Ozanimod Protects Against Hypoxia plus Glucose Deprivation‐Induced Morphological Changes and Improves Cell Health in Human Brain Vascular Smooth Muscle

Abstract

Vascular smooth muscle cells regulate arterial contractile tone, blood pressure, and flow in relation to specific metabolic demands during normal and pathophysiological (i.e. hypoxic or ischemic) conditions. The plasticity of vascular smooth muscle cells allows for phenotypic transitions from contractile to synthetic‐like dedifferentiated states during pathophysiological conditions which then can lead to the development and progression of vascular inflammation and blood vessel wall remodeling. Our previous studies demonstrated that hypoxia plus glucose deprivation (HGD), an in vitro model of ischemia, alters human vascular cell morphology and increases cell death. We further demonstrated that the selective sphingosine‐1‐phosphate receptor 1 (S1PR1) ligand, ozanimod, attenuated these HGD‐mediated responses. In this study, we characterized HGD mediated phenotypic transitions and assessed expression of proteins involved in contraction (myosin light chain kinase, MLCK) and structure formation (alpha‐smooth muscle actin, vimentin) following HGD exposure. We further investigated the effect of ozanimod HGD‐mediated phenotypic transitions by assessing protein levels and localization LC3B as an indicator of autophagocytosis. Using immunocytochemistry and standard immunoblotting, we determined the localization and expression levels of α‐smooth muscle actin, MLCK, vimentin, and LC3B protein during normoxia (21% O2, control) or HGD (1%O2) for 9 h in male human brain vascular smooth muscle cells (HBVSMC) at passage 6–7. We observed that LC3B protein, a vacuole marker associated with autophagy, is ubiquitously expressed in the cytoplasm of HBVSMC during normoxia. During HGD exposure, LC3B protein levels were increased and immunohistochemical staining demonstrated LC3B localization in what appears to be autophagic vacuole formation. Both responses were attenuated by ozanimod. Although alpha‐smooth muscle actin in HBVSMC was effective in visualization of morphological changes from spindle (contractile) to rhomboid (synthetic) shape following HGD, basal protein expression levels were not altered. Similarly, MLCK, an enzyme responsible for contractile protein phosphorylation, was not altered following HGD. Levels of the synthetic phenotype marker, vimentin, was basally expressed in HBVSMC, however, levels were not increased following HGD. In conclusion, although vacuole formation is prominent, indicating poor health, HBVSMCs retained a contractile phenotype during ischemic‐like conditions. Attenuation of HGD‐mediated LC3B via ozanimod suggests a novel mechanism associated with the beneficial effect of this selective S1PR1 ligand during ischemic‐like exposure.Support or Funding InformationAmerican Heart Association 19AIREA34480018 (RJG).