S1PR1 VSM paper 2020
The following data are included in a study we published in 2021 study. We investigated the effect of a selective S1PR type 1 ligand, ozanimod, on VSM phenotype expression and autophagy in primary human brain VSM cells following acute hypoxia plus glucose deprivation (HGD; in vitro ischemic-like injury). Cells were treated with ozanimod and exposed to normoxia or HGD. Crystal violet staining, standard immunoblotting, and immunocytochemical labeling techniques assessed cellular morphology, vacuolization, phenotype, and autophagic state. We observed that HGD temporally decreased VSM cell viability and concomitantly increased vacuolization, both of which ozanimod reversed. HGD induced a simultaneous elevation and reduction in levels of pro- and anti-autophagic proteins respectfully, and ozanimod attenuated this response. Protein levels of VSM phenotypic biomarkers, smoothelin and SM22, were decreased following HGD. Furthermore, we observed an HGD-induced epithelioid and synthetic morphological appearance accompanied by disorganized cytoskeletal filaments which was rescued by ozanimod. Thus, we conclude that ozanimod, a selective S1PR1 ligand, protects against acute HGD-induced phenotypic switching and promotes cell survival, in part, by attenuating HGD-induced autophagic flux thus improving vascular patency in response to acute ischemia-like injury. Citation: Wendt et al AJP Cell 2021
Steps to reproduce
Methodology Used: Brain vascular smooth muscle cell culture model: Vendor purchased cryopreserved subcultures of primary human brain VSM cells (HBVSMCs) (Cell Biologics, cat. no. H-6085, lot no. F11062014/17) of adult male origin were received at passage 3 and seeded in house at a density of 4 to 5×105/cm2. Western blot: Protein levels for VSM phenotype as well as for structural and autophagic markers were examined following normoxic or HGD exposure using standard western blotting described previously ( Am J Physiol Cell Physiol 314: C545-C553, 2018.) Polymerase Chain Reaction: RT-PCR was used to determine Acta2 and Edg1 gene expression to validate smooth muscle origin and presence of S1PR1 respectively as well as GAPDH, which served as a loading control marker. Briefly, RNA was extracted using the Qiagen kit (ThermoFisher, cat. no. 74034) according to the manufacturer’s instructions. Live Cell Count: Using an Olympus CKX41 inverted light microscope, total and live cell counts were obtained by a blinded analyzer. Crystal Violet Staining: HBVSMC structure and morphology were visualized using crystal violet staining. In brief, cells were seeded onto coverslips coated with attachment factor (Cell Systems; cat. no. 4Z0-201) within 6-well plates at passages 6-7, grown to confluency. ImageJ analysis of vacuole formations and immunocytochemical images: An enhanced version of ImageJ2, Fiji, was utilized to perform analysis on number and area of vacuole per cell using images from the crystal violet and immunocytochemical experiments. Reagents: All reagents were purchased from Sigma-Aldrich Corporation (St. Louis, MO) unless otherwise noted.
American Heart Association
19AIREA34480018 UA 4241520