Disruption Of Dll4/Notch Signaling In The Post Stroke Period Results In Enhanced Angiogenesis And Improved Outcome Following Stroke

Delta-like 4 (DLL4) is an endothelial transmembrane ligand for the Notch family receptors. Dll4 contributes to vascular development and angiogenesis, specifically as a negative regulator of capillary sprouting. We hypothesized endothelial-specific deletion of Dll4 or inhibition of Notch receptor activity in the post-stroke brain would result in enhanced reparative angiogenesis and improved functional outcome following permanent stroke in young and aged mice. Methods: We used the permanent distal middle cerebral artery occlusion (dMCAO) model to induce primary injury in the cortex with delayed secondary injury in the thalamus. DLL4 protein expression (IHC) and Notch receptor activity ( in vivo biosensor, CBF::H2B-Venus ) were determined through 2 weeks post-stroke. Tamoxifen-inducible, endothelial-specific Dll4 KO mice ( Dll4 iecKO ) and Dll4 fl/fl (“floxed-only” control) female mice (3 mos) were treated with tamoxifen (n=4 each) at 24 and 48 hrs post-stroke. In a separate cohort, young (3 mos, n=5 each) and aged (20 mos, n=3-4) male mice were treated with DAPT (Y-secretase inhibitor; 100 mg/kg) at 24 hours post-stroke to inhibit Notch activity. Functional angiogenesis was assessed histologically following in vivo perfusion of fluorescent lectin. Behavioral tests (foot fault) were conducted at post-stroke days 1, 3, 7, and 14. Results: Elevated Dll4 expression and Notch activation were observed in peri-infarct cortex (piCtx) and ipsilateral thalamus (iTh) by 24 hours after dMCAO. Post-stroke endothelial Dll4 deletion led to increased perfusion-competent microvessels (e.g. vessel area, length, etc.) at 2 weeks post-stroke in piCtx and iTh. Post-stroke DAPT treatment trended toward increased total microvascular length in piCtx (P=0.051 young/P=0.126 aged), but not iTh. Importantly, both treatment methods demonstrated significant improvement in neurological function at 7 and 14 days post-stroke. Conclusions: Deletion of endothelial Dll4 at 24 hours post-stroke leads to increased angiogenesis and improved functional recovery. Notch inhibition demonstrated improved outcome (young and aged), but increased angiogenesis was only suggestive in the cortex. DAPT treatment appears to have translational value as a delayed treatment option.