Differential Expression Profile of Sphingosine‐1‐Phosphate Receptors in Human Brain Vascular Smooth Muscle and Endothelial Cells Following Hypoxia Plus Glucose Deprivation

Abstract

Sphingosine‐1‐phosphate receptors (S1PRs) and their signaling pathways play an important role in mediating vascular health and function. Upon ligand activation, S1PRs 1‐5 couple with diverse heterotrimeric G‐protein subunits (Gαi, Gαq/11, Gα12/13) initiating multimodal downstream signaling pathways which result in various physiological outcomes in the vasculature, including cell proliferation and migration, barrier integrity preservation or loss, contraction, and inflammation. Specifically, S1PR2 activation has been linked to endothelial activation, barrier integrity loss, and inflammation, whereas S1PR1 activation contributes to barrier integrity preservation, vasodilation, and anti‐inflammatory properties. Although the role of S1PRs during pathophysiological conditions such as acute ischemic stroke is under current investigation, S1PR expression in the brain vasculature following ischemic injury has not yet been investigated. Therefore, the present study characterized the expression profile of S1PRs 1‐5 in human brain microvascular endothelial cells (ECs) and brain vascular smooth muscle cells (VSMCs) following hypoxia plus glucose deprivation (HGD). Male ECs and VSMCs were exposed to either HGD (1% O2) or normoxia (21% O2) for 3h. Semi‐quantitative RT‐PCR was used to examine gene expression and concomitantly, protein levels were assessed with standard western blot and/or FIJI‐mediated ICC analysis. We hypothesized that ECs and VSMCs differentially express S1PRs 1‐5 both basally and during ischemia‐like conditions. At the mRNA level, S1PRs 1‐5 gene expression was observed in VSMCs with S1PR4 and 5 being just above the level of detection. S1PRs 1‐4 expression was detected in ECs with S1PR4 having the lowest expression. During normoxic conditions, we observed that S1PRs 1 and 3 mRNA levels were most prominently expressed in both the ECs and VSMCs. In ECs, HGD exposure did not alter the gene expression of S1PRs 1‐3. At the protein level, we observed that HGD increased S1PR1 levels, however S1PR2 and 3 protein levels were unaltered. In VSMCs, HGD increased both mRNA and protein levels of S1PR2 but did not alter S1PRs 1 and 3 mRNA expression and protein levels. In conclusion, acute exposure to HGD appears to differentially regulate S1PRs in VSMCs and ECs at the transcriptional and translational level, respectively. In VSMCs, HGD increased S1PR2 gene expression and protein levels, whereas in ECs, HGD increased S1PR1 protein levels. The differential expression in S1PRs both basally and following HGD exposure may suggest differential signaling mechanisms within the two brain vascular cell types. Furthermore, differential expression of S1PR1 and 2 highlight the potential beneficial and/or detrimental involvement of S1PRs in the pathophysiology of ischemic injury within the cerebrovasculature. Further investigation will be necessary to determine the signaling role of S1PRs and their impact on cerebrovascular cell function following acute ischemic injury in the brain.