Homocysteine-induced Barrier Dysfunction and Hyperpermeability in the Blood-Brain and Blood-Retinal Barrier: Role of NLRP3 Inflammasome Pathway

Prolonged exposure to microgravity during long- and short-duration spaceflight has been found to produce many pathophysiological changes. Of these, a set constellation of changes called “Spaceflight Associated Neuro-ocular Syndrome” (SANS) induces neuro-ophthalmic changes. Recent evidence suggests astronauts who experienced SANS generally had higher concentrations of one-carbon pathway intermediate homocysteine (Hcy) prior to spaceflight. Hcy is a known inducer of inflammatory mediators and vascular endothelial dysfunction, leading to blood-brain barrier (BBB) breakdown. Mechanisms behind this dysfunction/hyperpermeability are not clearly known, but recent studies show evidence for loss of BBB integrity following microgravity/simulated microgravity. Recent studies from our lab suggest that activation of the nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor 3 (NLRP3) inflammasome plays a role in mediating BBB dysfunction and related microvascular hyperpermeability. Our main objective was to understand the mechanism(s) by which Hcy could induce BBB dysfunction/hyperpermeability, and potential association with SANS. We hypothesized that Hcy-mediated dysfunction/BBB hyperpermeability occurs via NLRP3 inflammasome activation and subsequent proteolytic breakdown of tight junction proteins. Understanding this pathway is critical to devise protection against SANS at a microvascular level. To study this relationship, human brain microvascular endothelial cells (HBMECs) were treated with various concentrations of Hcy. The monolayer permeability was evaluated using Transwell permeability assays with fluorescence tracer FITC-dextran (10-kDa). Matrixmetalloproteinase (MMP-9) and calpain activities were measured fluorometrically. Hcy induced endothelial monolayer hyperpermeability dose-dependently (p<0.05). NLRP3 inhibitor MCC950 decreased Hcy-induced hyperpermeability (p<0.05). Hcy treatment showed no significant effect on cell viability at lower concentration but decreased cell viability at higher concentrations. MCC950 inhibited Hcy-induced MMP-9 and calpain 1 activity. These results suggest a possible mechanism of BBB dysfunction/hyperpermeability by Hcy via activation of the NLRP3 inflammasome pathway. The proteolytic enzymes MMP-9 and calpain 1 are potential downstream mediators of Hcy-induced BBB dysfunction/hyperpermeability. Seeing that the blood-retinal barrier (BRB) shares structural-functional similarity with the BBB, human retinal microvascular endothelial cells (HRMEC) were examined for comparisons of barrier permeability. The results suggest Hcy-induced hyperpermeability in HRMEC monolayers and decreased expression of tight junction protein zonula occludens-1. Expression and effects were attenuated by NLRP3 inhibition. Our results suggest that NLRP3 inflammasome signaling may play an important role in promoting Hcy-induced barrier dysfunction and hyperpermeability in the blood-brain and blood-retinal barriers. Morehouse School of Medicine, Grady Hospital Surgery Dept, NASA This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.