Selective S1PR 1 Ligand Attenuates Autophagy Mediated Human Cerebrovascular Smooth Muscle Cell Death Secondary To In Vitro Ischemic Injury

Abstract

Cerebrovascular pathologies contribute significantly to acute ischemic stroke (AIS) outcome. Sphingosine-1-phosphate receptor (S1PR) modulators show beneficial effects against hypoxia plus glucose deprivation (HGD; in vitro ischemic-like model)-induced brain microvascular endothelial dysfunction and demonstrate clinical promise in improving cognitive AIS outcome in patients. However, the impact of S1PR modulation on vascular smooth muscle (VSM) health during ischemic injury has not been clearly elucidated. Therefore, the aim of this study was to investigate human brain VSM cell viability and autophagic responses following HGD. Adult male brain VSM cells were treated with ozanimod (selective S1PR type 1 ligand; 0.5nM) at P7 and exposed to normoxia (21% O2) or HGD (1% O2) for 9h, a timepoint based on cell survival studies conducted using trypan blue exclusion staining following HGD 3, 6, and 9h. Crystal violet staining determined vacuole presence and autophagic protein levels were examined using standard immunoblotting. Localization of autophagic markers were assessed via immunocytochemical labeling. We observed that HGD temporally decreased VSM cell viability and concomitantly increased vacuolization, and these responses were reversed by ozanimod. To characterize whether these vacuoles were of autophagosome phenotype, localization and punctation quantification of the upstream ubiquitin-like complex I mediator, Atg5 and its associated downstream autophagic marker, LC3B, were assessed. Following HGD insult, increased density of Atg5 and LC3B punctate clusters were visualized and expression levels were increased. In the presence of ozanimod, HGD-induced LC3B levels were attenuated suggesting that S1PR1modulation may regulate HGD-induced vacuole formation during autophagic flux. Concomitantly, basal levels of the anti-apoptotic and anti-autophagic protein Bcl-2 were significantly decreased following HGD, and ozanimod reversed this response. Further investigation revealed that while HGD did not alter LC3A/B-I (non-autophagic) levels, HGD-induced LC3A/B-II (autophagic) levels were increased but were attenuated by ozanimod. Autophagic protein levels of beclin-1 and Atg7 were not altered following HGD. However, in the presence of HGD plus ozanimod, we observed a decrease in Atg7 levels suggesting that ozanimod may have a protective effect against autophagy during HGD. While we did not observe alterations in beclin-1 protein levels across treatment groups, when combined with the decreased Bcl-2 levels, the availability of beclin-1 to mediate initiation of autophagy is enhanced following HGD via suppression of Bcl-2 inhibition. The rescue of Bcl-2 suggests that ozanimod may be suppressing the ability of beclin-1 to mediate initiation of autophagy following HGD. In conclusion, S1PRtype 1 ligands promote cell survival in part by attenuating HGD-induced autophagic flux in human brain VSM.