MMP9 was measured and its correlation with carotid artery or coronary artery plaques were determined. Results: Act-hMMP9 induced inflammatory response in HCASMCs in vitro , including increased gene and protein expressions of MCP-1, ICAM-1, VCAM-1, and enhanced macrophage adhesion. Exogenous act-hMMP9 increased inflammation and initiated atherosclerosis in KK mice at 2 and 10 weeks: increased vessel wall thickness, lipid accumulation, and Galectin-3+ macrophage infiltration into the carotid arteries. In newly diagnosed T2DM patients, serum MMP9 correlated with carotid artery plaque size with a possible threshold cutoff point. In addition, serum MMP9 correlated with number of mixed plaques and grade of lumen stenosis in coronary arteries of patients with drug naïve T2DM. Conclusions: MMP9 contributes to the initiation of atherosclerosis and may be a potential biomarker for the early identification of atherosclerosis in patients with diabetes

MiRNA-221 and -222 increase in exosomes upon insulin stimulation of human vascular smooth muscle cells lacking the insulin-like growth factor 1 receptor

As a result of increased vascular inflammation, patients with Type 2 diabetes experience a near 4-fold increase in their risk for heart attack and stroke. miR-221 and -222 are two microRNAs that are upregulated in the vasculature of Type 2 diabetic patients. miR-221/222 act by downregulating the cyclin-dependent kinase inhibitor, p27Kip1, in vascular smooth muscle cells (VSMCs) and exosome-mediated proinflammatory effects on endothelial cells and macrophages. Loss of the insulin-like growth factor receptor (IGFR) in VSMCs can unmask the insulin receptor (IR) and increase insulin signaling. We have demonstrated that the loss of IGFR, coupled with physiological insulin stimulation, promotes an increased expression of miR-221/-222 in murine VSMCs. Respectively, miR-221/222 content in VSMC-derived exosomes increases and p27Kip1 mRNA expression decreases in VSMCs. We asked whether the effect of loss of IGFR in human coronary artery smooth muscle cells (HCASMCs) would also lead to an increase in miR-221/-222 content of VSMC-derived exosomes. Transfection with a CRISPR-cas9 plasmid targeting IGFR resulted in a 50% decrease in IGFR mRNA in HCASMCs, when compared to transfection with negative control CRISPR plasmids. Droplet digital PCR was used to measure expression of miR-221 and miR-222 in the exosomes derived from both sets of HCASMCs. There was no change in the miR-221/222 content of exosomes derived from HCASMCs transfected with the negative control CRISPR following stimulation with 1nM insulin (7.27± 4.75 vs. 11.33 ± 1.09 copies of miR-221 and 6.43 ± 0.43 vs. 4.68 ± 0.02 copies of miR-222). In contrast, there was a 2 to 3-fold increase in miR-221/222 content in exosomes that were derived from HCASMCs with IGFR knockdown following stimulation with 1nM insulin (14.96 ± 0.3 vs. 35.66 ± 3.08 copies of miR-221 and 9.12 ± 1.19 vs.15.44 ± 5.36 copies of miR-222). We conclude that loss of IGFR coupled with physiological insulin stimulation increases the miR-221/222 content of exosomes of HCASMCs, recapitulating the effects of IGFR loss seen in murine VSMCs as well as the effects of diabetes on VSMCs. This model system will allow additional studies connecting the loss of IGFR to the cardiovascular complications of diabetes. Tulane University School of Medicine, Biomedical Sciences Program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2122: A Multiomic Timecourse Atlas Of Murine Vascular Disease Smooth Muscle Cell Transition Phenotypes And Their Perturbation With Tcf21 Knockout

