Human Gain-of-Function MC4R Variants Show Signaling Bias and Protect against Obesity.

The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor whose disruption causes obesity. We functionally characterized 61 MC4R variants identified in 0.5 million people from UK Biobank and examined their associations with body mass index (BMI) and obesity-related cardiometabolic diseases. We found that the maximal efficacy of ß-arrestin recruitment to MC4R, rather than canonical Gαs-mediated cyclic adenosine-monophosphate production, explained 88% of the variance in the association of MC4R variants with BMI. While most MC4R variants caused loss of function, a subset caused gain of function; these variants were associated with significantly lower BMI and lower odds of obesity, type 2 diabetes, and coronary artery disease. Protective associations were driven by MC4R variants exhibiting signaling bias toward ß-arrestin recruitment and increased mitogen-activated protein kinase pathway activation. Harnessing ß-arrestin-biased MC4R signaling may represent an effective strategy for weight loss and the treatment of obesity-related cardiometabolic diseases.

Unveiling the Role of the Most Impactful Cardiovascular Risk Locus through Haplotype Editing.

The 9p21.3 cardiovascular disease locus is the most influential common genetic risk factor for coronary artery disease (CAD), accounting for ∼10%-15% of disease in non-African populations. The ∼60 kb risk haplotype is human-specific and lacks coding genes, hindering efforts to decipher its function. Here, we produce induced pluripotent stem cells (iPSCs) from risk and non-risk individuals, delete each haplotype using genome editing, and generate vascular smooth muscle cells (VSMCs). Risk VSMCs exhibit globally altered transcriptional networks that intersect with previously identified CAD risk genes and pathways, concomitant with aberrant adhesion, contraction, and proliferation. Unexpectedly, deleting the risk haplotype rescues VSMC stability, while expressing the 9p21.3-associated long non-coding RNA ANRIL induces risk phenotypes in non-risk VSMCs. This study shows that the risk haplotype selectively predisposes VSMCs to adopt a cell state associated with CAD phenotypes, defines new VSMC-based networks of CAD risk genes, and establishes haplotype-edited iPSCs as powerful tools for functionally annotating the human genome.

Convergence of coronary artery disease genes onto endothelial cell programs.

Linking variants from genome-wide association studies (GWAS) to underlying mechanisms of disease remains a challenge1-3. For some diseases, a successful strategy has been to look for cases in which multiple GWAS loci contain genes that act in the same biological pathway1-6. However, our knowledge of which genes act in which pathways is incomplete, particularly for cell-type-specific pathways or understudied genes. Here we introduce a method to connect GWAS variants to functions. This method links variants to genes using epigenomics data, links genes to pathways de novo using Perturb-seq and integrates these data to identify convergence of GWAS loci onto pathways. We apply this approach to study the role of endothelial cells in genetic risk for coronary artery disease (CAD), and discover 43 CAD GWAS signals that converge on the cerebral cavernous malformation (CCM) signalling pathway. Two regulators of this pathway, CCM2 and TLNRD1, are each linked to a CAD risk variant, regulate other CAD risk genes and affect atheroprotective processes in endothelial cells. These results suggest a model whereby CAD risk is driven in part by the convergence of causal genes onto a particular transcriptional pathway in endothelial cells. They highlight shared genes between common and rare vascular diseases (CAD and CCM), and identify TLNRD1 as a new, previously uncharacterized member of the CCM signalling pathway. This approach will be widely useful for linking variants to functions for other common polygenic diseases.

LRG1 promotes atherosclerosis by inducing macrophage M1-like polarization.

Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by the accumulation of cholesterol-rich lipoproteins in macrophages. How macrophages commit to proinflammatory polarization under atherosclerosis conditions is not clear. Report here that the level of a circulating protein, leucine-rich alpha-2 glycoprotein 1 (LRG1), is elevated in the atherosclerotic tissue and serum samples from patients with coronary artery disease (CAD). LRG1 stimulated macrophages to proinflammatory M1-like polarization through the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways. The LRG1 knockout mice showed significantly delayed atherogenesis progression and reduced levels of macrophage-related proinflammatory cytokines in a high-fat diet-induced Apoe-/- mouse atherosclerosis model. An anti-LRG1 neutralizing antibody also effectively blocked LRG1-induced macrophage M1-like polarization in vitro and conferred therapeutic benefits to animals with ApoE deficiency-induced atherosclerosis. LRG1 may therefore serve as an additional biomarker for CAD and targeting LRG1 could offer a potential therapeutic strategy for CAD patients by mitigating the proinflammatory response of macrophages.

