SOX4 is a novel phenotypic regulator of endothelial cells in atherosclerosis revealed by single-cell analysis.

INTRODUCTION: Atherosclerotic complications represent the leading cause of cardiovascular mortality globally. Dysfunction of endothelial cells (ECs) often initiates the pathological events in atherosclerosis. OBJECTIVES: In this study, we sought to investigate the transcriptional profile of atherosclerotic aortae, identify novel regulator in dysfunctional ECs and hence provide mechanistic insights into atherosclerotic progression. METHODS: We applied single-cell RNA sequencing (scRNA-seq) on aortic cells from Western diet-fed apolipoprotein E-deficient (ApoE-/-) mice to explore the transcriptional landscape and heterogeneity of dysfunctional ECs. In vivo validation of SOX4 upregulation in ECs were performed in atherosclerotic tissues, including mouse aortic tissues, human coronary arteries, and human renal arteries. Single-cell analysis on human aortic aneurysmal tissue was also performed. Downstream vascular abnormalities induced by EC-specific SOX4 overexpression, and upstream modulators of SOX4 were revealed by biochemical assays, immunostaining, and wire myography. Effects of shear stress on endothelial SOX4 expression was investigated by in vitro hemodynamic study. RESULTS: Among the compendium of aortic cells, mesenchymal markers in ECs were significantly enriched. Two EC subsets were subsequently distinguished, as the 'endothelial-like' and 'mesenchymal-like' subsets. Conventional assays consistently identified SOX4 as a novel atherosclerotic marker in mouse and different human arteries, additional to a cancer marker. EC-specific SOX4 overexpression promoted atherogenesis and endothelial-to-mesenchymal transition (EndoMT). Importantly, hyperlipidemia-associated cytokines and oscillatory blood flow upregulated, whereas the anti-diabetic drug metformin pharmacologically suppressed SOX4 level in ECs. CONCLUSION: Our study unravels SOX4 as a novel phenotypic regulator during endothelial dysfunction, which exacerbates atherogenesis. Our study also pinpoints hyperlipidemia-associated cytokines and oscillatory blood flow as endogenous SOX4 inducers, providing more therapeutic insights against atherosclerotic diseases.

Yes-Associated Protein Targets the Transforming Growth Factor ß Pathway to Mediate High-Fat/High-Sucrose Diet-induced Arterial Stiffness.

BACKGROUND: Metabolic syndrome is related to cardiovascular diseases, which is attributed in part, to arterial stiffness; however, the mechanisms remain unclear. The present study aimed to investigate the molecular mechanisms of metabolic syndrome-induced arterial stiffness and to identify new therapeutic targets. METHODS: Arterial stiffness was induced by high-fat/high-sucrose diet in mice, which was quantified by Doppler ultrasound. Four-dimensional label-free quantitative proteomic analysis, affinity purification and mass spectrometry, and immunoprecipitation and GST (glutathione S-transferase) pull-down experiments were performed to explore the mechanism of YAP (Yes-associated protein)-mediated TGF (transforming growth factor) ß pathway activation. RESULTS: YAP protein was upregulated in the aortic tunica media of mice fed a high-fat/high-sucrose diet for 2 weeks and precedes arterial stiffness. Smooth muscle cell-specific YAP knockdown attenuated high-fat/high-sucrose diet-induced arterial stiffness and activation of TGFß-Smad2/3 signaling pathway in arteries. By contrast, Myh11CreERT2-YapTg mice exhibited exacerbated high-fat/high-sucrose diet-induced arterial stiffness and enhanced TGFß-activated Smad2/3 phosphorylation in arteries. PPM1B (protein phosphatase, Mg2+/Mn2+-dependent 1B) was identified as a YAP-bound phosphatase that translocates into the nucleus to dephosphorylate Smads (mothers against decapentaplegic homologs) in response to TGFß. This process was inhibited by YAP through removal of the K63-linked ubiquitin chain of PPM1B at K326. CONCLUSIONS: This study provides a new mechanism by which smooth muscle cell YAP regulates the TGFß pathway and a potential therapeutic target in metabolic syndrome-associated arterial stiffness.

Hypermethylation of mitochondrial DNA in vascular smooth muscle cells impairs cell contractility.

Vascular smooth muscle cell (SMC) from arterial stenotic-occlusive diseases is featured with deficiency in mitochondrial respiration and loss of cell contractility. However, the regulatory mechanism of mitochondrial genes and mitochondrial energy metabolism in SMC remains elusive. Here, we described that DNA methyltransferase 1 (DNMT1) translocated to the mitochondria and catalyzed D-loop methylation of mitochondrial DNA in vascular SMCs in response to platelet-derived growth factor-BB (PDGF-BB). Mitochondrial-specific expression of DNMT1 repressed mitochondrial gene expression, caused functional damage, and reduced SMC contractility. Hypermethylation of mitochondrial D-loop regions were detected in the intima-media layer of mouse carotid arteries subjected to either cessation of blood flow or mechanical endothelial injury, and also in vessel specimens from patients with carotid occlusive diseases. Likewise, the ligated mouse arteries exhibited an enhanced mitochondrial binding of DNMT1, repressed mitochondrial gene expression, defects in mitochondrial respiration, and impaired contractility. The impaired contractility of a ligated vessel could be restored by ex vivo transplantation of DNMT1-deleted mitochondria. In summary, we discovered the function of DNMT1-mediated mitochondrial D-loop methylation in the regulation of mitochondrial gene transcription. Methylation of mitochondrial D-loop in vascular SMCs contributes to impaired mitochondrial function and loss of contractile phenotype in vascular occlusive disease.

Black tea protects against hypertension-associated endothelial dysfunction through alleviation of endoplasmic reticulum stress.

Hypertensive patients have been found to be associated with elevated levels of homocysteine, known as hyperhomocysteinemia. Homocysteine (Hcy) can induce endoplasmic reticulum (ER) stress in endothelial cells. This study aims to investigate whether black tea (BT) protects against hypertension-associated endothelial dysfunction through alleviation of ER stress. Rat aortae and cultured rat aortic endothelial cells were treated with Hcy, BT extract, and theaflavin-3,3'-digallate (TF3). Male Sprague Dawley rats were infused with angiotensin II (Ang II) to induce hypertension and orally administrated with BT extract at 15 mg/kg/day for 2 weeks. Hcy impaired endothelium-dependent relaxations of rat aortae and led to ER stress in endothelial cells, which were ameliorated by co-incubation of BT extract and TF3. The blood pressure of Ang II-infused rats and plasma Hcy level were normalized by BT consumption. Impaired endothelium-dependent relaxations in renal arteries, carotid arteries and aortae, and flow-mediated dilatations in third-order mesenteric resistance arteries were improved. Elevations of ER stress markers and ROS level, plus down-regulation of Hcy metabolic enzymes in aortae from Ang II-infused rats were prevented by BT treatment. Our data reveal the novel cardiovascular benefits of BT in ameliorating vascular dysfunctions, providing insight into developing BT into beneficial dietary supplements in hypertensive patients.