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.

An atlas of genetic scores to predict multi-omic traits.

The use of omic modalities to dissect the molecular underpinnings of common diseases and traits is becoming increasingly common. But multi-omic traits can be genetically predicted, which enables highly cost-effective and powerful analyses for studies that do not have multi-omics1. Here we examine a large cohort (the INTERVAL study2; n = 50,000 participants) with extensive multi-omic data for plasma proteomics (SomaScan, n = 3,175; Olink, n = 4,822), plasma metabolomics (Metabolon HD4, n = 8,153), serum metabolomics (Nightingale, n = 37,359) and whole-blood Illumina RNA sequencing (n = 4,136), and use machine learning to train genetic scores for 17,227 molecular traits, including 10,521 that reach Bonferroni-adjusted significance. We evaluate the performance of genetic scores through external validation across cohorts of individuals of European, Asian and African American ancestries. In addition, we show the utility of these multi-omic genetic scores by quantifying the genetic control of biological pathways and by generating a synthetic multi-omic dataset of the UK Biobank3 to identify disease associations using a phenome-wide scan. We highlight a series of biological insights with regard to genetic mechanisms in metabolism and canonical pathway associations with disease; for example, JAK-STAT signalling and coronary atherosclerosis. Finally, we develop a portal ( https://www.omicspred.org/ ) to facilitate public access to all genetic scores and validation results, as well as to serve as a platform for future extensions and enhancements of multi-omic genetic scores.

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.

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.

Multimodal CRISPR perturbations of GWAS loci associated with coronary artery disease in vascular endothelial cells.

Genome-wide association studies have identified >250 genetic variants associated with coronary artery disease (CAD), but the causal variants, genes and molecular mechanisms remain unknown at most loci. We performed pooled CRISPR screens to test the impact of sequences at or near CAD-associated genetic variants on vascular endothelial cell functions. Using CRISPR knockout, inhibition and activation, we targeted 1998 variants at 83 CAD loci to assess their effect on three adhesion proteins (E-selectin, ICAM1, VCAM1) and three key endothelial functions (nitric oxide and reactive oxygen species production, calcium signalling). At a false discovery rate ≤10%, we identified significant CRISPR perturbations near 42 variants located within 26 CAD loci. We used base editing to validate a putative causal variant in the promoter of the FES gene. Although a few of the loci include genes previously characterized in endothelial cells (e.g. AIDA, ARHGEF26, ADAMTS7), most are implicated in endothelial dysfunction for the first time. Detailed characterization of one of these new loci implicated the RNA helicase DHX38 in vascular endothelial cell senescence. While promising, our results also highlighted several limitations in using CRISPR perturbations to functionally dissect GWAS loci, including an unknown false negative rate and potential off-target effects.

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).

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).

FHL5 Controls Vascular Disease-Associated Gene Programs in Smooth Muscle Cells.

BACKGROUND: Genome-wide association studies have identified hundreds of loci associated with common vascular diseases, such as coronary artery disease, myocardial infarction, and hypertension. However, the lack of mechanistic insights for many GWAS loci limits their translation into the clinic. Among these loci with unknown functions is UFL1-four-and-a-half LIM (LIN-11, Isl-1, MEC-3) domain 5 (FHL5; chr6q16.1), which reached genome-wide significance in a recent coronary artery disease/ myocardial infarction GWAS meta-analysis. UFL1-FHL5 is also associated with several vascular diseases, consistent with the widespread pleiotropy observed for GWAS loci. METHODS: We apply a multimodal approach leveraging statistical fine-mapping, epigenomic profiling, and ex vivo analysis of human coronary artery tissues to implicate FHL5 as the top candidate causal gene. We unravel the molecular mechanisms of the cross-phenotype genetic associations through in vitro functional analyses and epigenomic profiling experiments in coronary artery smooth muscle cells. RESULTS: We prioritized FHL5 as the top candidate causal gene at the UFL1-FHL5 locus through expression quantitative trait locus colocalization methods. FHL5 gene expression was enriched in the smooth muscle cells and pericyte population in human artery tissues with coexpression network analyses supporting a functional role in regulating smooth muscle cell contraction. Unexpectedly, under procalcifying conditions, FHL5 overexpression promoted vascular calcification and dysregulated processes related to extracellular matrix organization and calcium handling. Lastly, by mapping FHL5 binding sites and inferring FHL5 target gene function using artery tissue gene regulatory network analyses, we highlight regulatory interactions between FHL5 and downstream coronary artery disease/myocardial infarction loci, such as FOXL1 and FN1 that have roles in vascular remodeling. CONCLUSIONS: Taken together, these studies provide mechanistic insights into the pleiotropic genetic associations of UFL1-FHL5. We show that FHL5 mediates vascular disease risk through transcriptional regulation of downstream vascular remodeling gene programs. These transacting mechanisms may explain a portion of the heritable risk for complex vascular diseases.

