Inhibition Of Scramblase-1 Attenuates Endothelial Fluid Loss During Ischemia Reperfusion Injury

Abstract

Introduction: Scramblase 1(PLSCR-1) is perceived to trigger apoptosis in damaged cells by translocating phosphatidylserine (PS) from the inner leaflet to the outer leaflet of the lipid bilayer. The exposed PS is thought to target damaged cells for apoptosis. Though originally described in red blood cells, the existence of this system in endothelial cells is unknown. We hypothesized that PLSCR-1 plays a critical role during ischemia-reperfusion injury (IRI) by increasing endothelial cell surface PS leading to apoptosis and contributing to increased microvascular permeability. Our specific aims were: 1) to document inducible PS exposure on endothelial cell membranes, 2) to evaluate the impact of PLSCR-1 inhibition on microvascular permeability during IRI, and 3) to confirm protein knockdown of PLSCR-1 by RNA interference via Western Blot. Methods: Cultured bovine pulmonary artery endothelial cells (BPAECs) were exposed to calcium ionophore (A23187) to induce translocation of PS. PS exposure was assessed on BPAEC membranes by flow cytometry using the PS-binding molecule, Alexa 488 Di-annexin. Next, mesenteric venular microvascular permeability (Lp) during IRI was examined using an intra-vital micro-occlusion technique in rats. Prior to IRI, rats were pretreated with: 1) Dithioerythritol (DTE) an inhibitor of the cysteine residues essential for PLSCR-1 translocation (n=3), 2) 4,4'-diisothiocyano-stilbene-2,2'-disulfonic acid (DIDS), an inhibitor of the anion exchange mechanism necessary for PLSCR-1 function (n=3), 3) 2-bromopalmitate, an inhibitor of PLSCR-1 trafficking to the plasma membrane which renders PLSCR-1 nonfunctional (n=3), and 4) PLSCR-1 specific siRNA (n=3). Lp was measured throughout IRI for each group. PLSCR-1 knockdown was confirmed by Western blot (n=3). Results: Treatment of BPAECs with calcium ionophore increased Di-annexin binding in treated cells (N = 3) compared to controls (N = 3) (mean absorbance controls vs. treated = 109.4±42.1nm vs. 2765.5±848.5nm, p=0.04). In vivo, IRI increased Lp approximately 7-fold. This increase was attenuated 40% (p<0.01), 37% (p<0.01), and 55% (p< 0.01) by DTE, DIDS, and 2-BP respectively. The attenuation due to scramblase siRNA was most robust and attenuated Lp 70% during IRI (p<0.01). Western blots confirmed knockdown of PLSCR-1 as measured by mean peak band optical intensity units (OIU): PLSCR-1 siRNA = 2600±170 OIU, dsDNA control = 3100±66 OIU, p=0.02). Conclusions: The data demonstrates that endothelial cells have the capacity to translocate PS to the cell surface. The data also suggests that both non-specific (DTE, DIDS, and 2-BP) and specific (siRNA) inhibition of PLSCR-1 attenuate increases in microvascular venular permeability observed during IRI. Methods of targeting PLSCR-1 function and resultant PS exposure may be of potential therapeutic value against the development of microvascular permeability observed during IRI.