Dissecting the polygenic basis of atherosclerosis via disease-associated cell state signatures.

Coronary artery disease (CAD) is a pandemic disease where up to half of the risk is explained by genetic factors. Advanced insights into the genetic basis of CAD require deeper understanding of the contributions of different cell types, molecular pathways, and genes to disease heritability. Here, we investigate the biological diversity of atherosclerosis-associated cell states and interrogate their contribution to the genetic risk of CAD by using single-cell and bulk RNA sequencing (RNA-seq) of mouse and human lesions. We identified 12 disease-associated cell states that we characterized further by gene set functional profiling, ligand-receptor prediction, and transcription factor inference. Importantly, Vcam1+ smooth muscle cell state genes contributed most to SNP-based heritability of CAD. In line with this, genetic variants near smooth muscle cell state genes and regulatory elements explained the largest fraction of CAD-risk variance between individuals. Using this information for variant prioritization, we derived a hybrid polygenic risk score (PRS) that demonstrated improved performance over a classical PRS. Our results provide insights into the biological mechanisms associated with CAD risk, which could make a promising contribution to precision medicine and tailored therapeutic interventions in the future.

A Novel Macrophage Subpopulation Conveys Increased Genetic Risk of Coronary Artery Disease.

BACKGROUND: Coronary artery disease (CAD), the leading cause of death worldwide, is influenced by both environmental and genetic factors. Although over 250 genetic risk loci have been identified through genome-wide association studies, the specific causal variants and their regulatory mechanisms are still largely unknown, particularly in disease-relevant cell types such as macrophages. METHODS: We utilized single-cell RNA-seq and single-cell multiomics approaches in primary human monocyte-derived macrophages to explore the transcriptional regulatory network involved in a critical pathogenic event of coronary atherosclerosis-the formation of lipid-laden foam cells. The relative genetic contribution to CAD was assessed by partitioning disease heritability across different macrophage subpopulations. Meta-analysis of single-cell RNA-seq data sets from 38 human atherosclerotic samples was conducted to provide high-resolution cross-referencing to macrophage subpopulations in vivo. RESULTS: We identified 18 782 cis-regulatory elements by jointly profiling the gene expression and chromatin accessibility of >5000 macrophages. Integration with CAD genome-wide association study data prioritized 121 CAD-related genetic variants and 56 candidate causal genes. We showed that CAD heritability was not uniformly distributed and was particularly enriched in the gene programs of a novel CD52-hi lipid-handling macrophage subpopulation. These CD52-hi macrophages displayed significantly less lipoprotein accumulation and were also found in human atherosclerotic plaques. We investigated the cis-regulatory effect of a risk variant rs10488763 on FDX1, implicating the recruitment of AP-1 and C/EBP-ß in the causal mechanisms at this locus. CONCLUSIONS: Our results provide genetic evidence of the divergent roles of macrophage subsets in atherogenesis and highlight lipid-handling macrophages as a key subpopulation through which genetic variants operate to influence disease. These findings provide an unbiased framework for functional fine-mapping of genome-wide association study results using single-cell multiomics and offer new insights into the genotype-environment interactions underlying atherosclerotic disease.

Carbonylation of Runx2 at K176 by 4-Hydroxynonenal Accelerates Vascular Calcification.