Background: Vascular smooth muscle cells (SMC) contribute to heritable CAD risk and undergo cell changes to fibroblast-like (FMC) and osteochondrogenic (CMC) phenotypes. We perform molecular characterization of this transition process over the course of atherosclerosis development to better understand how causal SMC CAD genes such as TCF21 drive the phenotypic transition process to alter CAD risk. Methods/Results: Deep multiomic profiling of aortic roots from control atherosclerosis model ( Myh11Cre ERT2 , ROSA tdT/+ , ApoE -/- ) mice with SMC specific lineage tracing on high fat diet were harvested for 7 scRNA and 6 scATAC time points across 16 weeks. Similarly, aortic roots from Tcf21 knockout ( Myh11Cre ERT2 , Tcf21 ΔSMC/ΔSMC , ROSA tdT/+ , ApoE -/- ) were collected at 3 timepoints for paired scRNA/scATAC. Waddington-Optimal Transport (WOT) mapped transcriptional trajectories across SMC lineage clusters which terminate in FMC and CMC phenotype clusters. PANDO/CellOracle frameworks were used to create gene regulatory networks with the ability to model SMC transition perturbations in silico . These analyses generated a multiomic timecourse atlas demonstrating the lineage traced phenotypic progression of SMC to FMC and CMC. Upon knockout of Tcf21 , we discern notable shifts in the SMC to FMC transition characterized by differential scRNA transcriptional pathways and scATAC shifts in transcription factor (TF) motif accessibility which also overlap with WOT predicted TF enrichment for each cell state. These integrative analyses revealed Tcf21 regulation of Cebpb and the Tead1 -Hippo pathways as novel mechanisms for mediating its role in modulation of disease related processes such as inflammation, response to TGF- β signaling and cell proliferation. Further mechanistic validation is performed through a combination of TF binding site analysis with TEAD1 and CEBPB ChIP-Seq in human coronary artery SMCs, dual luciferase reporter assays and proximity ligation assays. Conclusion: We construct a single cell atherosclerosis timecourse to model mouse SMC cell states and perturb CAD gene Tcf21 to elucidate the interplay between epigenetic and transcriptional mechanisms which affect key SMC transition elements such as CEBPB and the TEAD1 -Hippo axis.

Abstract 3074: RNA Editing Enzyme ADAR1 In Vascular Smooth Muscle Controls Vascular Integrity And Progression Of Atherosclerosis

Adenosine-to-inosine (A-to-I) RNA editing is a common RNA modification catalyzed by ADAR enzymes. ADAR1 serves a crucial function to edit specific immunogenic double stranded RNA (dsRNA) to prevent the dsRNA sensor, MDA5 ( IFIH1 ), activating an interferon stimulated gene (ISG) response. Discoveries in human genetics now implicate deficient ADAR1 RNA editing as a causal mechanism for coronary artery disease (CAD) risk. Here, we show that human atherosclerotic smooth muscle cells (SMCs) express immunogenic RNA at markedly higher levels compared to all other cell types. We demonstrate that ADAR1 is the master regulator of RNA editing in primary human coronary artery SMC (HCASMC) and loss of RNA editing causes activation of MDA5, characterized by upregulation of ISG genes (i.e. ISG15 ) and others with known roles in SMC phenotypic transition and CAD risk (i.e. KLF4 ). ADAR1 and MDA5 regulate SMC phenotypic modulation and calcification in vitro . In a high fat diet atherosclerosis model, scRNAseq analysis revealed SMC to have increasing ISG expression with phenotypic modulation. To evaluate SMC RNA editing in vivo , we generated a conditional SMC specific Adar1 KD mouse ( Adar1 flox/flox , Myh11 CreERT2 , ROSA tdT/+ , ApoE -/- ). At 2 weeks following SMC- Adar1 KD, we observed 50% mortality with a severe phenotype of elastin degradation and disarray, intravascular hemorrhage, and inflammatory cell infiltration in the aortic wall. scRNAseq of aortic tissue showed that SMC- Adar1 KD causes ISG activation and coordinates a complex transcriptional response throughout the vessel wall with robust macrophage cell infiltration. Through evaluation of ligand-receptor interaction, we show that Ccl5:Ccr5 is the principal regulator of cell infiltration following SMC- Adar1 KD. Through study of SMC- Adar1 haploinsufficiency in the atherosclerosis mouse model, we further reveal that MDA5 activation occurs in SMC phenotypic modulation and accelerates formation of chondromyocytes — indicating a cell type and context specific requirement of RNA editing. Through this work, we describe a fundamental new mechanism of CAD, where RNA editing and sensing of dsRNA mediates disease progression, promoting our understanding of disease causality and CAD genetic risk.

