Ozanimod Attenuates Human Cerebrovascular Endothelial Derived MMP-9 Activity and Preserves Barrier Properties Following In Vitro Acute Ischemic Injury

Abstract

Endothelial integrity is critical in mitigating a vicious cascade of secondary injuries following acute ischemic stroke (AIS). Matrix metalloproteinase-9 (MMP-9), a contributor to endothelial integrity loss, is elevated during stroke and is associated with worsened stroke outcome. We investigated the FDA approved selective sphingosine-1-phosphate receptor 1 (S1PR1) ligand, ozanimod, on the regulation/activity of MMP-9 as well as endothelial barrier components (PECAM-1, claudin-5, and ZO-1) in human brain microvascular endothelial cells (HBMECs) following hypoxia plus glucose deprivation (HGD). We previously reported that S1PR1 activation improves HBMEC integrity; however, specific mechanisms underlying S1PR1 involvement in barrier integrity have not been clearly elucidated. We hypothesized that ozanimod would attenuate an HGD-induced increase in MMP-9 activity which would concomitantly attenuate the loss of integral barrier components. Male HBMECs were treated with ozanimod (0.5nM) or vehicle and exposed to 3h normoxia (21% O2) or HGD (1% O2). Immunoblotting, zymography, qRT-PCR, and immunocytochemical labeling techniques assessed processes related to MMP-9 and barrier markers. We observed that HGD acutely increased MMP-9 activity and reduced claudin-5 and PECAM-1 levels, and ozanimod attenuated these responses. In situ analysis via PROSPER, suggested that attenuation of MMP-9 activity may be a primary factor in maintaining these integral barrier proteins. We also observed that HGD increased intracellular mechanisms associated with augmented MMP-9 activation, however ozanimod had no effect on these targeted factors. Thus, we conclude that ozanimod has the potential to attenuate HGD mediated decreases in HBMEC integrity in part by decreasing MMP-9 activity as well as preserving barrier properties. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/526738v2_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@b4df43org.highwire.dtl.DTLVardef@addf76org.highwire.dtl.DTLVardef@129e86forg.highwire.dtl.DTLVardef@5ae8a0_HPS_FORMAT_FIGEXP M_FIG C_FIG