Modulation Of Smooth Muscle Cells Research Articles

Atherosclerosis Is a Smooth Muscle Cell-Driven Tumor-Like Disease.

Atherosclerosis, a leading cause of cardiovascular disease, involves the pathological activation of various cell types, including immunocytes (eg, macrophages and T cells), smooth muscle cells (SMCs), and endothelial cells. Accumulating evidence suggests that transition of SMCs to other cell types, known as phenotypic switching, plays a central role in atherosclerosis development and complications. However, the characteristics of SMC-derived cells and the underlying mechanisms of SMC transition in disease pathogenesis remain poorly understood. Our objective is to characterize tumor cell-like behaviors of SMC-derived cells in atherosclerosis, with the ultimate goal of developing interventions targeting SMC transition for the prevention and treatment of atherosclerosis. We used SMC lineage tracing mice and human tissues and applied a range of methods, including molecular, cellular, histological, computational, human genetics, and pharmacological approaches, to investigate the features of SMC-derived cells in atherosclerosis. SMC-derived cells in mouse and human atherosclerosis exhibit multiple tumor cell-like characteristics, including genomic instability, evasion of senescence, hyperproliferation, resistance to cell death, invasiveness, and activation of comprehensive cancer-associated gene regulatory networks. Specific expression of the oncogenic mutant KrasG12D in SMCs accelerates phenotypic switching and exacerbates atherosclerosis. Furthermore, we provide proof of concept that niraparib, an anticancer drug targeting DNA damage repair, attenuates atherosclerosis progression and induces regression of lesions in advanced disease in mouse models. Our findings demonstrate that atherosclerosis is an SMC-driven tumor-like disease, advancing our understanding of its pathogenesis and opening prospects for innovative precision molecular strategies aimed at preventing and treating atherosclerotic cardiovascular disease.

Abstract 18753: Dynamic Phenotypic Transition of Smooth Muscle Cells During Human Ascending Thoracic Aortic Disease Progression: From Compensation in Aortic Aneurysm to Decompensation in Aortic Dissection

Introduction: Although ascending thoracic aortic aneurysm (ATAA) and ascending thoracic aortic dissection (ATAD) share common pathologic features of aortic degeneration, the specific elements that lead to rapid disease progression in ATAD remain poorly understood. We examined the molecular and cellular dynamics in smooth muscle cells (SMCs) of aortic tissues from ATAA and ATAD patients and further examined mechanisms in a mouse model. Methods: Single-cell RNA sequencing (scRNA-seq) analyses were performed in ascending aortic tissues from individuals without aortic disease (n=8), patients with ATAA without dissection (n=9), and patients with acute ATAD (n=9). Further, scRNA-seq and scATAC-seq were performed in ascending aortic tissues from mice infused with angiotensin II (AngII) or control mice; scRNA-seq analyses were performed on SMC-specific Tgfbr2 -/- mice. Results: Six SMC subtypes were identified in human aortic tissues. Cluster proportion and trajectory analyses indicated a profound SMC transition from contractile in control to extracellular matrix (ECM)-contractile phenotype in ATAA, however, was reduced in ATAD tissues. Instead, ATAD showed significant SMC transition to inflammatory phenotype. Consistently, many genes in ECM organization and muscle contraction, collectively termed compensatory response, were upregulated in ATAA but downregulated in ATAD tissues compared with controls, whereas inflammatory response and cell death pathway genes were significantly upregulated in ATAD compared with control and ATAA tissues. Cell-cell interaction, regulatory network, and motif analysis suggested that TGF-beta-MEF2C signaling contributed to the induction of the ECM-contractile SMC phenotype. MEF2C + SMCs exhibited the capacity of transition towards ECM-producing SMCs in both mouse and human aorta. SMC-specific Tgfbr2 -/- mice showed compromised SMC transition to ECM-producing phenotype. Conclusions: Our study reveals SMC phenotypic transitions to ECM-producing SMCs in ATAA and to inflammatory and pro-death SMCs in ATAD, which represent a compensatory response in ATAA and a decompensatory status in ATAD. TGF-beta-MEF2C, a novel regulatory axis, promoted the compensatory response in SMCs of the ascending aorta.

Apelin is expressed in intimal smooth muscle cells and promotes their phenotypic transition

During atherosclerotic plaque formation, smooth muscle cells (SMCs) switch from a contractile/differentiated to a synthetic/dedifferentiated phenotype. We previously isolated differentiated spindle-shaped (S) and dedifferentiated rhomboid (R) SMCs from porcine coronary artery. R-SMCs express S100A4, a calcium-binding protein. We investigated the role of apelin in this phenotypic conversion, as well as its relationship with S100A4. We found that apelin was highly expressed in R-SMCs compared with S-SMCs. We observed a nuclear expression of apelin in SMCs within experimentally-induced intimal thickening of the porcine coronary artery and rat aorta. Plasmids targeting apelin to the nucleus (N. Ap) and to the secretory vesicles (S. Ap) were transfected into S-SMCs where apelin was barely detectable. Both plasmids induced the SMC transition towards a R-phenotype. Overexpression of N. Ap, and to a lesser degree S. Ap, led to a nuclear localization of S100A4. Stimulation of S-SMCs with platelet-derived growth factor-BB, known to induce the transition toward the R-phenotype, yielded the direct interaction and nuclear expression of both apelin and S100A4. In conclusion, apelin induces a SMC phenotypic transition towards the synthetic phenotype. These results suggest that apelin acts via nuclear re-localization of S100A4, raising the possibility of a new pro-atherogenic relationship between apelin and S100A4.