Abstract 1147: Crosstalk Between Alk5 And Mtorc1 Signaling Promotes VSMC Differentiation And The Therapeutic Effect Of Rapamycin

Background: Vascular smooth muscle cells (VSMC) phenotypic switching contributes to vascular repair and remodeling but also to pathologies including intimal hyperplasia. The mTORC1 inhibitor rapamycin is an effective drug-eluting stent agent that promotes VSMC differentiation. TGFβ also promotes VSMC differentiation through SMAD transcription factors. We investigated unexpected convergence between these pathways in VSMC plasticity. Methods: We assessed the interactions between rapamycin and the TGFβ1 (ALK5) signaling in human coronary artery SMCs and in vascular remodeling using inducible SMC-specific knockout mice (ALK5iKO). Results: Genome-wide histone H3K27 acetylation analysis unexpectedly identified SMAD binding elements as the motif most enriched after rapamycin treatment of hCASMCs. Treatment with rapamycin promoted rapid phosphorylation of SMAD2/3. Extensive signaling studies revealed that this phosphorylation required ALK5 activity but not TGF-β ligand. Surprisingly, ALK5 and SMAD2/3 were required for rapamycin-induced differentiation. We determined that rapamycin relieves FKBP12 inhibition of ALK5 to promote ligand independent Smad signaling, and FKBP12 knockdown was sufficient to induce contractile genes. Notably, rapamycin treatment induced an interaction between TET2 and SMAD2/3, and these SMADs were required for differentiation-associated chromatin remodeling, including DNA hydroxymethylation and H3K27 acetylation at contractile genes, suggesting that SMADs and TET2 function in concert at SBE- and CArG-containing promoter regions. Furthermore, ALK5iKO mice exhibited severe intimal hyperplasia after carotid artery injury compared to controls and were entirely resistant to the therapeutic effect of rapamycin, despite persistent inhibition of mTORC1. Consistent with in vitro findings, treatment with rapamycin elevated pSmad3 staining post-injury in the medial layer of control but not ALK5iKO mice. Conclusions: We report the surprising observation that rapamycin requires FKBP12/ALK5/SMAD2/3 signaling in order to promote TET2-dependent chromatin remodeling and SMC differentiation. Understanding these mechanisms may reveal new therapeutic strategies for treating vascular diseases.

