Biophysical Properties of Endothelial Mechanotransduction Mediated by Glycocalyx.

Endothelial surface glycocalyx (ESG) is a carbohydrate-rich layer found on the endothelial cell (EC) surface lining all blood vessels' inner lumen. Composed of membrane glycoproteins, glycosaminoglycans (GAGs), such as hyaluronan and heparan sulfate (HS), and proteoglycans, the ESG forms a bulky, gel-like layer serving critical functions in blood flow mechanotransduction, endothelial permeability maintenance, leukocyte adhesion, and inflammation control. ESG is known for sensing shear stress of blood flow and transducing the mechanical signal into nitric oxide (NO) production. NO is an essential signaling molecule to regulate vascular tone. To date, the molecular pathways of ESG-mediated mechanotransduction have not been completely clear. We utilize a custom-built AFM with fluorescence imaging capabilities to vertically stretch the ESG, which applies a pulling force to specific ESG components on the surface of a single live mouse brain endothelial cell (bEnd.3) and quantify the cell's NO production in real-time. NO production is quantified by the fluorescent dye DAF-FM. AFM probe is coated with monoclonal antibodies against HS or glypican-1 (a major core protein for HS) to exert pulling forces onto the HS or glypican-1 on bEnd.3. To ensure that DAF intensity is a signal of NO production, we treated the cells with L-NAME (an inhibitor of endothelial nitric oxide synthase). The DAF intensity was greatly reduced, which confirms that the DAF intensity is representing NO production. The AFM anti-glypican-1 coated probe showed an increase in DAF intensity, while the anti-HS probe is shown to have a slight rise in DAF intensity. Since HS may attach to other proteins besides glypican, the data suggest that glypican-1 is a more critical mechanotransducer. Our prior study indicated cation-permeable, transient receptor potential (TRP) ion channels mediate stretch-induced NO production. Probes were coated in glypican-1 while the cells were exposed to Amiloride or SKF96365, blockers of TRPP2 and TRPC1 channels, respectively. After the Amiloride-treated cells were mechanically stimulated, there was a reduction of NO production. However, the amount of NO output was still significant over the basal levels observed in cells. When the SKF96365-treated cells were mechanically stimulated, production was inhibited entirely, thus, demonstrating TRPC1 channels play a vital role in allowing the Ca into the cell to begin NO production cascade. These findings confirm the critical roles of HS, glypican-1, and TRP channels on the rapid NO production resulting from mechanical signaling and have important implications for ESG-related cardiovascular diseases such as diabetes, strokes, cardiac arrest, and atherosclerosis. It has been shown that intracellular Ca regulates eNOS activity. In future studies, we will measure the Ca intake mediated by TRP channels and delineate the interplay among mechanical stretch, TRP channel activation, Ca intake and NO production.