Modulation of Smooth Muscle Cell Phenotype for Translation of Tissue-Engineered Vascular Grafts.

Translation of small-diameter tissue-engineered vascular grafts (TEVGs) for the treatment of coronary artery disease (CAD) remains an unfulfilled promise. This is largely due to the limited integration of TEVGs into the native vascular wall-a process hampered by the insufficient smooth muscle cell (SMC) infiltration and extracellular matrix deposition, and low vasoactivity. These processes can be promoted through the judicious modulation of the SMC toward a synthetic phenotype to promote remodeling and vascular integration; however, the expression of synthetic markers is often accompanied by a decrease in the expression of contractile proteins. Therefore, techniques that can precisely modulate the SMC phenotypical behavior could have the potential to advance the translation of TEVGs. In this review, we describe the phenotypic diversity of SMCs and the different environmental cues that allow the modulation of SMC gene expression. Furthermore, we describe the emerging biomaterial approaches to modulate the SMC phenotype in TEVG design and discuss the limitations of current techniques. In addition, we found that current studies in tissue engineering limit the analysis of the SMC phenotype to a few markers, which are often the characteristic of early differentiation only. This limited scope has reduced the potential of tissue engineering to modulate the SMC toward specific behaviors and applications. Therefore, we recommend using the techniques presented in this review, in addition to modern single-cell proteomics analysis techniques to comprehensively characterize the phenotypic modulation of SMCs. Expanding the holistic potential of SMC modulation presents a great opportunity to advance the translation of living conduits for CAD therapeutics.

Single-cell transcriptomic data reveal the increase in extracellular matrix organization and antigen presentation abilities of fibroblasts and smooth muscle cells in patients with pelvic organ prolapse.

We aimed to explore the cellular properties of fibroblasts and smooth muscle cells (SMCs), the two major cell types of the vagina wall, in pelvic organ prolapse (POP) to improve the knowledge of the underlying molecular mechanisms of POP. The single-cell RNA sequencing (scRNA-seq) profile GSE151202 was downloaded from NCBI Gene Expression Omnibus, in which vaginal wall tissues were harvested from patients with anterior vaginal wall prolapse and control subjects respectively. The scRNA-seq data of samples (5 POP and 5 controls) were adopted for analysis. Cluster analysis was performed to identify the cell subclusters. Trajectory analysis was applied to construct the differentiation trajectories of fibroblasts and SMCs. Cellular communication analysis was carried out to explore the ligand-receptor interactions between fibroblasts/SMCs and immune cells. Ten subclusters were determined in both groups, among which fibroblasts and SMCs were the most abundant cell types. Compared with controls, fibroblasts increased whereas SMCs declined in POP. During the transition of fibroblasts and SMCs from a normal into a disease state, extracellular matrix organization and antigen presentation were heightened. The intercellular communications were altered in POP. Interactions between fibroblasts/SMCs and macrophages/natural killer/T cells were strengthened as more ligand-receptor pairs involved in antigen presentation pathways were gained in POP. Extracellular matrix organization and antigen presentation abilities of fibroblasts and SMCs were enhanced in POP.

Abstract 720: Integration Of Population Genetic And Human Knockout Studies With Smooth Muscle Cell Lineage Tracing In Mouse Models Identifies Novel Targets For Atherosclerosis And Cardiovascular Risk.

Despite lipid-lowering therapies, residual cardiovascular disease risk remains a major unmet clinical need. Large-scale genetic analyses have identified >250 loci for coronary artery disease (CAD) risk. Genes that function in vascular smooth muscle cells (SMC) are causal at several loci, yet the causal genes at most loci remain unknown. Using single cell profiling and SMC lineage tracing in mouse models, we and others have shown that SMC transition through an intermediate state to SMC-derived cells (SDC) with protective or harmful phenotypes for plaque stability. In this study, we integrated SMC lineage tracing in mouse models with ongoing analyses in > 1 million human participants with whole-genome (WGS) and whole-exome sequencing (WES) data, as well as rare variant analysis of human “knockouts” (homozygous for predicted loss-of-function variants) for a novel gene discovery pipeline (Figure 1). Through WES in 76,000 highly consanguineous participants in the Pakistan Genomic Resource (PGR), we assembled the largest global cohort of human KOs. At non-lipid CAD loci from a recent large multi-ethnic meta-analysis (~1.1 million participants), we prioritized genes as potentially causal in SMC/SDC as those that are expressed and regulated in human or mouse arteries and SMC in atherosclerosis progression. Through gene burden testing and analyses of rare predicted loss-of-function (pLoF) and damaging (pDMs) variants in the largest CAD WES/WGS meta-analyses we enriched for causal genes. We restricted to genes with at least 5 pLoF carriers in PGR to facilitate recall studies in Pakistan. Final prioritization incorporated state-of-the-art fine mapping and co-localization analyses as well as biological plausibility. From an initial pool of 1696 genes, our gene discovery pipeline prioritized 15 SMC/SDC genes, and initial recall in PGR identified multiple large pedigrees for mutations in candidate causal genes PDE3A, SERPINH1, HHIPL1, and ZEB2.