BACKGROUND: Vascular calcification, which is characterized by calcium deposition in arterial walls and the osteochondrogenic differentiation of vascular smooth muscle cells, is an actively regulated process that involves complex mechanisms. Vascular calcification is associated with increased cardiovascular adverse events. The role of 4-hydroxynonenal (4-HNE), which is the most abundant stable product of lipid peroxidation, in vascular calcification has been poorly investigated. METHODS: Serum was collected from patients with chronic kidney disease and controls, and the levels of 4-HNE and 8-iso-prostaglandin F2α were measured. Sections of coronary atherosclerotic plaques from donors were immunostained to analyze calcium deposition and 4-HNE. A total of 658 patients with coronary artery disease who received coronary computed tomography angiography were recruited to analyze the relationship between coronary calcification and the rs671 mutation in aldehyde dehydrogenase 2 (ALDH2). ALDH2 knockout (ALDH2-/-) mice, smooth muscle cell-specific ALDH2 knockout mice, ALDH2 transgenic mice, and their controls were used to establish vascular calcification models. Primary mouse aortic smooth muscle cells and human aortic smooth muscle cells were exposed to medium containing ß-glycerophosphate and CaCl2 to investigate cell calcification and the underlying molecular mechanisms. RESULTS: Elevated 4-HNE levels were observed in the serum of patients with chronic kidney disease and model mice and were detected in calcified artery sections by immunostaining. ALDH2 knockout or smooth muscle cell-specific ALDH2 knockout accelerated the development of vascular calcification in model mice, whereas overexpression or activation prevented mouse vascular calcification and the osteochondrogenic differentiation of vascular smooth muscle cells. In patients with coronary artery disease, patients with ALDH2 rs671 gene mutation developed more severe coronary calcification. 4-HNE promoted calcification of both mouse aortic smooth muscle cells and human aortic smooth muscle cells and their osteochondrogenic differentiation in vitro. 4-HNE increased the level of Runx2 (runt-related transcription factor-2), and the effect of 4-HNE on promoting vascular smooth muscle cell calcification was ablated when Runx2 was knocked down. Mutation of Runx2 at lysine 176 reduced its carbonylation and eliminated the 4-HNE-induced upregulation of Runx2. CONCLUSIONS: Our results suggest that 4-HNE increases Runx2 stabilization by directly carbonylating its K176 site and promotes vascular calcification. ALDH2 might be a potential target for the treatment of vascular calcification.

Harnessing tissue-specific genetic variation to dissect putative causal pathways between body mass index and cardiometabolic phenotypes.

Body mass index (BMI) is a complex disease risk factor known to be influenced by genes acting via both metabolic pathways and appetite regulation. In this study, we aimed to gain insight into the phenotypic consequences of BMI-associated genetic variants, which may be mediated by their expression in different tissues. First, we harnessed meta-analyzed gene expression datasets derived from subcutaneous adipose (n = 1257) and brain (n = 1194) tissue to identify 86 and 140 loci, respectively, which provided evidence of genetic colocalization with BMI. These two sets of tissue-partitioned loci had differential effects with respect to waist-to-hip ratio, suggesting that the way they influence fat distribution might vary despite their having very similar average magnitudes of effect on BMI itself (adipose = 0.0148 and brain = 0.0149 standard deviation change in BMI per effect allele). For instance, BMI-associated variants colocalized with TBX15 expression in adipose tissue (posterior probability [PPA] = 0.97), but not when we used TBX15 expression data derived from brain tissue (PPA = 0.04) This gene putatively influences BMI via its role in skeletal development. Conversely, there were loci where BMI-associated variants provided evidence of colocalization with gene expression in brain tissue (e.g., NEGR1, PPA = 0.93), but not when we used data derived from adipose tissue, suggesting that these genes might be more likely to influence BMI via energy balance. Leveraging these tissue-partitioned variant sets through a multivariable Mendelian randomization framework provided strong evidence that the brain-tissue-derived variants are predominantly responsible for driving the genetically predicted effects of BMI on cardiovascular-disease endpoints (e.g., coronary artery disease: odds ratio = 1.05, 95% confidence interval = 1.04-1.07, p = 4.67 × 10-14). In contrast, our analyses suggested that the adipose tissue variants might predominantly be responsible for the underlying relationship between BMI and measures of cardiac function, such as left ventricular stroke volume (beta = 0.21, 95% confidence interval = 0.09-0.32, p = 6.43 × 10-4).

Electrochemical impedance spectroscopy unmasks high-risk atherosclerotic features in human coronary artery disease.

