We have previously demonstrated the significance of endothelial cell-expressed alpha 5 beta 1 integrin in ischemic stroke, having shown that alpha 5 beta 1 integrin endothelial cell-selective knockout mice are significantly resistance to ischemic stroke injury via preservation of the tight junction protein claudin-5 and subsequent stabilization of the blood-brain barrier (BBB). In addition, inhibition of alpha 5 beta 1 by the small peptide noncompetitive integrin alpha 5 inhibitor, ATN-161, is beneficial in a mouse model of ischemic stroke through reduction of infarct volume, edema, stabilization of the BBB, and reduced inflammation and immune cell infiltration into the brain. In continuation with our previous findings, we have further evaluated the mechanistic role of ATN-161 in vitro and found that oxygen and glucose deprivation and reperfusion (OGD/R)-induced inflammation, oxidative stress, apoptosis, mitochondrial depolarization, and fibrosis attenuate tight junction integrity via induction of alpha 5, NLRP3, p-FAK, and p-AKT signaling in mouse brain endothelial cells. ATN-161 treatment (10 mu M) effectively inhibited OGD/R-induced extracellular matrix (ECM) deposition by reducing integrin alpha 5, MMP-9, and fibronectin expression, as well as reducing oxidative stress by reducing mitochondrial superoxide radicals, intracellular ROS, inflammation by reducing NLRP3 inflammasome, tight junction loss by reducing claudin-5 and ZO-1 expression levels, mitochondrial damage by inhibiting mitochondrial depolarization, and apoptosis via regulation of p-FAK and p-AKT levels. Taken together, our results further support therapeutically targeting alpha 5 integrin with ATN-161, a safe, well-tolerated, and clinically validated peptide, in ischemic stroke.
