Abstract TMP31: A Novel Shift of Notch Ligand, Dll4, Expression From Endothelial to Glial Cells in the Post-Stroke Brain May Contribute to Reparative Angiogenesis and Improved Functional Outcome

Abstract

Delta-like 4 (Dll4) is a Notch receptor ligand classically thought to be expressed exclusively in endothelial cells. However, little is known about the cellular expression or functional role of Dll4 following ischemic stroke. Here, we present novel findings that Dll4 expression is shifted from endothelial cells to glial cells in the sub-acute stroke phase. Method: The permanent distal middle cerebral artery occlusion (dMCAO) model was performed to induce stroke. To investigate expression patterns of Dll4 protein and mRNA, immunostaining as well as single-cell RNA sequencing and single-cell fluorescent in situ hybridization were performed. To verify the role of endothelial Dll4 , tamoxifen-inducible, endothelial-specific Dll4 KO mice (Dll4iecKO) and Dll4 fl/fl (“floxed-only”) female mice (3 mos) were injected with tamoxifen (n=4) at 24 and 48 hrs post-stroke. To determine the effect of Notch inhibition at different time points, DAPT (100 mg/kg) or vehicle was administered at 24 or 72 hours post-stroke (n=4-5). Behavioral tests (foot fault) were conducted at Post Stroke Day (PSD) 1, 3, 7, and 14 and functional angiogenesis was assessed at PSD 14. Results: Elevated expression of Dll4 protein was observed in the microvasculature of the peri-infarct cortex (piCtx) as early as PSD 1. By PSD 3, however, we discovered Dll4 expression in microglia within the piCtx, with robust expression by PSD 14. Post-stroke deletion of endothelial Dll4 resulted in significant improvement in neurological function and increased capillary density in piCtx[l1] . DAPT given at 24 hours post-stroke improved outcome and capillary density; however, DAPT given at 72 hours (when microglia Dll4 expression is evident) appeared to worsen outcome. Conclusions: After stroke, expression of Dll4 shifts from exclusively endothelial (PSD 1) to mixed endothelial/microglial (PSD 3) to primarily microglial (PSD 14). Notch inhibition beginning at 24 hours post-stroke or endothelial-only deletion of Dll4 leads to increased angiogenesis and improved functional recovery. In contrast, Notch inhibition initiated at 72 hours post-stroke leads to worse outcome[l2] . These findings suggest a potential novel role of microglia in supporting reparative angiogenesis through Dll4/Notch signaling.