Lineage-Specific Induced Pluripotent Stem Cell-Derived Smooth Muscle Cell Modeling Predicts Integrin Alpha-V Antagonism Reduces Aortic Root Aneurysm Formation in Marfan Syndrome Mice.

The role of increased smooth muscle cell (SMC) integrin αv signaling in Marfan syndrome (MFS) aortic aneurysm remains unclear. Herein, we examine the mechanism and potential efficacy of integrin αv blockade as a therapeutic strategy to reduce aneurysm progression in MFS. Induced pluripotent stem cells (iPSCs) were differentiated into aortic SMCs of the second heart field (SHF) and neural crest (NC) lineages, enabling in vitro modeling of MFS thoracic aortic aneurysms. The pathological role of integrin αv during aneurysm formation was confirmed by blockade of integrin αv with GLPG0187 in Fbn1C1039G/+ MFS mice. iPSC-derived MFS SHF SMCs overexpress integrin αv relative to MFS NC and healthy control SHF cells. Furthermore, integrin αv downstream targets (FAK [focal adhesion kinase]/AktThr308/mTORC1 [mechanistic target of rapamycin complex 1]) were activated, especially in MFS SHF. Treatment of MFS SHF SMCs with GLPG0187 reduced p-FAK/p-AktThr308/mTORC1 activity back to control SHF levels. Functionally, MFS SHF SMCs had increased proliferation and migration compared to MFS NC SMCs and control SMCs, which normalized with GLPG0187 treatment. In the Fbn1C1039G/+ MFS mouse model, integrin αv, p-AktThr308, and downstream targets of mTORC1 proteins were elevated in the aortic root/ascending segment compared to littermate wild-type control. Mice treated with GLPG0187 (age 6-14 weeks) had reduced aneurysm growth, elastin fragmentation, and reduction of the FAK/AktThr308/mTORC1 pathway. GLPG0187 treatment reduced the amount and severity of SMC modulation assessed by single-cell RNA sequencing. The integrin αv-FAK-AktThr308 signaling pathway is activated in iPSC SMCs from MFS patients, specifically from the SHF lineage. Mechanistically, this signaling pathway promotes SMC proliferation and migration in vitro. As biological proof of concept, GLPG0187 treatment slowed aneurysm growth and p-AktThr308 signaling in Fbn1C1039G/+ mice. Integrin αv blockade via GLPG0187 may be a promising therapeutic approach to inhibit MFS aneurysmal growth.

Abstract 15254: Acta2 Missense Variant Increases Atherosclerotic Plaque Burdern by Heat Shock Factor 1-Driven Augmented Phenotypic Modulation of Smooth Muscle Cells

ACTA2 encodes the contractile protein smooth muscle α-actin expressed in smooth muscle cells (SMCs). A heterozygous pathogenic variant in ACTA2 , p.R149C, predisposes to premature coronary artery disease (CAD) in the absence of risk factors. Recent studies have determined that SMCs contribute to atherosclerotic plaque formation after undergoing phenotypic modulation, characterized by de-differentiation and increased expression of macrophage, fibroblast, stem, and osteogenic markers. This SMC modulation is driven in part by exogenous cholesterol entering the endoplasmic reticulum (ER) and activating ER stress, specifically PERK signaling. To determine how a mutation in SM α-actin predisposes to atherosclerosis, Acta2 R149C/+ mice were crossed into Apoe -/- mice and fed a high fat diet for 3 months. Despite no differences in serum lipid levels, the Acta2 R149C/+ Apoe -/- mice have a 3-fold increased plaque burden compared to Apoe -/- mice. SMCs explanted from Acta2 R149C/+ mice have phenotypic modulation at baseline while WT SMCs do not. We determined that misfolding of the R149C SMα-actin activates heat shock factor 1 (HSF1), which increases endogenous cholesterol biosynthesis through increasing HMG-CoA reductase expression. The increase in endogenous cholesterol levels induces ER stress, activates PERK, and augments both modulation of SMCs and atherosclerotic plaque formation. ScRNA sequencing confirms increased HSF1 and PERK signaling and modulation in SMCs in the mutant mice. Importantly, pravastatin treatment dramatically lowers plaque burden to similar low levels of plaque burden in both the Acta2 R149C/+ Apoe -/- and Apoe -/- mice (Fig. 1a) . These studies identify a novel mechanism by which mutant SM α-actin increases endogenous cholesterol biosynthesis and drives atherosclerotic plaque formation, a mechanism that also informs as to why statins are effective in individuals at risk for CAD who do not have elevated plasma cholesterol levels (Fig. 1b) .