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

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

Platelet-Derived MicroRNAs Regulate Cardiac Remodeling After Myocardial Ischemia.

BACKGROUND: Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of microRNAs (miRNAs), which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. METHODS: In vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (light-sheet fluorescence microscopy, positron emission tomography and magnetic resonance imaging, speckle-tracking echocardiography) of myocardial inflammation and remodeling, and next-generation deep sequencing analysis of platelet miRNA expression. RESULTS: In mice with a megakaryocyte/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer, the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating left ventricular remodeling after myocardial I/R following transient left coronary artery ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial I/R. Worsened cardiac remodeling after myocardial infarction in mice with a platelet-specific Dicer deletion resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at day 28 post-myocardial infarction. Altogether, these observations culminated in an impaired left ventricular function and hampered long-term cardiac recovery after experimental myocardial infarction and reperfusion therapy. Treatment with the P2Y12 (P2Y purinoceptor 12) antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in DicerPf4∆/Pf4∆ mice. CONCLUSIONS: The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial I/R.

PAR4 Antagonism in Patients With Coronary Artery Disease Receiving Antiplatelet Therapies.

BACKGROUND: BMS-986141 is a novel potent highly selective antagonist of PAR (protease-activated receptor) type 4. PAR4 antagonism has been demonstrated to reduce thrombus formation in isolation and in combination with factor Xa inhibition in high shear conditions in healthy people. We sought to determine whether PAR4 antagonism had additive antithrombotic effects in patients with coronary artery disease who were receiving antiplatelet therapy. METHODS: Forty-five patients with stable coronary heart disease and 10 healthy volunteers completed a phase 2a open-label 4-arm single-center study. Patients were allocated to 1 of 3 treatment arms for 7 days: (1) ticagrelor (90 mg BID), (2) aspirin (75 mg QD), or (3) the combination of ticagrelor and aspirin. Agonist-induced platelet aggregation, platelet activation, and ex vivo thrombus formation were measured before and 2 and 24 hours after a single oral 4-mg dose of BMS-986141 on the first study visit day in all participants. RESULTS: BMS-986141 demonstrated highly selective inhibition of PAR4-AP (agonist peptide)-induced platelet aggregation, P-selectin expression, and platelet-monocyte aggregate expression (P≤0.001 for all), which were unaffected by concomitant antiplatelet therapies. PAR4 antagonism reduced ex vivo thrombus area in high shear conditions in healthy volunteers (-21%; P=0.001) and in patients receiving ticagrelor alone (-28%; P=0.001), aspirin alone (-23%; P=0.018), or both in combination (-24%; P≤0.001). Plasma concentration of BMS-986141 correlated with PAR4-AP-induced platelet responses (P≤0.001 for all) and total thrombus area under high shear stress conditions (P≤0.01 for all). CONCLUSIONS: PAR4 antagonism has additive antithrombotic effects when used in addition to ticagrelor, aspirin, or their combination, in patients with stable coronary heart disease. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT05093790.

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.

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.