Coronary plaque rupture remains the prominent mechanism of myocardial infarction. Accurate identification of rupture-prone plaque may improve clinical management. This study assessed the discriminatory performance of electrochemical impedance spectroscopy (EIS) in human cardiac explants to detect high-risk atherosclerotic features that portend rupture risk. In this single-center, prospective study, n = 26 cardiac explants were collected for EIS interrogation of the three major coronary arteries. Vessels in which advancement of the EIS catheter without iatrogenic plaque disruption was rendered impossible were not assessed. N = 61 vessels underwent EIS measurement and histological analyses. Plaques were dichotomized according to previously established high rupture-risk parameter thresholds. Diagnostic performance was determined via receiver operating characteristic areas-under-the-curve (AUC). Necrotic cores were identified in n = 19 vessels (median area 1.53 mm2) with a median fibrous cap thickness of 62 µm. Impedance was significantly greater in plaques with necrotic core area ≥1.75 mm2 versus <1.75 mm2 (19.8 ± 4.4 kΩ vs. 7.2 ± 1.0 kΩ, p = .019), fibrous cap thickness ≤65 µm versus >65 µm (19.1 ± 3.5 kΩ vs. 6.5 ± 0.9 kΩ, p = .004), and ≥20 macrophages per 0.3 mm-diameter high-power field (HPF) versus <20 macrophages per HPF (19.8 ± 4.1 kΩ vs. 10.2 ± 0.9 kΩ, p = .002). Impedance identified necrotic core area ≥1.75 mm2, fibrous cap thickness ≤65 µm, and ≥20 macrophages per HPF with AUCs of 0.889 (95% CI: 0.716-1.000) (p = .013), 0.852 (0.646-1.000) (p = .025), and 0.835 (0.577-1.000) (p = .028), respectively. Further, phase delay discriminated severe stenosis (≥70%) with an AUC of 0.767 (0.573-0.962) (p = .035). EIS discriminates high-risk atherosclerotic features that portend plaque rupture in human coronary artery disease and may serve as a complementary modality for angiography-guided atherosclerosis evaluation.

SREBP-1-mediated lipogenesis confers resistance to ferroptosis and improves endothelial injury.

Atherosclerosis refers to a disease characterized by the formation of lipid plaque deposits within arterial walls, leading to reduced blood flow or blockage of blood outflow. The process of endothelial injury induced by oxidized low-density lipoprotein (ox-LDL) is considered the initial stage of atherosclerosis. Ferroptosis is a form of iron-dependent, non-apoptotic cell death, and current research suggests its association with coronary artery disease (CAD). In this study, we observed a correlation between reduced expression of SREBP-1 and the occurrence of stable CAD. Additionally, during the process of endothelial injury induced by ox-LDL, we also noted decreased expression of the SREBP-1/SCD1/FADS2 and involvement in the ferroptosis process. Mechanistically, ox-LDL induced endothelial injury by inhibiting the lipid biosynthesis process mediated by the SREBP-1/SCD1/FADS2, thereby inducing lipid peroxidation and ferroptosis. On the contrary, overexpression of SREBP-1 or supplementation with monounsaturated fatty acids counteracted iron accumulation, mitochondrial damage, and lipid peroxidation-induced ferroptosis, thereby improving endothelial injury. Our study indicated that the decreased expression of peripheral blood SREBP-1 mRNA is an independent risk factor for stable CAD. Furthermore, in endothelial cells, the lipid biosynthesis process mediated by SREBP-1 could ameliorate endothelial injury by resisting ferroptosis. The study has been registered with the Chinese Clinical Trial Registry, which serves as a primary registry in the World Health Organization International Clinical Trials Registry Platform (ChiCTR2300074315, August 3rd, 2023).

Polygenic Risk Score Associates With Atherosclerotic Plaque Characteristics at Autopsy.

