In Vitro Acute Ischemic Injury Altered Human Brain Endothelial MMP‐9 Activity and Associated Cell Surface Adhesion Protein Expression in Part by S1PR Type 1 Activation

Abstract

Cerebrovascular endothelial cells are among the first cells to be impacted by acute ischemic stroke (AIS) which poses them as important therapeutic targets pivotal to the maintenance of blood brain barrier (BBB) integrity. Following AIS, matrix metalloproteinase 9 (MMP-9) proteolytic activity contributes to the loss in BBB integrity, increased hemorrhagic risk, and worse outcomes. We have previously shown that sphingosine-1-phosphate receptor (S1PR) ligands attenuated hypoxia plus glucose deprivation (HGD)-induced decreases in brain microvascular endothelial trans-endothelial resistance (TEER), suggesting a potential role for S1PR ligands on barrier function during AIS. Endothelial vascular cell adhesion molecule 1 (VCAM-1) is an inflammatory mediator and adhesion molecule that contributes to the loss of endothelial barrier integrity by serving as a scaffold for immune cell extravasation as well as a mediator for intracellular signals involved in MMP-9 activation. Cell surface adhesion receptor, CD44, likewise plays a key role in facilitating MMP-9 activity via co-localization at the endothelial surface. Thusly, we sought to investigate protein expression levels of VCAM-1, MMP-9, tight junctional proteins, (ZO-1 and claudin-5), as well as to characterize CD44 protein expression in cerebrovascular endothelial cells following HGD. Pediatric male human brain microvascular endothelial cells (HBMECs) were treated with ozanimod (selective S1PR type 1 ligand; 0.5 nM) at P7 and immediately exposed to normoxia (21% O2) or HGD (1% O2) for 3h, a timepoint determined from our previous TEER studies which revealed deterioration of the endothelial barrier as early as 3h. To verify S1PR type 1 dependency, selective blockade with antagonist W146 was applied to select experiments. We observed through standard immunoblotting that HGD induced a simultaneous decrease in ZO-1 and claudin-5 in parallel to increased expression of VCAM-1. Additionally, intracellular pro-MMP-9 (inactive enzymatic form) levels decreased following HGD and the ratio of active MMP-9 to pro-MMP-9 was increased. Ozanimod did not alter these responses. Using immunocytochemistry, anti-CD44 fluorescence was increased following HGD exposure, and ozanimod attenuated this response. S1PR type 1 blockade demonstrated receptor dependence. In conclusion, HGD induced endothelial activation characterized by increased adhesion molecule levels and involvement of MMP-9 activity potentially contributing to tight junction protein loss. Furthermore, S1PR type 1 ligands may in part attenuate HGD-induced BBB integrity loss by modulating the bioavailability of MMP-9 to disrupt endothelial barrier integrity. Mechanism(s) by which S1PR type 1 ligands regulate MMP-9 activity and its associated cell surface adhesion proteins (e.g. CD44 and VCAM-1) following ischemic-like injury warrants further investigation.