(193p) Engineering the Endothelial Glycocalyx to Restore Its Structure and Function

The glycocalyx (GCX) coats and protects the vascular endothelial cell (EC) layer and mediates EC function [1, 2, 3]. However, the specific pathways by which GCX regulates EC function remain to be clarified. Studies suggest that the EC GCX transmits signals, via the cytoskeleton, to cell-to-cell junctions where biological responses occur [1, 2]. Of the cell-to-cell junctions, gap junctions are potentially an important downstream player in GCX-mediated signaling, because gap junctions transport ions and signaling molecules which regulate numerous vessel functions [4, 5, 6] that rely on the GCX. The goal of our current research is to test the hypotheses that (i) GCX controls gap junction protein expression and gap junction coupling of neighboring ECs, and (ii) repair of damaged GCX can reverse dysfunctional EC communication. We studied EC with intact GCX, heparinase III enzyme degraded GCX (to disrupt GCX HS, the most abundant GCX component), or regenerated GCX (by replacing and stabilizing GCX HS). Inter-endothelial gap junction communication was evaluated using immunofluorescence confocal microscopy to examine gap junction protein (connexin43) expression and localization and by quantifying Lucifer yellow dye spread via gap junctions in EC monolayers. The results demonstrated gap junction communication dependence on GCX function. In addition, GCX repair by treating cells with exogenous HS and a co-factor restored gap junction protein placement which translated to the reactivation of gap junction channel activity. The results of this study are encouraging and will lead to future development of new therapies targeted at the GCX to address endothelial-dependent aspects of vascular disease. References: [1] Tarbell and Ebong, Sci Signal, 2008; [2] Thi et al., P Natl Acad Sci USA, 2004; [3] Yao et al., Am J Physiol Heart Circ Physiol, 2007; [4] Segal and Beny, Am J Physiol, 1992; [5] Figueroa and Duling, Antioxid Redox Signal, 2009; and [6] Ebong et al., Pflugers Arch, 2013.