BACKGROUND: Polygenic risk scores (PRSs) for coronary artery disease (CAD) potentially improve cardiovascular risk prediction. However, their relationship with histopathologic features of CAD has never been examined systematically. METHODS: From 4327 subjects referred to CVPath by the State of Maryland Office Chief Medical Examiner for sudden death between 1994 and 2015, 2455 cases were randomly selected for genotyping. We generated PRS from 291 known CAD risk loci. Detailed histopathologic examination of the coronary arteries was performed in all subjects. The primary study outcome measurements were histopathologic plaque features determining severity of atherosclerosis, including %stenosis, calcification, thin-cap fibroatheromas, and thrombotic CAD. RESULTS: After exclusion of cases with insufficient DNA sample quality or with missing data, 954 cases (mean age, 48.8±14.7 years; 75.7% men) remained in the final study cohort. Subjects in the highest PRS quintile exhibited more severe atherosclerosis compared with subjects in the lowest quintile, with greater %stenosis (80.3%±27.0% versus 50.4%±38.7%; adjusted P<0.001) and a higher frequency of calcification (69.6% versus 35.8%; adjusted P=0.004) and thin-cap fibroatheroma (26.7% versus 9.5%; adjusted P=0.007). Even after adjustment for traditional CAD risk factors, subjects within the highest PRS quintile had higher odds of severe atherosclerosis (ie, ≥75% stenosis; adjusted odds ratio, 3.77 [95% CI, 2.10-6.78]; P<0.001) and plaque rupture (adjusted odds ratio, 4.05 [95% CI, 2.26-7.24]; P<0.001). Moreover, subjects within the highest quintile had higher odds of CAD-associated cause of death, especially among those aged ≤50 years (adjusted odds ratio, 4.08 [95% CI, 2.01-8.30]; P<0.001). No statistically significant associations were observed with plaque erosion after adjusting for covariates. CONCLUSIONS: This is the first autopsy study investigating associations between PRS and atherosclerosis severity at the histopathologic level in subjects with sudden death. Our pathological analysis suggests PRS correlates with plaque burden and features of advanced atherosclerosis and may be useful as a method for CAD risk stratification, especially in younger subjects.

Subclinical Atherosclerosis: Part 1: What Is it? Can it Be Defined at the Histological Level?

While coronary artery disease remains a major cause of death, it is preventable. Therefore, the focus needs to shift to the early detection and prevention of atherosclerosis. Asymptomatic atherosclerosis is widely termed subclinical atherosclerosis, which is an early indicator of atherosclerotic burden, and understanding this disease is important because timely intervention could prevent future cardiovascular morbidity and mortality. We histologically recognize the earliest lesion of atherosclerosis as pathological intimal thickening, which is characterized by the presence of lipid pools. The difference between clinical atherosclerosis and subclinical atherosclerosis is whether the presence of atherosclerosis results in the clinical symptoms of ischemia, such as stroke, myocardial infarction, or chronic limb-threatening ischemia. In the absence of thrombosis, there are various types of histological plaque that encompass subclinical atherosclerosis: pathological intimal thickening, fibroatheroma, thin-cap fibroatheroma, plaque rupture, healed plaque ruptures, and fibrocalcific plaque. Plaque morphology that is most frequently responsible for acute coronary thrombosis is plaque rupture. Calcification of coronary arteries is the hallmark of atherosclerosis and is a predictor of future coronary events. Atherosclerosis occurs in other vascular beds and is most frequent in arteries of the lower extremity, followed by carotid, aorta, and coronary arteries, and the mechanisms leading to clinical symptoms are unique for each location.

Whole-Blood Transcriptome Unveils Altered Immune Response in Acute Myocardial Infarction Patients With Aortic Valve Sclerosis.

BACKGROUND: Aortic valve sclerosis (AVSc) presents similar pathogenetic mechanisms to coronary artery disease and is associated with short- and long-term mortality in patients with coronary artery disease. Evidence of AVSc-specific pathophysiological traits in acute myocardial infarction (AMI) is currently lacking. Thus, we aimed to identify a blood-based transcriptional signature that could differentiate AVSc from no-AVSc patients during AMI. METHODS: Whole-blood transcriptome of AVSc (n=44) and no-AVSc (n=66) patients with AMI was assessed by RNA sequencing on hospital admission. Feature selection, differential expression, and enrichment analyses were performed to identify gene expression patterns discriminating AVSc from no-AVSc and infer functional associations. Multivariable Cox regression analysis was used to estimate the hazard ratios of cardiovascular events in AVSc versus no-AVSc patients. RESULTS: This cross-sectional study identified a panel of 100 informative genes capable of distinguishing AVSc from no-AVSc patients with 94% accuracy. Further analysis revealed significant mean differences in 143 genes, of which 30 genes withstood correction for age and previous AMI or coronary interventions. Functional inference unveiled a significant association between AVSc and key biological processes, including acute inflammatory responses, type I IFN (interferon) response, platelet activation, and hemostasis. Notably, patients with AMI with AVSc exhibited a significantly higher incidence of adverse cardiovascular events during a 10-year follow-up period, with a full adjusted hazard ratio of 2.4 (95% CI, 1.3-4.5). CONCLUSIONS: Our findings shed light on the molecular mechanisms underlying AVSc and provide potential prognostic insights for patients with AMI with AVSc. During AMI, patients with AVSc showed increased type I IFN (interferon) response and earlier adverse cardiovascular outcomes. Novel pharmacological therapies aiming at limiting type I IFN response during or immediately after AMI might improve poor cardiovascular outcomes of patients with AMI with AVSc.