Novel Role of YAP1 in Regulating Blood Pressure and Vascular Tone

Introduction: Hypertension is a major risk factor for occlusive vascular diseases, myocardial infarction, heart failure, stroke, and chronic kidney disease. In the U.S., ~30% of adults suffer from hypertension. Merely half of the individuals with hypertension have their blood pressure under control, despite the wide range of antihypertensive medications available, indicating an urgent need for a better understanding of the underlying causes of hypertension to identify novel therapeutic strategies for its treatment and prevention. Recent genome-wide association study (GWAS) has identified an unexpected association between a loss-of-function SNP in the YAP1 gene locus and decreased blood pressure (BP). Moreover, de novo analysis of publicly available RNAseq data revealed that the vascular tissues of hypertensive mice treated with Ang-II exhibit increased YAP1 expression. Suggesting a novel role of YAP1 in blood pressure regulation. However, whether Vascular smooth muscle cell (VSMC) expressed YAP1 has a specific role in VSMC contraction and BP regulation remains completely unknown. Therefore, we hypothesized that YAP1 could play a key role in regulating blood pressure. Methodology: We have generated a novel inducible SM-specific- Yap1 Knockout ( YAP1 iKO) mouse model and employed a pharmacological inhibitor of YAP1, Verteporfin, to delineate the functional role of VSMC-expressed YAP1 in hypertension in regulating vascular tone and BP. Vascular reactivity experiments were performed using wire myography. BP was measured in ambulant mice via radiotelemetry. In vitro gain- and loss-of-function studies utilizing human coronary artery SMCs were conducted to examine the impact of YAP1 on contractility signaling components using western blotting. Results: Consistent with GWAS and RNAseq data, we found that YAP1 is upregulated in vascular tissues of spontaneously hypertensive rats (SHRs). Using aortic rings from WT mice, we discovered that Verteporfin significantly reduced vasoconstrictive responses to Phenylephrine or Serotonin but did not alter endothelium-dependent vasorelaxant responses to Acetylcholine, suggesting a specific role of YAP1 in VSMCs. Consistently, YAP1 iKO exhibited attenuated responses to vasoconstrictors in isolated conduit and resistance vessels. Importantly, radiotelemetry studies demonstrated for the first time that YAP1 iKO mice exhibit a hypotensive phenotype. Mechanistically, YAP1 gain- and loss-of-function studies in human VSMCs showed that YAP1 activates multiple signaling pathways that play a key role in VSMC contractility including RhoA/ROCK1/actin polymerization and PKC/ERK signaling pathways. Summary/Conclusion: This is the first study to demonstrate that inhibition of SM-specific-Yap1 expression regulates BP and vascular tone, likely via the regulation of contractile machinery components. Together, our data suggest that YAP1 is a promising novel therapeutic target for ameliorating hypertension. This work is supported by a grant from National Heart, Lung, and Blood Institute (R00HL153896). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Matrix metallopeptidase 9 contributes to the beginning of plaque and is a potential biomarker for the early identification of atherosclerosis in asymptomatic patients with diabetes.

To determine the roles of matrix metallopeptidase-9 (MMP9) on human coronary artery smooth muscle cells (HCASMCs) in vitro, early beginning of atherosclerosis in vivo in diabetic mice, and drug naïve patients with diabetes. Active human MMP9 (act-hMMP9) was added to HCASMCs and the expressions of MCP-1, ICAM-1, and VCAM-1 were measured. Act-hMMP9 (n=16) or placebo (n=15) was administered to diabetic KK.Cg-Ay/J (KK) mice. Carotid artery inflammation and atherosclerosis measurements were made at 2 and 10 weeks after treatment. An observational study of newly diagnosed drug naïve patients with type 2 diabetes mellitus (T2DM n=234) and healthy matched controls (n=41) was performed and patients had ultrasound of carotid arteries and some had coronary computed tomography angiogram for the assessment of atherosclerosis. Serum MMP9 was measured and its correlation with carotid artery or coronary artery plaques was determined. In vitro, act-hMMP9 increased gene and protein expressions of MCP-1, ICAM-1, VCAM-1, and enhanced macrophage adhesion. Exogenous act-hMMP9 increased inflammation and initiated atherosclerosis in KK mice at 2 and 10 weeks: increased vessel wall thickness, lipid accumulation, and Galectin-3+ macrophage infiltration into the carotid arteries. In newly diagnosed T2DM patients, serum MMP9 correlated with carotid artery plaque size with a possible threshold cutoff point. In addition, serum MMP9 correlated with number of mixed plaques and grade of lumen stenosis in coronary arteries of patients with drug naïve T2DM. MMP9 may contribute to the initiation of atherosclerosis and may be a potential biomarker for the early identification of atherosclerosis in patients with diabetes. https://clinicaltrials.gov, identifier NCT04424706.

