INVESTIGATING THE MOLECULAR MECHANISMS OF SHEAR STRESS INDUCED ENDOTHELIAL DYSFUNCTION AND PROGRESSION OF ATHEROSCLEROSIS

Abstract

Background: Atherosclerotic vascular disease, such as coronary artery disease (CAD) and ischemic stroke is the leading cause of mortality and disability worldwide. Therefore, understanding the mechanisms underlying atherosclerosis is crucial for prevention and treatment. Atherosclerosis, characterized by plaque buildup in arteries, is influenced by factors such as disturbed blood flow, endothelial dysfunction, inflammation, and dyslipidemia. Endothelial cells (ECs) play a pivotal role in vascular health, responding to shear stress and maintaining homeostasis. In the vascular system, shear stress on ECs was generated by mainly two types of blood flow: "laminar" and "disturbed". ECs that are exposed to laminar flow exhibit healthy cellular functions including cell alignment, enhanced barrier function, nitric oxide (NO) production, and autophagy. However, disturbed flow induces endothelial dysfunction, inflammation, oxidative stress, and atherosclerosis in presence of cardiovascular risk factors which could be hypercholesterolemia (HC), hyperglycemia, or hypertension. Additionally, elevated blood leptin is also considered to be related to risk for cardiovascular diseases (CVDs). Distinct behavior of ECs in response to shear stress is a key stage for the development of vascular diseases and therefore an important research area. In vitro models, such as orbital shakers, provide valuable tools for studying shear stress-induced endothelial responses and investigating potential therapeutic targets. On the other side, exploring biomarkers for atherosclerosis is significant for early diagnosis and prediction of prognosis of the disease. Omics studies, with its holistic examination of biological samples, offer a significant advantage over conventional single-biomarker studies to gain insights into the molecular mechanisms of atherosclerosis and facilitate biomarker discovery. Objectives: The study involves two independent, yet interrelated aims directed towards (1) unraveling the mechanisms underlying disturbed flow-induced endothelial dysfunction and (2) identifying potential biomarkers for atherosclerosis progression. To address those aims, we established an in vitro flow system set up to study endothelial dysfunction. We examined intracellular protein alterations of EC line and rat aorta tissue, in response to shear stress. We also conducted proteomics and metabolomics profiling in patients with atherosclerosis and dyslipidemia. Methods: The in vitro and ex vivo arms of our study were conducted on human ECs and rat aortic tissues, respectively, and our proteomics and metabolomics studies were done on human blood plasma. The in vitro experiments involved EC culture, exposure to laminar and disturbed flow using an orbital shaker and the ex vivo experiments involved utilizing isolated rat aorta for examining the effect of shear stress on vascular cell behavior. Proteomics data were analyzed using multiple statistical methods. The data generated using Slow Off-rate Modified Aptamer (SOMAmer)-based protein array, was provided by Qatar Biobank. Our cross-sectional study involved healthy controls (n=45), and patients diagnosed with HC (n=51), or with CAD (n=32). Our metabolomics study employed a total of 221 participants including healthy controls, CAD patients, and HC patients with complications (i.e. hypertension and diabetes). A linear regression model was used to demonstrate how variations in the ordered categorical groups (stages of atherosclerosis progression) influenced changes in metabolite levels. Results: Shear stress experiments conducted in vitro showed distinct alignment and differential gene expression patterns in ECs exposed to either laminar flow or disturbed flow conditions. Along this, our findings from the in vitro studies also revealed leptin involvement in EC response to disturbed flow. Similar changes were observed using the ex vivo model too, particularly in the inner curve of the rat aorta, where the blood flow was disturbed. Our proteomics analysis, which was conducted pairwise comparisons among the study groups, revealed a total of 65 proteins differentially expressions, as well as strong diagnostic value for CAD and HC with area under the curve (AUC) values exceeding 0.75. Notably, among these proteins, 14 showed significant correlations with blood cholesterol levels. Additionally, 22 of the identified proteins were associated with CAD or HC pathways, with nine proteins including Apo E, Apo E3, MMP-3, PCSK9, SDF-1, Apo B, PAFAH, HSP 60, and TAK1-TAB1being common to both conditions. Our metabolomics analysis revealed differential metabolite levels associated with CAD progression, with notable changes in carbohydrate and lipid metabolism linked to CAD risk. Specifically, mannitol/sorbitol, mannose, glucose, and ribitol exhibited positive associations, while pregnenediol sulfate, oleoylcarnitine, and quinolinate were negatively associated with an increased risk of CAD. Correlation analysis further elucidated the relationship between clinical traits and metabolites, providing insights into the underlying mechanisms of CAD progression. Conclusion: In conclusion, this study provides comprehensive insights into the complex interplay between flow conditions, EC responses, inflammatory processes, and metabolic alterations in the context of CVD. By elucidating these molecular mechanisms, the study paves the way for the development of novel diagnostic tools and therapeutic strategies aimed at combating atherosclerosis in specific and reducing the burden of CVD in general.