Single-Cell RNA Sequencing Reveals an Immune Landscape of CD4+ T Cells in Coronary Culprit Plaques With Acute Coronary Syndrome in Humans-Brief Report.

BACKGROUND: Acute coronary syndrome (ACS) involves plaque-related thrombosis, causing primary ischemic cardiomyopathy or lethal arrhythmia. We previously demonstrated a unique immune landscape of myeloid cells in the culprit plaques causing ACS by using single-cell RNA sequencing. Here, we aimed to characterize T cells in a single-cell level, assess clonal expansion of T cells, and find a therapeutic target to prevent ACS. METHODS: We obtained the culprit lesion plaques from 4 patients with chronic coronary syndrome (chronic coronary syndrome plaques) and the culprit lesion plaques from 3 patients with ACS (ACS plaques) who were candidates for percutaneous coronary intervention with directional coronary atherectomy. Live CD45+ immune cells were sorted from each pooled plaque samples and applied to the 10× platform for single-cell RNA sequencing analysis. We also extracted RNA from other 3 ACS plaque samples and conducted unbiased TCR (T-cell receptor) repertoire analysis. RESULTS: CD4+ T cells were divided into 5 distinct clusters: effector, naive, cytotoxic, CCR7+ (C-C chemokine receptor type 7) central memory, and FOXP3 (forkhead box P3)+ regulatory CD4+ T cells. The proportion of central memory CD4+ T cells was higher in the ACS plaques. Correspondingly, dendritic cells also tended to express more HLAs (human leukocyte antigens) and costimulatory molecules in the ACS plaques. The velocity analysis suggested the differentiation flow from central memory CD4+ T cells into effector CD4+ T cells and that from naive CD4+ T cells into central memory CD4+ T cells in the ACS plaques, which were not observed in the chronic coronary syndrome plaques. The bulk repertoire analysis revealed clonal expansion of TCRs in each patient with ACS and suggested that several peptides in the ACS plaques work as antigens and induced clonal expansion of CD4+ T cells. CONCLUSIONS: For the first time, we revealed single cell-level characteristics of CD4+ T cells in patients with ACS. CD4+ T cells could be therapeutic targets of ACS. REGISTRATION: URL: https://upload.umin.ac.jp/cgi-open-bin/icdr_e/ctr_view.cgi?recptno=R000046521; Unique identifier: UMIN000040747.

Coronary Artery Disease Risk Variant Dampens the Expression of CALCRL by Reducing HSF Binding to Shear Stress Responsive Enhancer in Endothelial Cells In Vitro.

BACKGROUND: CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of CALCRL expression in endothelial cells. METHODS: To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene CALCRL, we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq [transposase-accessible chromatin with sequencing], chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells. RESULTS: We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of CALCRL expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and CALCRL expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. CALCRL knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial nitric oxide synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production. CONCLUSIONS: Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates CALCRL expression. A better understanding of CALCRL gene regulation and the role of single-nucleotide polymorphisms in the modulation of CALCRL expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.

Spatial Transcriptomic Approach to Understanding Coronary Atherosclerotic Plaque Stability.