Abstract 15469: Activation of Sting Pathway Promotes Epigenetic Induction of Smooth Muscle Cell Phenotypic Alterations in Aortic Wall

Introduction: Smooth muscle cell phenotypic alteration is a prominent feature of aortic aneurysms and dissections (AAD) that causes the loss of functional, contractile, smooth muscle cells (SMCs) and subsequent aortic dysfunction. However, SMC phenotypic alteration in AAD and the underlying mechanism is poorly understood. Hypothesis: Stress on the aorta induces a phenotypic switch in SMCs from a contractile role to a pro-inflammatory role that promotes cell dysfunction and death. Methods: We performed single-cell RNA and ATAC sequencing (scRNA-seq, scATAC-seq) of ascending aortic tissues from patients with sporadic ascending AAD and organ donor controls, alongside murine aortic tissues from a sporadic AAD model and wild-type mice. The role of the STING pathway in SMC phenotypic alterations was studied in human aortic SMCs. Chromatin immunoprecipitation assays were used to study epigenetic regulation Results: Significant SMC transformation from a contractile phenotype to a pro-inflammatory, pro-fibroblast, and pro-death phenotype was seen in scRNA-seq of aortic tissues from AAD patients and a sporadic AAD murine tissue. Our sc-ATACseq indicated that SMC transformation was partially controlled by chromatin remodeling of these genes, and IRF3 was identified as a key transcriptional factor for the reduction of chromatin accessibility of contractile genes, but induction of chromatin accessibility of inflammatory genes. In cultured SMCs, cytosolic DNA, through STING-TBK1 signaling, activated IRF3, which directly bound cis-elements of contractile genes, and recruited EZH2 to induce repressive H3K27me3 modification, leading to SMC gene suppression. Activated IRF3 also induced inflammatory gene expression. SMC-Sting deficiency prevented proinflammatory phenotypic switch in the sporadic AAD mouse model and restored contractile phenotype. Conclusion: We highlight an epigenetic pathway responsible for SMC dysfunction that induced SMC gene suppression and SMC phenotype changes and identified STING-TBK-IRF3-EZH2 as key signaling that suppresses SMC genes. To the best of our knowledge, this is the first study reporting that the STING pathway contributes to AAD development by SMC transition from a contractile to an inflammatory phenotype.

Abstract 11748: Prophylactic Aortic Arch Replacement in Patients With Loeys-Dietz Syndrome: Surgical Outcomes and Molecular Rationale

Introduction: Loeys-Dietz syndrome (LDS) patients typically develop focal aortic root aneurysm but have substantial risk of distal aortic events (arch aneurysm and type B dissection) after root replacement. We offer prophylactic arch replacement to LDS patients of all genotypes undergoing elective valve-sparing aortic root replacement (VSARR) to prevent late aortic events. We hypothesized that non-dilated LDS aortic arch tissue demonstrates ongoing deterioration at the time of VSARR. Methods: Aortic tissue was anatomically segmented (root, ascending, arch) then examined via histology and single-cell RNA sequencing (‘scRNAseq’, n=4 patients) to comprehensively profile cellular phenotypes. Results: LDS patients (n=8, mean age 37.9 years, 87.5% male) had root aneurysm (mean 45.1 ± 8.9mm) without ascending (35.1± 7.8mm) or arch dilation (28.7 ± 2.8mm) and underwent concurrent VSARR and branched graft total arch replacement. There were no perioperative deaths or strokes and no distal aortic events or reoperations after median 1.26 years follow-up. Histologically, elastin fragmentation and collagen deposition were detectable in each aortic segment (n=4). scRNAseq identified a continuum of smooth muscle cell (SMC) phenotype modulation associated with progressively reduced contractile gene expression. Pathway analysis of 260 genes enriched during SMC modulation identified heightened integrin signaling, transforming growth factor-beta-responsiveness, cell adhesion, and extracellular matrix remodeling. Histologic confirmation using RNA probes specific for modulated SMCs (TGF-beta responsive gene SERPINE1 and glycoprotein TNFRSF11B ) identified these cells in the tunica media layer of each aortic segment, with greatest density in the root aneurysm but extending to the arch. In age-matched donor aortas, TNFRSF11B + cells were restricted to the atherosclerotic tunica intima, confirming specificity of medial SMC modulation in aneurysm. Conclusions: Total arch replacement during elective VSARR is feasible without substantial morbidity. In LDS patients with root aneurysm, matrix deterioration and SMC phenotype modulation are present in the non-dilated aortic arch, providing molecular rationale for aggressive arch replacement.

