Brain endothelial cell barrier function is compromised by autophagy depletion

Abstract

Age‐associated cerebral hypoperfusion and heightened blood brain barrier (BBB) permeability contribute importantly to vascular cognitive impairment. Earlier we reported that mRNA and protein expression of autophagy markers is repressed (p<0.05) in endothelial cells (ECs) from older (24‐month) mice and older (> 68 y) male subjects that exhibit concurrent peripheral arterial dysfunction. Of note, adult mice with inducible EC specific depletion of autophagy related gene 3 (Atg3) display endothelium‐dependent dysfunction (p<0.05) of femoral and middle cerebral arteries, whereas smooth muscle function is intact in both vascular beds. While these findings indicate repressed EC autophagy associates strongly with arterial dysfunction, the impact of EC autophagy on BBB permeability is unknown. Because EC autophagy repression mimics an aging phenotype, we hypothesized that BBB permeability is heightened in mice with EC specific Atg3 depletion (Atg3EC‐/‐) vs. wild type littermates (WT). In support of this, Evans blue dye was more abundant (p<0.05) in brains from Atg3EC‐/‐ vs. WT littermates (n=6 per group). Further, fluorescence assisted cell sorting results indicated a greater number (p<0.05) of white blood cells, microglia, and neutrophils in brains from Atg3EC‐/‐ vs. WT mice (n=4 per group). Using a reductionist approach we tested whether human brain microvascular ECs (HBMVECs) with autophagy depletion exhibit heightened permeability. Results from electric cell‐substrate impedance sensing procedures indicate that permeability was greater (p<0.05) in HBMVECs after : (i) IL‐1β (positive control) vs. vehicle‐treatment; (ii) 3‐methyladenine (autophagy initiation inhibitor) vs. vehicle‐treatment; and (iii) CRISPR‐cas9 mediated Atg3 knock‐down (Atg3 KD) vs. transfection with negative sgRNA (control) (p<0.05, 24 wells per treatment x three repetitions). In addition, Atg3 KD HBMVECs displayed reduced protein expression of: (i) Atg3; (ii) zonula occludens‐1; (iii) platelet endothelial cell adhesion molecule‐1; (iv) vascular endothelial cadherin; and (v) glucose transporter 1 (p<0.05, n=6). These data provide evidence that Atg3 depletion jeopardizes BBB integrity.