BACKGROUND: Coronary atherosclerotic plaques susceptible to acute coronary syndrome have traditionally been characterized by their surrounding cellular architecture. However, with the advent of intravascular imaging, novel mechanisms of coronary thrombosis have emerged, challenging our contemporary understanding of acute coronary syndrome. These intriguing findings underscore the necessity for a precise molecular definition of plaque stability. Considering this, our study aimed to investigate the vascular microenvironment in patients with stable and unstable plaques using spatial transcriptomics. METHODS: Autopsy-derived coronary arteries were preserved and categorized by plaque stability (n=5 patients per group). We utilized the GeoMx spatial profiling platform and Whole Transcriptome Atlas to link crucial histological morphology markers in coronary lesions with differential gene expression in specific regions of interest, thereby mapping the vascular transcriptome. This innovative approach allowed us to conduct cell morphological and spatially resolved transcriptional profiling of atherosclerotic plaques while preserving crucial intercellular signaling. RESULTS: We observed intriguing spatial and cell-specific transcriptional patterns in stable and unstable atherosclerotic plaques, showcasing regional variations within the intima and media. These regions exhibited differential expression of proinflammatory molecules (eg, IFN-γ [interferon-γ], MHC [major histocompatibility complex] class II, proinflammatory cytokines) and prothrombotic signaling pathways. By using lineage tracing through spatial deconvolution of intimal CD68+ (cluster of differentiation 68) cells, we characterized unique, intraplaque subpopulations originating from endothelial, smooth muscle, and myeloid lineages with distinct regional locations associated with plaque instability. In addition, unique transcriptional signatures were observed in vascular smooth muscle and CD68+ cells among plaques exhibiting coronary calcification. CONCLUSIONS: Our study illuminates distinct cell-specific and regional transcriptional alterations present in unstable plaques. Furthermore, we characterize spatially resolved, in situ evidence supporting cellular transdifferentiation and intraplaque plasticity as significant contributors to plaque instability in human coronary atherosclerosis. Our results provide a powerful resource for the identification of novel mediators of acute coronary syndrome, opening new avenues for preventative and therapeutic treatments.

Single-Nucleus Transcriptomic Atlas of Human Pericoronary Epicardial Adipose Tissue in Normal and Pathological Conditions.

BACKGROUND: Pericoronary epicardial adipose tissue (EAT) is a unique visceral fat depot that surrounds the adventitia of the coronary arteries without any anatomic barrier. Clinical studies have demonstrated the association between EAT volume and increased risks for coronary artery disease (CAD). However, the cellular and molecular mechanisms underlying the association remain elusive. METHODS: We performed single-nucleus RNA sequencing on pericoronary EAT samples collected from 3 groups of subjects: patients undergoing coronary bypass surgery for severe CAD (n=8), patients with CAD with concomitant type 2 diabetes (n=8), and patients with valvular diseases but without concomitant CAD and type 2 diabetes as the control group (n=8). Comparative analyses were performed among groups, including cellular compositional analysis, cell type-resolved transcriptomic changes, gene coexpression network analysis, and intercellular communication analysis. Immunofluorescence staining was performed to confirm the presence of CAD-associated subclusters. RESULTS: Unsupervised clustering of 73 386 nuclei identified 15 clusters, encompassing all known cell types in the adipose tissue. Distinct subpopulations were identified within primary cell types, including adipocytes, adipose stem and progenitor cells, and macrophages. CD83high macrophages and FOSBhigh adipocytes were significantly expanded in CAD. In comparison to normal controls, both disease groups exhibited dysregulated pathways and altered secretome in the primary cell types. Nevertheless, minimal differences were noted between the disease groups in terms of cellular composition and transcriptome. In addition, our data highlight a potential interplay between dysregulated circadian clock and altered physiological functions in adipocytes of pericoronary EAT. ANXA1 (annexin A1) and SEMA3B (semaphorin 3B) were identified as important adipokines potentially involved in functional changes of pericoronary EAT and CAD pathogenesis. CONCLUSIONS: We built a complete single-nucleus transcriptomic atlas of human pericoronary EAT in normal and diseased conditions of CAD. Our study lays the foundation for developing novel therapeutic strategies for treating CAD by targeting and modifying pericoronary EAT functions.

CircTOP1 targeted regulation of PTBP1 expression promotes the progression of coronary artery calcification.

Coronary artery calcification (CAC) is a hallmark event in the pathogenesis of cardiovascular disease, involving the phenotypic transformation of vascular smooth muscle cells (VSMC) towards an osteogenic state. Despite this understanding, the molecular mechanisms governing the VSMC osteogenic switch remain incompletely elucidated. Here, we sought to examine the potential role of circular RNA (circRNA) in the context of CAC. Through transcriptome analysis of circRNA-seq, we identified circTOP1 as a potential candidate circRNA in individuals with CAC. Furthermore, we observed that overexpression of circTOP1 exacerbated vascular calcification in a CAC model. Subsequent pull-down assays revealed an interaction between circTOP1 and PTBP1, a putative target gene of circTOP1 in the context of CAC. In both in vivo and in vitro experiments, we observed heightened expression of circTOP1 and PTBP1 in the CAC model, and noted that reducing circTOP1 expression effectively reduced calcium salt deposits and mineralized nodules in model mice. Additionally, in vitro experiments demonstrated that overexpression of PTBP1 reversed the weakening of signaling caused by silencing circTOP1, thereby exacerbating the osteogenic transition and calcification of VSMC. Collectively, our findings suggested that circTOP1 promotes CAC by modulating PTBP1 expression to mediate VSMC transdifferentiation.

