(636h) Flow-Induced Interendothelial Communication & the Glycocalyx

Vascular endothelial cells (EC) are the first defense against atherosclerosis. EC are healthy and lesions are rare at vascular sites where blood flow is uniform [Chiu and Chien, Physiol Rev, 2011]. EC dysfunction and plaque formation coincide with vascular sites where blood flow is altered (disturbed) [Chiu and Chien, Physiol Rev, 2011]. The mechanisms of blood flow regulation of EC function and atherogenesis are unclear. Studies have implicated the EC surface sugar coat—the glycocalyx (GCX), which is shed in atherosclerosis—as a prime candidate mechanotransducer that transmits flow derived forces, via the cytoskeleton, to cell-to-cell junctions where biological responses occur [Thi et al, Proc Acad Sci USA, 2004; Tarbell and Ebong, Sci Signal, 2008]. Of the cell-to-cell junctions that connect endothelial cells, gap junctions transport <1-1.5 kDa ions and signaling molecules that are involved in vascular tone and other vessel functions [Segal and Beny, Am J Physiol, 1992; Figueroa and Duling, Antioxid Redox Signal, 2009]. In addition, gap junctions are notably altered in atherosclerosis [Pfenniger et al, Biochim Biophys Acta, 2013]. Here, we test the hypothesis that GCX mediates flow-regulated EC gap junction function. To test our hypothesis, we first verify the presence of an intact GCX on rat fat pad EC (RFPEC) and human aortic EC (HAEC), by immunolabeling the well-integrated bovine serum albumin (BSA) component of the GCX. We also confirm immunocytochemically that these cells express heparan sulfate (HS) as part of their GCX. RFPEC and HAEC with intact GCX or heparinase III enzyme degraded GCX (to disrupt GCX HS) are exposed to physiological flows generated in a parallel-plate flow chamber. Flow-induced and GCX mediated inter-endothelial communication via gap junctions is evaluated by: (i) using immunofluorescent confocal microscopy to examine gap junction protein expression and localization and (ii) quantifying Lucifer yellow dye spread via gap junctions in monolayers of gap junction-coupled EC. Our preliminary results confirm that our EC are well covered with GCX, as indicated by BSA staining, and demonstrate that this GCX contains abundant HS. We are successfully degrading the HS component enzymatically, and we have observed that 15 mU/ml of heparinase III degrades almost 54% of heparan sulfate. The effect of the intact or degraded HS on flow-induced gap junctional communication is still under investigation. We look forward to elucidating the glycocalyx mediated mechanisms involved in EC conversion of fluid stress into gap junctional activity. Our findings promise to inform the development of new anti-atherogenic approaches.