Poster U20 - Basal CD44 / hyaluronic acid and shear stress dependent endothelial cell remodeling

Introduction: The endothelial glycocalyx is a transmembrane grass-like structure that encompasses endothelial cells (ECs) serving to transduce extracellular signals, regulate vascular permeability, and inhibit the adhesion of leukocytes. Proteoglycans and glycoproteins serve as the surface receptors for various carbohydrate chains known as glycosaminoglycans (GAGs). The glycocalyx along the lumen of the vessel is referred to as the apical glycocalyx, which is directly exposed to shear due to blood flow while the basal glycocalyx is present at the smooth muscle cell interface. Historically, the apical and basal glycocalyx have been independently investigated due to their differences in their local environment and stimuli. The apical glycocalyx has been at the forefront of research efforts due to its direct role in the mechanotransduction of shear forces due to blood flow. However, shear forces due to flow are transmitted from the apical glycocalyx through the cell cytoskeleton to basal glycocalyx components promoting alternate signaling pathways and influencing cell migration and morphology. Yet, the basal glycocalyx remains largely unexplored.

CD44 is a transmembrane glycoprotein serving as a surface receptor for hyaluronic acid (HA), a known fluid shear sensor in the apical glycocalyx shown to influence the development of cardiovascular disease. The role of basal CD44 and HA in mechanotransduction and EC remodeling is unclear. Here we aim to examine the relationship between shear stress and CD44 and HA-instigated EC remodeling by visualizing basal CD44 and HA expression and cytoskeletal alignment under normal and pathophysiological conditions.

Materials and

Methods: Human lung microvascular endothelial cells (HUVECs) were maintained in Microvascular Endothelial Cell Growth Medium at 37 °C in static culture. Passage five HLMVECs were plated at 100,000 cells/mL on a 24 mm x 60 mm fibronectin-coated (10 ug/mL) glass cover slip. Once confluent the slide was placed in a simple continuous flow loop parallel plate flow chamber. Media flowing through the chamber exposed the HLMVECs to 10 dynes/cm2 of shear stress for 30 minutes. This shear stress is within the normal reported range for microvascular endothelial cells. To determine shear stress rate and time dependence the effects of physiologically low and high shear rates as well as increased exposure time will be investigated.

Slides were fixed with 4% paraformaldehyde and 2% sucrose for membrane stabilization. A blocking solution containing 10% goat serum and 3% BSA was supplemented with 0.1% saponin for permeabilization. Immunostaining of CD44 and HA was conducted using CD44 antibody (1:250, MA5 44271 Thermo) and hyaluronic acid binding protein (1:100, 50 ug/mL, 385911 Sigma) as primary antibodies and Rb IgG (H+L) Alexa-flour 488 (1:250) and Alexa-flour 488 conjugated anti-biotin as secondary antibodies respectively. The slides were mounted with Flouromount DAPI Mounting Media. Z-stack images were taken with a 0.1 µm slice thickness on a Leica STELLARIS 8 laser scanning confocal microscope. Sum projections of the apical and basal cell and Z-direction orthogonal views were generated to visualize the coverage and thickness of CD44 and HA in the apical and basal cell.

Results, Conclusions, and Discussions: Visualization of CD44 (Fig. 1) and HA (Fig. 2) in the apical (A) and basal (C) HLMVEC after 30 minutes of exposure to physiologically normal fluid shear stress was as shown in Figure 1 and Figure 2 respectively. In Figure 1A and Figure 1C distributed expression of CD44 can be seen throughout the apical and basal cell and on-cell processes. CD44 signal is seen encompassing the nuclei, and cell boundaries can be seen in Figure 1B. Apical and basal HA signal is shown in Figure 2A and Figure 2C respectively. A distinct separation of the apical and basal cell HA signal can be seen in Figure 2B.

The presence of CD44 and HA in both the apical and basal cell suggests that CD44 in the apical and basal glycocalyx cell, through their connection to the cytoskeleton, form a whole cell force transduction network that allows the cell to respond to complex extracellular forces such as shear forces due to blood flow as other models have suggested. Future experiments will examine how CD44 and HA expression and cytoskeletal alignment change with alterations in shear rates and exposure times. Changes in CD44 and HA expression (cell coverage and florescent intensity) and glycocalyx component thickness (orthogonal view) will be quantified. The individual roles of CD44 and HA will also be examined using enzyme degradation and cell knock-out models. The role of the cytoskeleton in CD44 and HA expression will also be investigated by blocking actin polymerization. The results of this experiment will expose the shear stress-dependent role of CD44 in regulating EC remodeling.

Future studies will examine how basal CD44 and HA affect integrin clustering and modulate crucial mechanotransduction pathways such as YAP/TAZ and Rho which modulate EC proliferation and cytoskeletal remodeling. The cumulative knowledge from this study will be used to further complete existing models of EC shear force transduction which historically neglect the role of the basal glycocalyx. Finally, the characterization of basal CD44 will provide insight into basal glycocalyx function in vascular physiology and disease allowing for the identification and development of new therapeutic targets.

Acknowledgements (Optional): This research was supported with startup funds from Worcester Polytechnic Institute awarded to Dr. Mensah.