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, 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 NO 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.

Translatome profiling reveals Itih4 as a novel smooth muscle cell-specific gene in atherosclerosis.

AIMS: Vascular smooth muscle cells (SMCs) and their derivatives are key contributors to the development of atherosclerosis. However, studying changes in SMC gene expression in heterogeneous vascular tissues is challenging due to the technical limitations and high cost associated with current approaches. In this paper, we apply Translating Ribosome Affinity Purification sequencing (TRAP-Seq) to profile SMC-specific gene expression directly from tissue. METHODS AND RESULTS: To facilitate SMC-specific translatome analysis, we generated SMCTRAP mice, a transgenic mouse line expressing EGFP-tagged ribosomal protein L10a (EGFP-L10a) under the control of the SMC-specific αSMA promoter. These mice were further crossed with the atherosclerosis model Ldlr-/-, ApoB100/100 to generate SMCTRAP-AS mice and used to profile atherosclerosis-associated SMCs in thoracic aorta samples of 15-month-old SMCTRAP and SMCTRAP-AS mice. Our analysis of SMCTRAP-AS mice showed that EGFP-L10a expression was localized to SMCs in various tissues, including the aortic wall and plaque. The TRAP fraction demonstrated high enrichment of known SMC-specific genes, confirming the specificity of our approach. We identified several genes, including Cemip, Lum, Mfge8, Spp1, and Serpina3, that are known to be involved in atherosclerosis-induced gene expression. Moreover, we identified several novel genes not previously linked to SMCs in atherosclerosis, such as Anxa4, Cd276, Itih4, Myof, Pcdh11x, Rab31, Serpinb6b, Slc35e4, Slc8a3, and Spink5. Among them, we confirmed the SMC-specific expression of Itih4 in atherosclerotic lesions using immunofluorescence staining of mouse aortic roots and spatial transcriptomics of human carotid arteries. Furthermore, our more detailed analysis of Itih4 showed its link to coronary artery disease (CAD) through the colocalization of GWAS, splice-QTL, and protein-QTL signals. CONCLUSIONS: We generated a SMC-specific TRAP mouse line to study atherosclerosis and identified Itih4 as a novel SMC-expressed gene in atherosclerotic plaques, warranting further investigation of its putative function in extracellular matrix stability and genetic evidence of causality.

ErbB signaling is a potential therapeutic target for vascular lesions with fibrous component.

Background: Sporadic venous malformation (VM) and angiomatosis of soft tissue (AST) are benign, congenital vascular anomalies affecting venous vasculature. Depending on the size and location of the lesion, symptoms vary from motility disturbances to pain and disfigurement. Due to the high recurrence of the lesions, more effective therapies are needed. Methods: As targeting stromal cells has been an emerging concept in anti-angiogenic therapies, here, by using VM/AST patient samples, RNA-sequencing, cell culture techniques, and a xenograft mouse model, we investigated the crosstalk of endothelial cells (EC) and fibroblasts and its effect on vascular lesion growth. Results: We report, for the first time, the expression and secretion of transforming growth factor A (TGFA) in ECs or intervascular stromal cells in AST and VM lesions. TGFA induced secretion of vascular endothelial growth factor (VEGF-A) in paracrine fashion, and regulated EC proliferation. Oncogenic PIK3CA variant in p.H1047R, a common somatic mutation found in these lesions, increased TGFA expression, enrichment of hallmark hypoxia, and in a mouse xenograft model, lesion size, and vascularization. Treatment with afatinib, a pan-ErbB tyrosine-kinase inhibitor, decreased vascularization and lesion size in a mouse xenograft model with ECs expressing oncogenic PIK3CA p.H1047R variant and fibroblasts. Conclusions: Based on the data, we suggest that targeting of both intervascular stromal cells and ECs is a potential treatment strategy for vascular lesions having a fibrous component. Funding: Academy of Finland, Ella and Georg Ehnrooth foundation, the ERC grants, Sigrid Jusélius Foundation, Finnish Foundation for Cardiovascular Research, Jane and Aatos Erkko Foundation, GeneCellNano Flagship program, and Department of Musculoskeletal and Plastic Surgery, Helsinki University Hospital.

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.

Nondestructive Evaluation of Mechanical and Histological Properties of the Human Aorta With Near-Infrared Spectroscopy.

INTRODUCTION: Ascending aortic dilatation is a well-known risk factor for aortic rupture. Indications for aortic replacement in its dilatation concomitant to other open-heart surgery exist; however, cut-off values based solely on aortic diameter may fail to identify patients with weakened aortic tissue. We introduce near-infrared spectroscopy (NIRS) as a diagnostic tool to nondestructively evaluate the structural and compositional properties of the human ascending aorta during open-heart surgeries. During open-heart surgery, NIRS could provide information regarding tissue viability in situ and thus contribute to the decision of optimal surgical repair. MATERIALS AND METHODS: Samples were collected from patients with ascending aortic aneurysm (n = 23) undergoing elective aortic reconstruction surgery and from healthy subjects (n = 4). The samples were subjected to spectroscopic measurements, biomechanical testing, and histological analysis. The relationship between the near-infrared spectra and biomechanical and histological properties was investigated by adapting partial least squares regression. RESULTS: Moderate prediction performance was achieved with biomechanical properties (r = 0.681, normalized root-mean-square error of cross-validation = 17.9%) and histological properties (r = 0.602, normalized root-mean-square error of cross-validation = 22.2%). Especially the performance with parameters describing the aorta's ultimate strength, for example, failure strain (r = 0.658), and elasticity (phase difference, r = 0.875) were promising and could, therefore, provide quantitative information on the rupture sensitivity of the aorta. For the estimation of histological properties, the results with α-smooth muscle actin (r = 0.581), elastin density (r = 0.973), mucoid extracellular matrix accumulation(r = 0.708), and media thickness (r = 0.866) were promising. CONCLUSIONS: NIRS could be a potential technique for in situ evaluation of biomechanical and histological properties of human aorta and therefore useful in patient-specific treatment planning.