Therapeutic peptides for coronary artery diseases: in silico methods and current perspectives.

Many drug formulations containing small active molecules are used for the treatment of coronary artery disease, which affects a significant part of the world's population. However, the inadequate profile of these molecules in terms of therapeutic efficacy has led to the therapeutic use of protein and peptide-based biomolecules with superior properties, such as target-specific affinity and low immunogenicity, in critical diseases. Protein‒protein interactions, as a consequence of advances in molecular techniques with strategies involving the combined use of in silico methods, have enabled the design of therapeutic peptides to reach an advanced dimension. In particular, with the advantages provided by protein/peptide structural modeling, molecular docking for the study of their interactions, molecular dynamics simulations for their interactions under physiological conditions and machine learning techniques that can work in combination with all these, significant progress has been made in approaches to developing therapeutic peptides that can modulate the development and progression of coronary artery diseases. In this scope, this review discusses in silico methods for the development of peptide therapeutics for the treatment of coronary artery disease and strategies for identifying the molecular mechanisms that can be modulated by these designs and provides a comprehensive perspective for future studies.

Single-nucleus transcriptomics of epicardial adipose tissue from female pigs reveals effects of exercise training on resident innate and adaptive immune cells.

BACKGROUND: Coronary artery disease (CAD) is a leading cause of death in women. Epicardial adipose tissue (EAT) secretes cytokines to modulate coronary artery function, and the release of fatty acids from EAT serves as a readily available energy source for cardiomyocytes. However, despite having beneficial functions, excessive amounts of EAT can cause the secretion of proinflammatory molecules that increase the instability of atherosclerotic plaques and contribute to CAD progression. Although exercise mitigates CAD, the mechanisms by which exercise impacts EAT are unknown. The Yucatan pig is an excellent translational model for the effects of exercise on cardiac function. Therefore, we sought to determine if chronic aerobic exercise promotes an anti-inflammatory microenvironment in EAT from female Yucatan pigs. METHODS: Sexually mature, female Yucatan pigs (n = 7 total) were assigned to sedentary (Sed, n = 3) or exercise (Ex, n = 4) treatments, and coronary arteries were occluded (O) with an ameroid to mimic CAD or remained non-occluded (N). EAT was collected for bulk (n = 7 total) and single nucleus transcriptomic sequencing (n = 2 total, 1 per exercise treatment). RESULTS: Based on the bulk transcriptomic analysis, exercise upregulated S100 family, G-protein coupled receptor, and CREB signaling in neurons canonical pathways in EAT. The top networks in EAT affected by exercise as measured by bulk RNA sequencing were SRC kinase family, fibroblast growth factor receptor, Jak-Stat, and vascular endothelial growth factor. Single nucleus transcriptomic analysis revealed that exercise increased the interaction between immune, endothelial, and mesenchymal cells in the insulin-like growth factor pathway and between endothelial and other cell types in the platelet endothelial cell adhesion molecule 1 pathway. Sub-clustering revealed nine cell types in EAT, with fibroblast and macrophage populations predominant in O-Ex EAT and T cell populations predominant in N-Ex EAT. Unlike the findings for exercise alone as a treatment, there were not increased interactions between endothelial and mesenchymal cells in O-Ex EAT. Coronary artery occlusion impacted the most genes in T cells and endothelial cells. Genes related to fatty acid metabolism were the most highly upregulated in non-immune cells from O-Ex EAT. Sub-clustering of endothelial cells revealed that N-Ex EAT separated from other treatments. CONCLUSIONS: According to bulk transcriptomics, exercise upregulated pathways and networks related to growth factors and immune cell communication. Based on single nucleus transcriptomics, aerobic exercise increased cell-to-cell interaction amongst immune, mesenchymal, and endothelial cells in female EAT. Yet, exercise was minimally effective at reversing alterations in gene expression in endothelial and mesenchymal cells in EAT surrounding occluded arteries. These findings lay the foundation for future work focused on the impact of exercise on cell types in EAT.