VASCULAR REMODELING IN HYPERTENSION: IN-VITRO/ EX-VIVO MECHANICAL STRETCH-INDUCED ALTERATIONS AND OMICS ANALYSIS

Abstract

Hypertension, being a primary vasculature disorder, contributes significantly to premature death globally. Over the years, numerical figures have evidenced a dramatic increase in hypertension’s global prevalence, affecting approximately 1.3 billion individuals, according to the World Health Organization (WHO). Hypertension mainly develops as a result of excessive mechanical stretch exerted by the pulsatile blood flow on the vessel wall and endothelial cells (ECs), lining the inner face of the blood vessel, chiefly respond to these hemodynamic mechanical forces. Although physiological levels of mechanical stretch are crucial for maintaining vascular homeostasis, excessive levels, such as those reported in hypertension conditions, can induce pathological consequences, leading to structural and functional alterations in the blood vessel, a phenotype referred to as 'vascular remodeling'. In this study, we aim to (1) characterize structural and functional alterations occurring in ECs in response to mechanical stretch using an in-vitro model, (2) unravel the role of ECs in mechanical stretch-induced vascular remodeling using an ex-vivo model, and (3) uncover potential biomarkers that serve as plausible indicators for hypertension development through omics analysis. To address this, immortalized human umbilical vein endothelial cells (EA.hy 926) and Rat portal Vein (RPV) were subjected to mechanical stretch for 24 hours. Actin cytoskeleton remodeling, monocytes adhesion, and reactive oxygen species (ROS) generation were evaluated using immunofluorescence staining. Real-time PCR was employed to assess mRNA levels of inflammatory cytokines, adhesion molecules, and adipokines. Moreover, proteomics and metabolomics data of hypertensive patients, obtained from Qatar Biobank, were analyzed. The outcome of this study firstly, revealed significant morphological changes in ECs exposed to mechanical stretch, characterized by actin filament alignment perpendicular to the stretching axis and an increase in the F to G actin ratio. Interestingly, inhibition of Rho-associated protein kinase (ROCK), using Y-27632, prevented this orientation and the increase in F-actin. Furthermore, mechanical stretch significantly upregulated the transcript levels of several inflammatory cytokines including IL8, IL6, and IL1B, as well as adhesion molecules, including ICAM1, VCAM1, and E selectin, along with NFκB activation. Increased mRNA expression of leptin and adiponectin was also observed. Moreover, mechanical stretch of ECs stimulated the adhesion of THP-1 monocytes and increased CCL2 expression. ROS production was also significantly elevated under mechanical stretch conditions. Furthermore, denudation of the endothelium from the RPV was able to inhibit mechanical stretch-induced vascular hypertrophy, oxidative stress, and leptin synthesis. Secondly, proteomics analysis revealed a group of proteins, including the proto-oncogene tyrosine-protein kinase Src (SRC) family, calcium/calmodulin-dependent protein kinase 2 beta and delta subunits (CAMK2B and CAMK2D), Tec Protein Tyrosine Kinase (TEC), Glycogen synthase kinase-3 (GSK3), Vav Guanine Nucleotide Exchange Factor 1 (VAV1), and Ras-related C3 botulinum toxin substrate 1 (RAC1), markedly upregulated in patients with hypertension compared to those with prehypertension. Pathway analysis showed that the majority of these proteins play a role in actin cytoskeleton remodeling. Thirdly, metabolomics analysis identified six metabolites including stearidonate, hexadecadienoate, N6-carbamoylthreonyladenosine, 9 and 13-S-hydroxyoctadecadienoic acid (HODE), 2,3-dihydroxy-5-methylthio-4-pentenoate (DMTPA), and linolenate associated with an increased risk of developing hypertension. Taken together, our findings illustrate significant structural and functional changes in ECs subjected to mechanical stretch, mimicking the hypertension condition, and demonstrate a chief role of ECs in mechanical stretch-induced vascular remodeling. Additionally, our findings also identify novel proteins and metabolites involved in hypertension progression, shedding further insight into the underlying pathological mechanisms involved in hypertension and paving the way for novel diagnostic and therapeutic approaches for the treatment of hypertension and its associated complications.