Wall Shear Stress Predicts Media Degeneration and Biomechanical Changes in Thoracic Aorta.

Objectives: In thoracic aortic aneurysm (TAA) of the ascending aorta (AA), AA is progressively dilating due to the weakening of the aortic wall. Predicting and preventing aortic dissections and ruptures in TAA continues to be challenging, and more accurate assessment of the AA dilatation, identification of high-risk patients, and timing of repair surgery are required. We investigated whether wall shear stress (WSS) predicts pathological and biomechanical changes in the aortic wall in TAA. Methods: The study included 12 patients with bicuspid (BAV) and 20 patients with the tricuspid aortic valve (TAV). 4D flow magnetic resonance imaging (MRI) was performed a day before aortic replacement surgery. Biomechanical and histological parameters, including assessing of wall strength, media degeneration, elastin, and cell content were analyzed from the resected AA samples. Results: WSSs were greater in the outer curves of the AA compared to the inner curves in all TAA patients. WSSs correlated with media degeneration of the aortic wall (ρ = -0.48, p < 0.01), elastin content (ρ = 0.47, p < 0.01), and aortic wall strength (ρ = -0.49, p = 0.029). Subsequently, the media of the outer curves was thinner, more rigid, and tolerated lower failure strains. Failure values were shown to correlate with smooth muscle cell (SMC) density (ρ = -0.45, p < 0.02), and indicated the more MYH10+ SMCs the lower the strength of the aortic wall structure. More macrophages were detected in patients with severe media degeneration and the areas with lower WSSs. Conclusion: The findings indicate that MRI-derived WSS predicts pathological and biomechanical changes in the aortic wall in patients with TAA and could be used for identification of high-risk patients.

Large Vessel Cell Heterogeneity and Plasticity: Focus in Aortic Aneurysms.

Smooth muscle cells and endothelial cells have a remarkable level of plasticity in vascular pathologies. In thoracic and abdominal aortic aneurysms, smooth muscle cells have been suggested to undergo phenotypic switching and to contribute to degradation of the aortic wall structure in response to, for example, inflammatory mediators, dysregulation of growth factor signaling or oxidative stress. Recently, endothelial-to-mesenchymal transition, and a clonal expansion of degradative smooth muscle cells and immune cells, as well as mesenchymal stem-like cells have been suggested to contribute to the progression of aortic aneurysms. What are the factors driving the aortic cell phenotype changes and how vascular flow, known to affect aortic wall structure and to be altered in aortic aneurysms, could affect aortic cell remodeling? In this review, we summarize the current literature on aortic cell heterogeneity and phenotypic switching in relation to changes in vascular flow and aortic wall structure in aortic aneurysms in clinical samples with special focus on smooth muscle and endothelial cells. The differences between thoracic and abdominal aortic aneurysms are discussed.

Hypoxia-Mediated Regulation of Histone Demethylases Affects Angiogenesis-Associated Functions in Endothelial Cells.

OBJECTIVE: Previous studies have demonstrated that the expression of several lysine (K)-specific demethylases (KDMs) is induced by hypoxia. Here, we sought to investigate the exact mechanisms underlying this regulation and its functional implications for endothelial cell function, such as angiogenesis. Approach and Results: We analyzed the expression changes of KDMs under hypoxia and modulation of HIF (hypoxia-inducible factor) expression using GRO-Seq and RNA-Seq in endothelial cells. We provide evidence that the majority of the KDMs are induced at the level of nascent transcription mediated by the action of HIF-1α and HIF-2α. Importantly, we show that transcriptional changes at the level of initiation represent the major mechanism of gene activation. To delineate the epigenetic effects of hypoxia and HIF activation in normoxia, we analyzed the genome-wide changes of H3K27me3 using chromosome immunoprecipitation-Seq. We discovered a redistribution of H3K27me3 at ≈2000 to 3000 transcriptionally active loci nearby genes implicated in angiogenesis. Among these, we demonstrate that vascular endothelial growth factor A (VEGFA) expression is partly induced by KDM4B- and KDM6B-mediated demethylation of nearby regions. Knockdown of KDM4B and KDM6B decreased cell proliferation, tube formation, and endothelial sprouting while affecting hundreds of genes associated with angiogenesis. These findings provide novel insights into the regulation of KDMs by hypoxia and the epigenetic regulation of VEGFA-mediated angiogenesis. CONCLUSIONS: Our study describes an additional level of epigenetic regulation where hypoxia induces redistribution of H3K27me3 around genes implicated in proliferation and angiogenesis. More specifically, we demonstrate that KDM4B and KDM6B play a key role in modulating the expression of the major angiogenic driver VEGFA.