Poster T20 - Vascular Endothelial Heparan Sulfate Cooperates with Endothelin B Receptor to Promote Endothelin-1 Synthesis

Introduction: The endothelial glycocalyx (GCX), a layer of proteins coating the luminal surface of endothelial cells (ECs), plays a crucial role in vascular permeability and endothelial integrity. Its functions have only recently become the subject of in-depth research. Comprised of various transmembrane proteins connected to the cytoskeleton serving as mechanotransducers for cellular responses, the GCX can influence many biochemical activities. Diseases like atherosclerosis or hypertension can lead to damage to the GCX, adversely affecting vasculature.
Endothelin-1 (ET-1) is a potent vasoconstrictor that plays a significant role in many cardiovascular-related conditions. It may function to regulate contraction and dilation of blood vessels in response to changes in blood flow. Elevated ET-1 has been noted in patients with moderate-to-severe hypertension and atherosclerosis, implicating ET-1 in the pathogenesis of these conditions. ET-1 is produced by ECs and secreted into the extracellular space to bind to either the endothelin B receptor (ETB) on ECs, stimulating vasodilation, or the endothelial A receptor on smooth muscle cells, stimulating vasoconstriction. Due to proximity and the related roles of GCX and ETB, it is hypothesized that ETB cooperates with the GCX to trigger the production and release of ET-1 via disruptions in the cell cytoskeleton. Reports suggest that the expression of ET-1 is shear stress magnitude and time-dependent and that the disruption of cytoskeletal structures could mediate the shear stress-induced production of ET-1. This cooperation between ETB, ET-1 and GCX is yet to be clarified, a question that needs further investigation, which is the goal of this project.

Materials and

Methods: To measure the expression of ETB on the endothelial cell surface, Human Lung Microvascular ECs (HLMVEC) were subjected to laminar flow in a parallel-plate flow chamber and expression changes of ET-1, ETB, and Heparan Sulfate (HS), a major component of the glycocalyx, were measured. The cells were exposed to fluid flow at controlled rates and durations to impart a shear stress of 5, 15, and 25 dynes/cm2 on the cells for 30 minutes. A static control not subjected to fluid flow was included.
After exposure to flow, cells were fixed using 4% formaldehyde and immunostaining was performed to fluorescently tag ET-1, ETB, and HS. A laser-scanning confocal microscope was used to capture Z-stack images for quantification. The sum projection of these images was generated using ImageJ FIJI, and analysis of fluorescent intensity was performed in CellProfiler. Fluorescent intensity was calculated per-cell area, and a fold-change calculation was performed to compare the change in fluorescent intensity between the flow conditions and the static control.

Results, Conclusions, and Discussions: Preliminary results show that ETB expression is reduced, and HS expression is slightly increased when exposed to fluid flow at 5, 15, and 25 dynes/cm2 for 30 minutes. ET-1 expression may be reduced in the presence of low shear stress (5 dynes/cm2), though there is variability in our preliminary data, but is increased under higher shear stress, 15 and 25 dynes/cm2.
Long-term exposure to fluid flow is known to upregulate ETB, but little is known about ETB’s shear stress response to short-term exposure. HS is known to exhibit increased expression when exposed to shear stress – its natural condition in the vasculature. ET-1 expression has been shown to be increased during short-term exposure to low to high shear stress with a peak between 0.5-2 hours of exposure. Our preliminary results align with the literature, demonstrating the accuracy of our model.
Ongoing experiments include further quantification of gene expression profiles using qPCR to confirm our results match the literature. The increased expression of HS demonstrates glycocalyx health under physiological conditions. We will begin to establish the relationship between ETB, ET-1 and the GCX by inducing GCX damage by cleaving HS with a Heparinase-III treatment prior to short-term flow exposure. This will clarify the relationship between the molecules by showing the response of ETB and ET-1 to GCX damage.
During longer term exposure (6-12 hours), ET-1 expression has been demonstrated to decrease. Further experiments will include 12- and 24-hour flow exposure durations to study long-term shear stress exposure and western blotting for further gene expression profile quantification.
The GCX is known to play a vital role in vascular health and function. Understanding this role is a recent development, and the scale of GCX influence in the body is not well understood. ET-1 and ETB also play a major role in vascular function. Based on our preliminary data on expression changes when exposed to shear stress, ET-1, ETB, and GCX may lead a cooperative response to changes in vascular condition.

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