Abstract 12293: Retinoic Acid Signaling Regulates Smooth Muscle Cell Phenotypic Switching in Atherosclerosis Through Epigenetic Modulation of Gene Expression

Background: Smooth muscle cells (SMCs) substantially contribute to atherosclerosis through “phenotypic switching.” Our previous work identified an intermediate SMC-derived cell type, termed “SEM” cells, which was multipotent and activated in inflammatory response. Activation of retinoic acid (RA) signaling by all-trans retinoic acid (ATRA) attenuated atherosclerosis in mice coincident with dramatic suppression of SEM cell formation from SMCs. However, the regulatory mechanisms by which RA signaling modulates SMC transition to SEM cells are largely unknown. Methods: We employed molecular and cell biology techniques, SMC-linage tracing and atheroprone mouse models, and next-generation sequencing (e.g., RNA-seq, ChIP-seq) to reveal how RA signaling modulates SMC transition to SEM cells. Results: Activation of RA signaling with ATRA significantly reduced SEM cells in established atherosclerosis, as well as downregulated the expression of SEM cell marker genes (e.g., Ly6a , Ly6c1 ) and repressed inflammatory response in ex vivo SEM cells, whereas inhibition of the signaling with antagonist, BMS49334, showed opposite results. RARα occupied the promoter regions of SEM cell marker genes, and ATRA treatment significantly increased the enrichment of NCOR1 at promoters of these genes. These findings suggest that RA signaling suppresses SMC transition to SEM cells via directly repressing the expression of SEM cell marker genes. Furthermore, we found EZH2, one of the subunits of PRC2, physically interacted with RARα in SMCs and occupied the promoters of SEM cell marker genes, and its methyltransferase activity at the promoter regions was responsible for the repression of SEM cell maker genes. Moreover, activation of RA signaling inhibited SEM cell inflammatory response through LXR-mediated suppression of a series of inflammatory genes. Finally, multiple epigenetic signatures (e.g., H3K27me3, H3K27ac, H3K4me3, etc.) at TSS of SEM cell marker genes and inflammatory genes were extensively altered in response to the activation of RA signaling. Conclusions: Our findings indicate that RA signaling modulates maintenance of SEM cell identity and inflammatory function in atherosclerosis by epigenetic regulation of gene expression.

Lack of PI 3-kinase isoform p110alpha impairs SMC differentiation and proliferation and promotes aortic aneurysm formation

Abstract Background Proliferation and phenotypic modulation of vascular smooth muscle cells (SMCs) significantly contribute to the functionality of the aortic wall. Dysregulation of underlying signal transduction pathways impairs the vessel wall structure and promote the development and progression of abdominal aortic aneurysms (AAA). The PI 3-kinase (PI3K) isoform p110α is activated downstream of receptor tyrosine kinases (RTKs) and represents the most relevant PI3K isoform in SMCs. Aim This project follows the hypothesis that p110α deficiency impairs proliferation and phenotypic modulation of SMCs as well as the structure of the extracellular matrix (ECM) and therefore promotes the development and progression of AAA. It was investigated how p110α deficiency affects the plasticity of SMCs, the production and structure of ECM components, and the formation of AAA. Methods and results Western blot analyses showed that SMCs isolated from smooth muscle specific p110α−/− (sm-p110α−/−) mice were characterized by decreased expression of the differentiation markers sm-α-actin, calponin and sm-MHC. Mechanistically, phosphorylation of key modulators of the SMC phenotype – AKT1, AKT2, FOXO1, -3 and -4 as well as GSK3β – was impaired in p110α−/− SMCs after RTK stimulation. These findings indicate that phenotypic modulation of p110α−/− SMCs is restricted. In addition, protein expression of elastin and fibrillin was reduced in p110α−/− SMCs. In silico analysis (MatLab macro CT-FIRE and Curvalign) of the ECM produced by SMCs in vitro revealed a significantly reduced elastin fiber length and width in p110α−/− SMCs compared to fibers produced by WT SMCs (p<0.05). Consistently, aortas from sm-p110α−/− mice showed a significantly higher number of elastic fiber breaks specifically in the thoracic section than WT controls (289±31 mmm–2 versus 190±9 mmm–2, n=5, p=0.015). Aortic aneurysms in sm-p110α−/− mice and wild-type littermates were analyzed using the established porcine pancreatic elastase (PPE) model. PPE was perfused into the infrarenal aorta to induce AAA formation. Ultrasound examination of the aorta revealed an enlarged aortic diameter in all PPE-treated mice. However, the increase in aortic diameter in sm-p110α−/− mice (70.16±10.82% mm, n=9) was significant more pronounced compared to wild-type animals (42.44±5.99%, n=10) (p<0.05). Three days after PPE perfusion, the number of elastic fiber breaks was significantly increased, and amount of proliferating SMCs were decreased in the infrarenal aorta of sm-p110α−/− mice compared to WT controls. Conclusion p110α deficiency in SMCs impairs aortic wall structure and promotes the development and progression of aortic aneurysms. Mechanistically, p110α activity maintains a differentiated SMC phenotype as well as the expression and assembly of ECM components. These data identify p110α signaling as a modifiable target for preventive and therapeutic strategies for aortic aneurysms. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

Embryologic Origin Influences Smooth Muscle Cell Phenotypic Modulation Signatures in Murine Marfan Syndrome Aortic Aneurysm.

