ChE PhD Dissertation Defense: Ronodeep Mitra

Name:
Ronodeep Mitra

Title:

Glycocalyx Therapy to Restore Anti-Atherosclerotic Endothelial Cell Function

Date:
9/11/2024

Time:
9:00:00 AM

Committee Members:
Prof. Eno Ebong (Advisor)
Prof. Mansoor Amiji
Prof. Rebecca Carrier
Prof. Arthur J. Coury
Prof. Jessica M. Oakes

Location:
EXP 610 and Zoom

Abstract:
The endothelial cell (EC) glycocalyx (GCX) is a negatively charged complex sugar-rich layer that lines the endothelium. It is an important contributor to the physical and biochemical health of the vasculature and endothelium, while mediating mechanotransduction and vascular signaling. For example, when exposed to physiological (unidirectional and uniform in magnitude) levels of shear stress from the mechanical force of blood flow, the GCX is abundant and aids in the production of vasodilator nitric oxide (NO), which regulates vascular tone. Furthermore, the dynamics of the flow-regulated GCX determine the structural integrity of connexin proteins that comprises interendothelial gap junctions and control the flow of communication between neighboring ECs. Finally, the GCX acts as a physical barrier to numerous components in circulating blood, including low-density lipoproteins (LDLs) and inflammatory cells such as monocytes that differentiate into macrophages and platelets.

Loss of the EC GCX can be attributed to disturbed vasculature blood flow patterns. This condition renders the endothelium as adhesive and permeable, resulting in infiltration of the vessel walls by blood circulating LDLs, compromising active EC-EC communication via interendothelial gap junctions, and reduction in NO production, leading to vasoconstriction. These phenotypes lead to vascular dysfunction, atherosclerosis, and other serious secondary cardiovascular events, such as myocardial infarctions and strokes. Hence, we propose either repurposing therapies that were
not originally indicated for GCX therapy or the development of novel GCX therapies and hypothesize that targeting the EC GCX will restore vascular function and prevent further downstream cardiovascular events, such as atherosclerosis.

We first tested our hypothesis by assessing the efficacy of repurposing diosmin, a flavanone glycoside of diosmetin, which is a nutraceutical used to currently treat chronic venous insufficiency. Previous studies have shown diosmin’s potent anti-inflammatory and anti-oxidant properties on the endothelium. Hence, we wanted to determine if diosmin would repair mechanically damaged endothelial GCX in regions of disturbed flow (DF) patterns and restore anti-atherosclerotic endothelium mechanotransduction function. For this study, we utilized a unique murine in vivo DF model, where the left carotid artery (LCA) is partially ligated, while the right carotid artery (RCA) is not surgically intervened and was the designated uniform flow (UF) control for each mouse. Diosmin treatment elevated activated endothelial NO synthase level (p-eNOS), inhibited inflammatory cell uptake, decreased vessel wall thickness and increased vessel diameter, and increased GCX coverage on the endothelium in ligated LCA. This corroborated support that diosmin protects endothelial GCX integrity and preserves complex endothelial function.

Next, in vitro and in vivo DF models were used to assess a novel therapy, combining sphingosine-1-phosphate (S1P), a bioactive lipid mediator, and heparin in regenerating the endothelial GCX. We used a parallel-plate flow chamber to simulate flow conditions in vitro on human coronary arterial endothelial cells (HCAECs) and a partial carotid ligation murine model to mimic DF in vivo, as mentioned above. In vitro data showed that heparin/S1P therapy improved the function of DF-conditioned ECs by restoring the GCX and promoting EC alignment and elevated p-eNOS expression. Furthermore, heparin/S1P treatment restored GCX in the LCA, enhancing GCX thickness and coverage of the blood vessel wall and reducing vessel wall thickness, demonstrating advances in a novel therapeutic that regenerates EC GCX and restores complex vascular function in DF conditions.

This research work is an excellent step towards the development of repurposed or novel therapeutics that can be applied to replace, stabilize, or protect the GCX and restore GCX-mediated EC mechanotransduction, particularly in DF conditions. These prospective mechano-therapeutics could represent breakthrough solutions for preventing cardiovascular diseases such as atherosclerosis in the future.