Abstract TP120: Inhibition of α5β1 Integrin With the Small Peptide ATN-161 Reduces Inflammation, Stabilizes the Blood-Brain Barrier, and is Neuroprotective After Experimental Ischemic Stroke

Abstract

Endothelial cell integrin receptors, specifically the β 1 subtype, play a direct role in inflammation and blood-brain barrier (BBB) dysfunction, both implicated in poor ischemic stroke outcomes. We hypothesize that inhibition of a particular β1 integrin subtype, α5β1, (a pro-angiogenic fibronectin receptor acutely expressed after ischemic stroke) could be therapeutic in ischemic stroke via BBB stabilization. Methods: 3-month-old male, C57BL/J6 wildtype mice underwent transient middle cerebral artery occlusion for 1 hour. Mice were treated intraperitoneally with the small peptide α5β1 integrin inhibitor ATN-161 (1 mg/kg) upon reperfusion, post stroke day (PSD) 1 and PSD2. On PSD3, infarct volume (TTC and T2-weighted MRI) and vasogenic edema (MRI) were assessed. Neuroscore, an 11-point functional assessment, was performed on PSD1-14. Expression of MMP-9, claudin-5 and IL-1β levels were determined by qPCR. On PSD3, immunohistochemical analyses of α5 integrin, collagen IV, claudin-5, CD45, and DAPI expression were performed. In vitro oxygen-glucose deprivation (OGD) experiments on brain endothelial (bEND3) cells were also performed, evaluating ATN-161 administration (10 μM) effects on BEND3 layer permeability via FITC-dextran migration, and on immunocytochemistry of α5 integrin and claudin-5 expression. Results: Acute post-stroke administration of ATN-161 significantly decreased infarct volume and vasogenic edema, and improved functional recovery. Additionally, ATN-161 appeared to stabilize the BBB by increasing expression of the tight junction protein claudin-5, while decreasing expression of the extracellular matrix proteinase, MMP-9, inflammatory cytokine (IL-1β), and infiltrating leukocytes (CD45). Additionally, permeability was conserved with ATN-161 administration following OGD. Conclusion: Administration of the α5β1 inhibitor ATN-161 stabilizes the BBB barrier, and reduces inflammatory infiltration, while reducing infarct volume, vasogenic edema, and behavioral dysfunction following experimental stroke. Collectively, these results suggest inhibition of α5β1 via ATN-161 could represent a promising novel therapeutic approach for ischemic stroke.