Aortic root smooth muscle cells (SMC) develop from both the second heart field (SHF) and neural crest. Disparate responses to disease-causing Fbn1 variants by these lineages are proposed to promote focal aortic root aneurysm formation in Marfan syndrome (MFS), but lineage-stratified SMC analysis in vivo is lacking. We generated SHF lineage-traced MFS mice and performed integrated multiomic (single-cell RNA and assay for transposase-accessible chromatin sequencing) analysis stratified by embryological origin. SMC subtypes were spatially identified via RNA in situ hybridization. Response to TWIST1 overexpression was determined via lentiviral transduction in human aortic SMCs. Lineage stratification enabled nuanced characterization of aortic root cells. We identified heightened SHF-derived SMC heterogeneity including a subset of Tnnt2 (cardiac troponin T)-expressing cells distinguished by altered proteoglycan expression. MFS aneurysm-associated SMC phenotypic modulation was identified in both SHF-traced and nontraced (neural crest-derived) SMCs; however, transcriptomic responses were distinct between lineages. SHF-derived modulated SMCs overexpressed collagen synthetic genes and small leucine-rich proteoglycans while nontraced SMCs activated chondrogenic genes. These modulated SMCs clustered focally in the aneurysmal aortic root at the region of SHF/neural crest lineage overlap. Integrated RNA-assay for transposase-accessible chromatin analysis identified enriched Twist1 and Smad2/3/4 complex binding motifs in SHF-derived modulated SMCs. TWIST1 overexpression promoted collagen and SLRP gene expression in vitro, suggesting TWIST1 may drive SHF-enriched collagen synthesis in MFS aneurysm. SMCs derived from both SHF and neural crest lineages undergo phenotypic modulation in MFS aneurysm but are defined by subtly distinct transcriptional responses. Enhanced TWIST1 transcription factor activity may contribute to enriched collagen synthetic pathways SHF-derived SMCs in MFS.

Preventing Cholesterol-Induced Perk (Protein Kinase RNA-Like Endoplasmic Reticulum Kinase) Signaling in Smooth Muscle Cells Blocks Atherosclerotic Plaque Formation.

Background:Vascular smooth muscle cells (SMCs) undergo complex phenotypic modulation with atherosclerotic plaque formation in hyperlipidemic mice, which is characterized by de-differentiation and heterogeneous increases in the expression of macrophage, fibroblast, osteogenic, and stem cell markers. An increase of cellular cholesterol in SMCs triggers similar phenotypic changes in vitro with exposure to free cholesterol due to cholesterol entering the endoplasmic reticulum, triggering endoplasmic reticulum stress and activating Perk (protein kinase RNA-like endoplasmic reticulum kinase) signaling.Methods:We generated an SMC-specific Perk knockout mouse model, induced hyperlipidemia in the mice by AAV-PCSK9DY injection, and subjected them to a high-fat diet. We then assessed atherosclerotic plaque formation and performed single-cell transcriptomic studies using aortic tissue from these mice.Results:SMC-specific deletion of Perk reduces atherosclerotic plaque formation in male hyperlipidemic mice by 80%. Single-cell transcriptomic data identify 2 clusters of modulated SMCs in hyperlipidemic mice, one of which is absent when Perk is deleted in SMCs. The 2 modulated SMC clusters have significant overlap of transcriptional changes, but the Perk-dependent cluster uniquely shows a global decrease in the number of transcripts. SMC-specific Perk deletion also prevents migration of both contractile and modulated SMCs from the medial layer of the aorta.Conclusions:Our results indicate that hypercholesterolemia drives both Perk-dependent and Perk-independent SMC modulation and that deficiency of Perk significantly blocks atherosclerotic plaque formation.

Abstract 220: Smad3 Regulates Smooth Muscle Cell Fate And Governs Adverse Remodeling And Calcification Of Atherosclerotic Plaque

Atherosclerotic plaques consist mostly of smooth muscle cells (SMC), and genes that influence SMC phenotype can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD, and is associated with expression of SMAD3 in SMC , however the mechanism by which this gene modifies CAD risk remains poorly understood. SMC-specific deletion of Smad3 in a murine atherosclerosis model resulted in greater plaque burden, more positive remodeling, and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of Smad3 altered SMC transition cell state toward two fates: a novel SMC phenotype that governs both vascular remodeling and recruitment of inflammatory cells, as well as a chondromyocyte fate. The remodeling population was marked by uniquely high Mmp3 and Cxcl12 expression, and its appearance correlated with higher risk plaque features such as increased positive remodeling and macrophage content. Further, investigation of transcriptional mechanisms by which Smad3 alters SMC cell fate revealed novel roles for Hox and Sox transcription factors whose direct interaction with Smad3 regulate an extensive transcriptional program balancing remodeling and vascular extracellular matrix. These findings have significant implications for atherosclerotic and Mendelian aortic aneurysmal diseases. Together, these data suggest that Smad3 expression in SMC inhibits the emergence of specific SMC phenotypic transition cells that mediate adverse plaque features, including positive remodeling, monocyte recruitment, and vascular calcification.

