Abstract
The loss of vascular integrity at the level of the blood brain barrier leads to a vicious cascade of secondary injuries following acute ischemic stroke (AIS). Elevated MMP-9 activity within the cerebral vasculature has been implicated with severe degradation of the vascular basement membrane leading to abnormal cerebrovascular permeability and detrimental stroke outcome. Sphingosine-1-phosphate receptor (S1PR) modulation improves stroke outcome in AIS patients, however the influence of selective S1PR1ligands, such as ozanimod, on brain endothelial health and MMP-9 activity following AIS has not been investigated. Thus, the aim of this study was to determine the impact of acute ischemic injury on MMP-9 activity in both the rat and the human cerebrovasculature as well as the vascular specific role of ozanimod on human endothelial health and MMP-9 activity. Using an in vivo thromboembolic stroke model, cerebral vessels were isolated from male Wistar rats that underwent a right middle cerebral artery (MCA) thrombin injection. Sham-operated animals underwent the same surgical procedure; however, nothing was injected. Vascular MMP-9 enzymatic activity of was evaluated at 3, 6 and 24h post injury using zymography. Using an in vitro ischemic injury model (HGD; hypoxia plus glucose deprivation), male human brain microvascular endothelial cells (HBMECs; P7) were treated with ozanimod (0.5nM) or vehicle (DMSO) and then exposed to normoxia (21% O2) or HGD (1% O2). In some experiments, W146 (a selective antagonist), verified S1PR1 dependence. Morphology and vacuole formations were assessed using crystal violet staining. Enzymatic activity of MMP-9 was evaluated via zymography and the extracellular H2O2 concentration, inducer of MMP-9 activity, was measured using a colorimetric assay. Following in vivo thromboembolic occlusion, ipsilateral MMP-9 activity increased at 6h post injury and returned to baseline when compared to sham. In comparison to the contralateral side, thromboembolic occlusion induced a time dependent modulation of ipsilateral MMP-9 activity compared to the contralateral side with the highest activity peaking at 6h. In our in vitro HBMEC stroke model, MMP-9 enzymatic activity was increased following 3h HGD exposure, and this response was attenuated by ozanimod. Concomitantly, during HGD, H2O2 production, a partial inducer of MMP-9 activity, was increased in a time dependent manner when compared to normoxic controls. Furthermore, HGD induced an increase in HBMEC vacuole formations and a decrease in cell viability at 3h. Loss of cell viability was rescued with ozanimod. In conclusion, increased MMP-9 activity, in part due to H2O2, is an acute response to ischemic injury in the cerebrovasculature. Specifically, ozanimod's ability to attenuate endothelial MMP-9 activity may play an important beneficial role in mitigating blood brain barrier integrity loss following an acute ischemic injury.
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