EGR1 transcriptionally regulates SVEP1 to promote proliferation and migration in human coronary artery smooth muscle cells.

A low-frequency variant of sushi, von Willebrand factor type A, EGF, and pentraxin domain-containing protein 1 (SVEP1) is associated with the risk of coronary artery disease, as determined by a genome-wide association study. SVEP1 induces vascular smooth muscle cell proliferation and an inflammatory phenotype to promote atherosclerosis. In the present study, qRT‒PCR demonstrated that the mRNA expression of SVEP1 was significantly increased in atherosclerotic plaques compared to normal tissues. Bioinformatics revealed that EGR1 was a transcription factor for SVEP1. The results of the luciferase reporter assay, siRNA interference or overexpression assay, mutational analysis and ChIP confirmed that EGR1 positively regulated the transcriptional activity of SVEP1 by directly binding to its promoter. EGR1 promoted human coronary artery smooth muscle cell (HCASMC) proliferation and migration via SVEP1 in response to oxidized low-density lipoprotein (ox-LDL) treatment. Moreover, the expression level of EGR1 was increased in atherosclerotic plaques and showed a strong linear correlation with the expression of SVEP1. Our findings indicated that EGR1 binding to the promoter region drive SVEP1 transcription to promote HCASMC proliferation and migration.

Intralesional pentraxin 3 increases with atherosclerotic disease progression, but may protect from thrombosis: Friend or foe?

AimTo investigate the role of pentraxin 3 (PTX3) in atherosclerotic disease progression and plaque destabilization, as well as in coronary restenosis after directional coronary atherectomy (DCA). Materials and methodsPTX3 contents of early and advanced atherosclerotic lesions of the aorta obtained at autopsy were determined by ELISA and Western blot. Also, coronary plaques of patients with acute coronary syndrome (ACS) or stable angina pectoris (SAP) obtained by DCA were analyzed by immunohistochemistry for PTX3. The effects of PTX3 on smooth muscle cells (SMCs) and thrombogenesis were investigated with cultured human coronary artery SMCs and a flow chamber system, respectively. ResultsAdvanced atherosclerotic lesions contained a significantly larger amount of PTX3 than early lesions (ELISA: 9.96 ± 2.77 ng/100 mg tissue, n = 8 vs 0.24 ± 0.18 ng/100 mg tissue, n = 6, P = 0.0097). Also, ACS plaques contained a significantly larger amount of PTX3 than SAP plaques (PTX3 immunohistochemistry–positive area percentage: 2.88 ± 0.53 %, n = 22 vs 0.67 ± 0.27 %, n = 23, P = 0.0009). Curiously, the patients who would remain free of post-DCA restenosis (n = 19) had plaques with a significantly higher PTX3 immunohistochemistry–positive area percentage than those who would develop restenosis (n = 12) (2.32 ± 0.49 % vs 0.49 ± 0.17 %, P = 0.002). In the mechanistic part of the study, PTX3 inhibited SMC proliferation and migration. PTX3 also inhibited platelet thrombus formation in the condition simulating arterial blood flow. ConclusionsPTX3 is increased in advanced (vs early) atherosclerotic lesions and unstable (vs stable) coronary plaques. The inhibitory effects of PTX3 on SMCs and thrombogenesis suggest that intraplaque PTX3 might have atheroprotective effects.

Endothelial-derived microvesicles promote pro-migratory cross-talk with smooth muscle cells by a mechanism requiring tissue factor and PAR2 activation.