Homocysteine concentration in coronary artery disease and severity of coronary lesions.

Our previous study reckons that the impact of the rs1801133 variant of 5,10-methylenetetrahydrofolate reductase (MTHFR) on coronary artery disease (CAD) is possibly mediated by cardiometabolic disorder. This study is performed to verify this hypothesis. Four hundred and thirty CAD patients and 216 CAD-free individuals were enrolled in this case-control study. The rs1801133 variant was genotyped by PCR-RFLP. Severity of coronary lesions was evaluated by number of stenotic coronary vessels and extent of coronary stenosis. The rs1801133 T allele significantly increased homocysteine levels in patients with CAD and CAD-free individuals. Individuals with the T allele of rs1801133 had an increased risk of developing CAD. In contrast, individuals with the TT genotype of rs1801133 were at high risk of multiple vessel lesions. The carriers of CT genotype had higher levels of systolic blood pressure (SBP), low-density lipoprotein cholesterol (LDL-C), and high-sensitivity C-reactive protein (hs-CRP), and lower levels of apolipoprotein A1 (APOA1) than those with CC genotype in male patients with CAD. The receiver operating characteristic (ROC) curve and precision-recall (PR) curve indicated that hyperhomocysteinemia was sensitive to predict the severity of CAD. Multivariate logistic regression revealed that homocysteine, rs1801133, age, smoking, weight, body mass index (BMI), lipoprotein(a) [Lp(a)], and hs-CRP were independent risk factors for CAD. The increased risk of CAD and severity of coronary lesions associated with rs1801133 in the Chinese Han population were attributed, at least partly, to high homocysteine levels. Hyperhomocysteinemia had a high predictive value for severe CAD or multiple vessel lesions.

lncRNA CDKN2B-AS1 regulates collagen expression.

The long noncoding RNA CDKN2B-AS1 harbors a major coronary artery disease risk haplotype, which is also associated with progressive forms of the oral inflammatory disease periodontitis as well as myocardial infarction (MI). Despite extensive research, there is currently no broad consensus on the function of CDKN2B-AS1 that would explain a common molecular role of this lncRNA in these diseases. Our aim was to investigate the role of CDKN2B-AS1 in gingival cells to better understand the molecular mechanisms underlying the increased risk of progressive periodontitis. We downregulated CDKN2B-AS1 transcript levels in primary gingival fibroblasts with LNA GapmeRs. Following RNA-sequencing, we performed differential expression, gene set enrichment analyses and Western Blotting. Putative causal alleles were searched by analyzing associated DNA sequence variants for changes of predicted transcription factor binding sites. We functionally characterized putative functional alleles using luciferase-reporter and antibody electrophoretic mobility shift assays in gingival fibroblasts and HeLa cells. Of all gene sets analysed, collagen biosynthesis was most significantly upregulated (Padj=9.7 × 10- 5 (AUC > 0.65) with the CAD and MI risk gene COL4A1 showing strongest upregulation of the enriched gene sets (Fold change = 12.13, Padj = 4.9 × 10- 25). The inflammatory "TNFA signaling via NFKB" gene set was downregulated the most (Padj=1 × 10- 5 (AUC = 0.60). On the single gene level, CAPNS2, involved in extracellular matrix organization, was the top upregulated protein coding gene (Fold change = 48.5, P < 9 × 10- 24). The risk variant rs10757278 altered a binding site of the pathogen responsive transcription factor STAT1 (P = 5.8 × 10- 6). rs10757278-G allele reduced STAT1 binding 14.4% and rs10757278-A decreased luciferase activity in gingival fibroblasts 41.2% (P = 0.0056), corresponding with GTEx data. CDKN2B-AS1 represses collagen gene expression in gingival fibroblasts. Dysregulated collagen biosynthesis through allele-specific CDKN2B-AS1 expression in response to inflammatory factors may affect collagen synthesis, and in consequence tissue barrier and atherosclerotic plaque stability.