Abstract 222: The Molecular Mechanisms Of Coronary Artery Disease Risk At The Pdgfd Locus

Background: Atherosclerotic cardiovascular diseases, including coronary artery disease (CAD), are the worldwide leading cause of death. Recent genome wide association studies (GWAS) have now identified over 250 genetic loci that are associated with CAD, and efforts in this lab and others are investigating the mechanisms of association at these loci. The CAD GWAS locus at 11q22.3 is of potential interest because it encodes the PDGFD gene, a member of the platelet-derived growth factor family whose expression is up-regulated in atherosclerosis. But whether disease allelic variation regulates PDGFD expression and how this growth factor might influence the risk of CAD has not been studied. Methods: The causal variant and gene were investigated with colocalization and in vitro transcription studies in cultured human coronary artery smooth muscle cells (SMC). Single-cell transcriptomic profiling (scRNAseq) and histological analyses were conducted in SMC-specific lineage tracing in an in vivo Pdgfd knockout mouse model of atherosclerosis. Results: The causal variant rs2019090 was linked to PDGFD expression in SMC. In silico analysis of transcription factor binding site prediction and in vitro haplotype-specific chromatin immunoprecipitation combined with qPCR analysis revealed another causal CAD factor forkhead box c1 (FOXC1) preferentially binds to the A-allele of variant rs2019090 and controls the expression level of PDGFD . scRNAseq data identified that loss of Pdgfd resulted in a marked increase in differentiated SMC but a decrease in fibromyocyte (FMC) and chondromyocyte (CMC) transition populations, as well as a significant decrease in plaque macrophages. Differentially expressed gene analysis in SMC lineage cells was highly enriched for CMC genes such as Col2a1 and Ibsp. Lesion histology analysis identified an increase in medial SMC, but a decrease in fibrous cap SMC, with decreased Cd68 and Col2a1 stained area. Conclusion: PDGFD is a causal CAD gene that increases disease risk by promoting SMC transition primarily to the CMC phenotype, and recruitment of monocyte-macrophage lineage cells to disease lesions. These findings support suppression of PDGFD as a possible therapy for human atherosclerotic disease.

Abstract 360: Epigenomic Changes Govern Smooth Muscle Cell Phenotype Shift In Marfan Syndrome Aortic Root Aneurysm

Background: Vascular smooth muscle cell (SMC) phenotype modulation produces a mixed proteolytic/collagen synthetic state in Marfan syndrome (MFS) aortic root aneurysm. While the transcriptomic changes associated with this process are established, upstream regulators governing this plasticity are poorly characterized. Methods/Results: We performed concurrent single-cell RNA sequencing (scRNAseq) and assay for transposase-accessible chromatin using sequencing (scATACseq) on aortic root aneurysm tissue from adult Fbn1 C1041G/+ (MFS) mice and littermate controls. MFS/control scRNAseq data analysis identified four SMC subtypes including MFS-specific ‘modulated’ cells (modSMCs) which enriched for extracellular matrix organization and collagen synthesis pathway activation. These SMC clusters were projected onto the MFS/control scATAC dataset via integrative label transfer to study dynamic chromatin accessibility during SMC phenotype modulation. We compared DNA accessibility in modSMCs versus mature SMCs, finding 336 enriched and 29 suppressed peaks, suggesting increased open chromatin during modulation. Using chromVAR, we identified 242 enriched transcription factor motifs overrepresented in modSMCs. Motifs representing central but nonspecific transcription factor families including numerous AP-1 (FOS/JUN/ATF) heterodimers and TEAD family members showed highest enrichment, while enriched TWIST1, HAND2, and SMAD2:SMAD3:SMAD4 complex motifs suggested more specific functions. To functionally validate these findings, we examined TWIST1 as a potential regulator of SMC modulation in vitro via lentiviral overexpression in MFS patient-derived aortic SMCs.In SMCs with forced TWIST1 overexpression, we found heightened promoter region DNA accessibility (via bulk ATACseq) and increased mRNA expression (via RT-PCR) for specific modSMC markers (e.g., COL1A1 and LUM confirming TWIST1 promotes collagen synthesis. Conclusions: Integrated single-cell transcriptomic/epigenomic analysis permits identification of critical upstream regulatory signals promoting disease-specific cell phenotype changes. TWIST1 is a potential driver of SMC modulation and a target for therapeutic agent design in MFS aortic aneurysm.