Microvesicles (MV) released by endothelial cells (EC) following injury or inflammation contain tissue factor (TF) and mediate communication with the underlying smooth muscle cells (SMC). Ser253-phosphorylated TF co-localizes with filamin A at the leading edge of migrating SMC. In this study, the influence of endothelial-derived TF-MV, on human coronary artery SMC (HCASMC) migration was examined. MV derived from human coronary artery EC (HCAEC) expressing TFWt accelerated HCASMC migration, but was lower with cytoplasmic domain-deleted TF. Furthermore, incubation with TFAsp253-MV, or expression of TFAsp253 in HCASMC, reduced cell migration. Blocking TF-factor VIIa (TF-fVIIa) procoagulant/protease activity, or inhibiting PAR2 signaling on HCASMC, abolished the accelerated migration. Incubation with fVIIa alone increased HCASMC migration, but was significantly enhanced on supplementation with TF. Neither recombinant TF alone, factor Xa, nor PAR2-activating peptide (SLIGKV) influenced cell migration. In other experiments, HCASMC were transfected with peptides corresponding to the cytoplasmic domain of TF prior to stimulation with TF-fVIIa. Cell migration was suppressed only when the peptides were phosphorylated at position of Ser253. Expression of mutant forms of filamin A in HCASMC indicated that the enhancement of migration by TF but not by PDGF-BB, was dependent on the presence of repeat-24 within filamin A. Incubation of HCASMC with TFWt-MV significantly reduced the levels of Smoothelin-B protein, and upregulated FAK expression. In conclusion, Ser253-phosphorylated TF and fVIIa released as MV-cargo by EC, act in conjunction with PAR2 on SMC to promote migration and may be crucial for normal arterial homeostasis as well as, during development of vascular disease.

Phosphoglycerate Dehydrogenase Overexpression Inhibits Ferroptosis to Repress Calcification of Human Coronary Artery Vascular Smooth Muscle Cells via the P53/SLC7A11 Pathway.

Coronary artery calcification (CAC) is in almost all patients with coronary artery disease and requires more effective therapies. We aim to explore the effects of phosphoglycerate dehydrogenase (PHGDH) on CAC. We identified the differentially expressed genes through bioinformatic analysis and selected PHGDH for further verification. Human coronary artery smooth muscle cells (HCASMCs) cultured with calcifying medium were used as models of CAC in vitro. Erastin was administered to induce ferroptosis. We determined the cell viability by the cell count kit-8 assay. The alkaline phosphatase activity, calcium content, and the expression of glutathione were evaluated by the corresponding detection kits. The calcification level was detected by alizarin red staining. Then we performed Western blot to examine the expression of runt-related transcription factor 2, bone morphogenetic protein 2, cyclooxygenase 2, glutathione peroxidase 4, P53, and solute carrier family 7a member 11 (SLC7A11). We acquired 201 differentially expressed genes and selected PHGDH to verify. In calcifying medium-induced HCASMCs, PHGDH overexpression increased the cell viability and decreased the alkaline phosphatase activity, calcium content, calcification level, and the expression of bone morphogenetic protein 2 and runt-related transcription factor 2. Additionally, we found higher levels of glutathione, glutathione peroxidase 4, and SLC7A11 and lower levels of cyclooxygenase 2 and P53 after up-regulating PHGDH. Erastin reversed the effects of PHGDH on calcification of HCASMCs. PHGDH overexpression suppresses the calcification level of HCASMCs by inhibiting ferroptosis through the P53/SLC7A11 signaling pathway, suggesting PHGDH as a promising therapeutic target of CAC.

Abstract 17720: The Role of Soluble Guanylyl Cyclase/NLRP3/RHOA/ROCK Axis in Coronary Artery Spasm: A Potential Biomarker and Therapeutic Target

