Endothelial Glycocalyx‐Mediated Nitric Oxide Production in Response to Selective AFM Pulling

The inhibition of nitric oxide (NO) production due to endothelial damage contributes to cardiovascular disease. The glycocalyx, a thin layer of glycolipids, glycoproteins, and proteoglycans on the surface of ECs, senses extracellular stimuli and initiates subsequent intracellular signaling pathways. Previous studies from our laboratory highlighted the glycocalyx as a significant player in NO production; degradation of the endothelial glycocalyx drastically reduced EC production of NO in response to fluid shear stress. However, the specific glycocalyx components involved in this process are not known. Recent work using shRNA approaches in vitro suggested that the proteoglycan glypican‐1, not syndecan‐1, is the dominant core protein mediating shear‐induced NO production (Ebong et al., Integr Biol 2014). In the present study we apply atomic force microscopy (AFM) combined with fluorescence microscopy to observe how components of the endothelial glycocalyx, including proteoglycans and glycosaminoglycans, contribute to the force‐induced production of NO. AFM tipless cantilevers are functionalized using antibodies for proteoglycans in the glycocalyx and their isotype controls. Rat fat pad ECs (RFPECs) are cultured to confluence and incubated with a cell‐permeable fluorescent molecule, DAF‐2, used to detect real time changes in NO concentration. Fluorescent microscopy is used to monitor DAF‐2 levels in response to applied tensile stress on specific glycocalyx components to determine those critical to initiating NO production. Strong surface binding of glypican‐1 and syndecan‐1 relative to their isotype controls has been observed (Fig1). These studies will allow us to determine the mechanosenor(s) for shear‐induced NO production. Supported by NIH Grant HL094889