Abstract 532: Dioxin Modifies The Transcriptomic And Epigenetic Landscape Of Smooth Muscle Cells Leading To Adverse Remodeling Of Atherosclerosis

Background: Environmental exposure to dioxin, an active component of cigarette smoke and air pollution 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. We have previously found strong dioxin response element activity in modulated SMC of atherosclerotic lesion. In this study, we aimed to understand the effect of dioxin on vascular SMC and atherosclerosis phenotype. Methods: Primary human coronary artery SMC (HCASMC) treated in culture with 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) or vehicle control were used to perform RNA-Seq and ATAC-Seq. ChIP-Seq was performed with antibodies against Aryl-hydrocarbon receptor (AHR) and TCF21. Tamoxifen inducible SMC-lineage tracing reporter mice were used to assess the in-vivo effect of TCDD on aortic SMC. After 8 weeks of high fat diet (HFD), followed by 8 weeks of TCDD injection and HFD, the aortic sinus was collected for histology and single-cell sequencing (10X Genomics). Results: Analysis of the RNA-Seq from TCDD treated HCASMC showed differential enrichment of pathways related to cell migration and vascular development. Further, ATAC-Seq data showed a significant enrichment for pathways regulating vascular development, cell migration, and apoptosis. There was an overall increase in chromatin accessibility suggesting transcriptional activation with TCDD. We observed enrichment for AHR ChIP-Seq peaks in the open chromatin regions along with a significant reciprocal reduction in TCF21 occupancy. The scRNA-Seq of mice treated with TCDD exposure showed differential gene expression of SMC-lineage cells enriching for extracellular matrix (ECM) organization, inflammation, unfolded protein response, and apoptotic process, and histological analysis of the aortic sinus showed increased lesion area with increased presence of SMC-lineage cells and Cd68 + cells compared to control. Conclusion: Dioxin adversely remodels atherosclerotic plaque affecting pathways of ECM organization, inflammation and unfolded protein response in SMC, partly by modifying chromatin accessibility and occupancy of key transcription factors of phenotypic modulation.

Smad3 regulates smooth muscle cell fate and mediates adverse remodeling and calcification of the atherosclerotic plaque.

Atherosclerotic plaques consist mostly of smooth muscle cells (SMC), and genes that influence SMC phenotype can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD and is associated with the expression of SMAD3 in SMC. However, the mechanism by which this gene modifies CAD risk remains poorly understood. Here we show that SMC-specific deletion of Smad3 in a murine atherosclerosis model resulted in greater plaque burden, more outward remodelling and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of Smad3 altered SMC transition cell state toward two fates: a SMC phenotype that governs both vascular remodelling and recruitment of inflammatory cells, as well as a chondromyocyte fate. Together, the findings reveal that Smad3 expression in SMC inhibits the emergence of specific SMC phenotypic transition cells that mediate adverse plaque features, including outward remodelling, monocyte recruitment, and vascular calcification.

Abstract 11990: Insulin-Like Growth Factor Binding Protein 2 is a Novel Marker of Early Smooth Muscle Cell Phenotype Modulation in Thoracic Aortic Aneurysm

Introduction: Single cell RNA sequencing (scRNAseq) enables high resolution analysis of vascular smooth muscle cell (SMC) phenotype modulation in thoracic aortic aneurysm. Insulin-like growth factor binding protein-2 ( Igfbp2 ) expression by SMCs is activated early in a murine Marfan syndrome (MFS) aortic aneurysm model. IGFBP2 modulates IGF1 bioavailability and interacts directly with SMCs via integrins. We hypothesize that IGFBP2 is a marker of early SMC modulation in human aortopathy. Methods: Surgically resected aortic aneurysm tissue was collected from patients spanning the clinical spectrum of monogenic aortopathies ( ACTA2, FBN1, TGFBR1 (n=2), TGFB2, or SMAD3 variants). Tissues were digested and processed for scRNAseq. SMC clusters from all patients were jointly analyzed with pseudotemporal trajectory analysis to reconstruct the SMC phenotypic continuum. Primary MFS human (n=5) aortic root SMCs underwent IGFBP2 silencing via siRNA followed by bulk RNAseq. Results: scRNAseq identified two main SMC clusters, comprising 39% of all cells in the dataset, including a quiescent contractile SMCs and a modulated, synthetic subset. Pseudotemporal ordering generated a continuous ‘pseudotime’ scale inversely correlated with contractile gene (e.g., CNN1, MYH11) expression, modeling gradual de-differentiation in vivo . We identified similar markers of late SMC modulation (e.g., TNFRSF11B , MMP2, CYTL1, COL1A1 ) in patients of all genotypes. IGFBP2 expression showed comparatively earlier activation and peak with reduced expression late in pseudotime. Silencing IGFBP2 in human MFS SMCs in vitro significantly depressed contractile gene expression. Conclusions: SMC modulation occurs in consistent pattern across hereditary aortopathy subtypes. IGFBP2 activation occurs early in this trajectory, marking cells in phenotypic transition. IGFBP2 may promote contractile phenotype maintenance, presenting a novel pathway for potential therapies.