Background: Coronary artery spasm (CAS) can lead to potentially life-threatening acute coronary syndrome. The second messenger cyclic guanosine 3',5'-monophosphate (cGMP), which is regulated by BNP-pGC-cGMP and nitric oxide (NO)-soluble guanylate cyclase (sGC)-cGMP pathways, regulate not only vasodilation but also the functions of immune cells. However, the significance of sGC, as a primary receptor for NO, is unclear in CAS. Methods: We investigated sGC and downstream signaling in patient monocyte-derived macrophages (PMDMs) and human coronary artery smooth muscle cells (HCASMCs) using computational biology and bioinformatics to correlate macrophage polarization subsets, Lp(a), and inflammasome signaling axes with CAS. Results: Plasma sGC levels were significantly higher in 30 CAS patients compared to 20 non-CAS controls (P<0.001). RNA-sequencing analysis revealed a significant association of sGC expression with NLR Family Pyrin Domain Containing (NLRP)-3 in HCASMCs and induced expression of inflammatory cytokines-specific surface markers. Lp(a) induction promoted the sGC-NLRP3 mediated lipid metabolism axis in PMDMs at both mRNA and protein levels, with elevated M1 polarization. sGC inhibition in HCASMCs reduced RhoA-GTP activity and its downstream effector ROCK, mitigating the Lp(a) effect, while sGC-specific modulator 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-l-one (ODQ) treatment decreased Lp(a)-induced expression of sGC-NLRP3-inflammasome mediators and induced caspase-1, as well as subsequent cleavage of proIL-1β and proIL-18 in VSMCs. Conclusion: sGC/GC molecule in conjunction with the RHOA/ROCK pathway is an early biomarker for CAS. The sGC-NLRP3-inflammasome mediators signaling axis plays an important role in Lp(a)-regulated macrophage polarization and CAS development.

Abstract 12901: ADAR1 RNA Editing in Vascular Smooth Muscle Maintains Vascular Integrity, Regulates Cell State Transition, and is a Novel Mechanism of Coronary Artery Disease Risk

Adenosine-to-inosine (A-to-I) RNA editing is a common RNA modification catalyzed by ADAR (adenosine deaminase acting on RNA) enzymes. ADAR1 serves a crucial function to edit specific immunogenic double stranded RNA (dsRNA) molecules to prevent the dsRNA sensor, MDA5 ( IFIH1 ), activating an interferon stimulated gene (ISG) response. Discoveries in human genetics now implicate deficient ADAR1 RNA editing as a causal mechanism in coronary artery disease (CAD). Here, we reveal in human atherosclerosis, smooth muscle cells (SMCs) express immunogenic dsRNA at markedly higher levels compared to all other cell types. We demonstrate that ADAR1 is the master regulator of RNA editing in primary human coronary artery SMC (HCASMC). Loss of RNA editing through ADAR1 KD in HCASMC causes an ISG response characterized by upregulation of ISG genes (i.e. ISG15, IFI6, IFIH1 ) in addition to genes with known roles in SMC phenotypic transition and CAD risk (i.e. KLF4 , TCF21 , and ZEB2 ). ADAR1 and MDA5 regulate SMC phenotypic modulation and calcification in vitro and in atherosclerosis prone mice. scRNAseq analysis reveals SMC to have increasing ISG expression as cells transition from mature SMC to a more calcified chondromyocyte phenotype. To further evaluate the role for SMC RNA editing in vivo , we generated a conditional SMC specific Adar1 KD mouse ( Adar1 flox/flox , Myh11 CreERT2 , ROSA tdT/+ , ApoE -/- ). At 2 weeks following SMC- Adar1 KD, we observe 50% mortality, where mice demonstrate a severe phenotype of elastin degradation and disarray, intravascular hemorrhage, and inflammatory cell infiltration in the aortic wall. This effect was not seen with an endothelial cell specific deletion model ( Adar1 flox/flox , Cx40 CreERT2 ). scRNAseq of aortic tissue shows that SMC- Adar1 KD causes ISG activation throughout the vessel wall, SMCs show loss of mature SMC markers, and there is robust macrophage and T cell infiltration. Through evaluation of ligand-receptor interaction, we show that Ccl5:Ccr5 is the principal regulator of cell infiltration following SMC- Adar1 KD. Here we provide key evidence to support the observations from human genetics that the ADAR1-dsRNA-MDA5 axis is a causal mechanism in vascular disease and warrants further investigation.