The Contribution of Hyperhomocysteinemia to Aβ‐Induced Endothelial Cell Apoptosis, Blood Brain Barrier Dysfunction, and Angiogenic Deficits

Abstract

AbstractBackgroundCerebrovascular dysfunction has been implicated as a major contributor to Alzheimer’s Disease (AD) pathology, with particularly endothelial cell (EC) stress promoting the focal ischemia, cerebral blood flow impairments, and blood brain barrier (BBB) permeability that are pathologically characteristic in AD. Recent evidence has emerged suggesting a link between cardiovascular (CV) diseases and AD pathology, particularly showing that CV/cerebrovascular risk factors, including hyperhomocysteinemia (Hhcy), contribute to AD pathology and risk. Despite this, the underlying molecular mechanisms for this interaction remain unclear. Previously, our lab has demonstrated that amyloid beta (Aβ), particularly Aβ40‐Q22 (the vasculotropic Dutch mutant), promotes TRAIL death receptor (DR)‐mediated apoptosis, barrier permeability, and angiogenic impairment within human cerebral ECs. We tested the hypothesis that Hhcy exacerbates Aβ‐induced cerebral EC TRAIL DR‐mediated apoptosis, barrier dysfunction, and angiogenesis defects.MethodHuman cerebral microvascular ECs (HCMECs) were challenged with AβQ22 and/or homocysteine (Hcy). Apoptotic mediator expression, caspase activation, cytochrome c release, and DNA fragmentation were measured to assess apoptosis. Junction protein expression, trans‐endothelial‐electrical‐resistance (TEER), and inflammatory mediators were measured to assess EC barrier integrity. Angiogenesis inhibition and activation (cytokine expression, pVEGFR2Y1175 expression/localization, VEGF‐A ELISA) assays were utilized to measure EC angiogenic capability and wound healing and actin polymerization assays were utilized to measure EC wound healing ability.ResultCombined challenge of ECs with AβQ22 and Hcy potentiated increases in TRAIL DR‐related apoptotic mediators' expression, caspase activity, cytochrome c release, and DNA fragmentation and potentiated decreases in anti‐apoptotic mediators’ expression at differential timepoints. Treatment of ECs with AβQ22 and Hcy additively decreased TEER and, at certain timepoints, potentiated decreased junction protein expression, increased inflammatory mediator expression, and phosphorylated BBB protein expression. Combined treatment of ECs with AβQ22 and Hcy additively decreased angiogenesis progression, angiogenesis‐stimulating cytokine expression, wound healing capability, and actin polymerization levels. Additionally, Hcy decreased HCMEC soluble VEGF‐A levels.ConclusionThe presence of high Hcy exacerbates Aβ induced‐EC apoptosis, barrier dysfunction, and angiogenic impairment. This study revealed several of the specific molecular mechanism through which amyloidosis and CV risk factors may additively act to produce EC dysfunction and death in AD.