Manipulating cerebral endothelial cell autophagy impacts outcomes of acute ischemic stroke in mice

Outcomes of acute ischemic stroke (AIS) are worsened by aging, but precise mechanisms are unclear. Earlier we reported that autophagic flux in non-cerebral endothelial cells (ECs) is blunted in mice and humans. EC autophagy is an intrinsic cytoprotective process that is heightened in response to nutrient-stress, and the accrual of damaged or dysregulated intracellular organelles, both of which occur in response to a sudden reduction in cerebral perfusion. Here we tested the hypothesis that repressed cerebral EC autophagy contributes to worse AIS outcomes in older vs. adult mice. Adult (~4-mo) and older (~24-mo) male mice completed 60-min transient middle cerebral artery occlusion (tMCAO) + 23-h reperfusion (R) to evoke AIS. Ipsilesional reperfusion blood flow measured via laser speckle contrast imaging (LSCI), neurobehavioral (Bederson test) and motor (Grip test) performance, and neuronal viability (NeuN staining) were worse (p< 0.05) in older vs. adult mice (n=10 per group), substantiating results from others. In additional cohorts that completed tMCAO+R we isolated cerebral ECs from ipsilesional hemispheres and confirmed their purity via immunoblotting i.e., robust CD31 yet sparse α-SMA, NeuN in the EC-enriched fraction and reciprocal results from the non-EC fraction. Indexes of autophagy assessed via immunoblotting (e.g., LC3-II/GAPDH) and immunofluorescence (e.g., Atg3:CD31 colocalization) were lower (p< 0.05) in cerebral EC fractions isolated from older vs. adult mice (n=3 per group). Next, we reasoned that if repressed cerebral EC autophagy is responsible for worse stroke outcomes in older mice, then adult male mice with inducible depletion of cerebral autophagy related gene 3 (Atg3EC-/-) should be more vulnerable to AIS. In support, AIS -induced reductions in reperfusion blood flow, tissue injury, neurobehavioral and motor performance, and neuronal death were more severe in Atg3EC-/- vs. wild-type (WT) mice (p< 0.05, n=10 per group). Underscoring the important role for cerebral EC autophagy, adult male Atg3EC-/- mice were refractory to benefits (p< 0.05) afforded by mTORC1 inhibition using rapamycin, observed in WT mice concerning AIS outcomes (n=5 per group). Because results to this point indicate defective cerebral EC autophagy contributes to worse AIS outcomes in older mice, we reasoned that boosting cerebral EC autophagy in older mice might be protective. Older male mice received a cerebral EC-specific adeno-associated viral vector (AAV-BR1) to deliver Atg3 or Null virus. In one cohort we verified increased (p< 0.05) Atg3 gene expression in cerebral EC but not non-EC -enriched fractions from mice that received AAV-BR1-Atg3 vs. Null virus (n=3 per group). A second cohort of older male mice completed tMCAO+R 21-days after receiving the respective virus. Ipsilesional reperfusion blood flow was greater, neuronal viability heightened, and infarct volume and neurobehavioral and motor deficits were less severe (p< 0.05) in older mice that received AAV-BR1-Atg3 vs. Null virus. (n=10 per group). Collectively, these results support that cerebral EC autophagy confers intrinsic cytoprotection in the context of AIS and should be considered further as a therapeutic target for treating AIS. Funding: NIH R21AG081780-01A1 to JDS and RC, AHA23PRE1025910 and University of Utah Graduate Research Fellowship to SM This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.