Selective Sphingosine‐1‐Phosphate Receptor‐1 Ligand Protects Against Ischemia‐Induced Inflammation and Dysfunction in Human Brain Microvascular Endothelial Cells

Abstract

Cerebrovascular inflammation and endothelial cell dysfunction are key contributors to brain damage following acute ischemic stroke. In preclinical and clinical studies, sphingosine‐1‐phosphate receptor (S1PR) ligands are emerging as exciting therapeutic strategies to treat acute ischemic stroke. We previously observed that selective S1PR1 modulation reduced lesion volume following transient middle cerebral artery occlusion (tMCAO). However, S1PR1 expression and the influence of a selective S1PR1 ligand on brain endothelial health during ischemic injury has not been investigated. Therefore, the objective of this study was to determine human and murine brain‐derived endothelial cell S1PR1 expression profiles in response to in vitro ischemic‐like conditions or in vivo ischemia respectively. In adult C57BL/6blk male and female mice, S1PR1 expression was examined in cell suspensions from whole brain 24h post 60‐min tMCAO via flow cytometric (BD FACSARIA) analysis. Quantitative expression was determined in gated cell sub‐types using an anti‐mouse S1PR1/EDG‐1 conjugated fluorescent antibody. S1PR1 expression levels were also examined in primary male human brain microvascular endothelial cells (HBMEC) at passage 7 using standard immunoblotting. Additionally, the impact of RP101075 (novel selective S1PR1 ligand) on cyclooxygenase‐2 (COX‐2) levels was assessed following hypoxia plus glucose deprivation (HGD; 3h at 1% O2) or normoxia (21% O2). Brightfield microscopy was used to visualize HBMEC density and morphology, and proliferation was determined via bromodeoxyuridine and flow cytometric analysis. Basal levels of S1PR1 expression were detected in both human and murine brain‐derived endothelial cells. HGD and tMCAO resulted in increased endothelial S1PR1 expression levels. RP101075 partly preserved HGD‐induced morphological changes in HBMEC and elicited a trend in attenuating HGD‐induced HBMEC viability. Equally importantly, RP10175 attenuated HGD‐induced increases in COX‐2 levels, a recognized marker of ischemia‐induced vascular inflammation. In conclusion, selective S1PR1 ligands may serve as important beneficial mediators against ischemic damage in the brain endothelium through S1PR1 receptor upregulation and by attenuating brain endothelial cell inflammation and dysfunction.Support or Funding InformationUniversity of Arizona Valley Research Partnership Grant VRP37 P2 (RJG, FS).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.