Cerebrovascular dysfunction has been implicated as a major contributor to Alzheimers Disease (AD) pathology, with cerebral endothelial cell (cEC) stress promoting ischemia, cerebral-blood flow impairments and blood-brain barrier (BBB) permeability. Recent evidence suggests that cardiovascular (CV)/cerebrovascular risk factors, including hyperhomocysteinemia (Hhcy), exacerbate AD pathology and risk. Yet, the underlying molecular mechanisms for this interaction remain unclear. Our lab has demonstrated that amyloid beta 40 (Aβ40) species, and particularly Aβ40-E22Q (vasculotropic Dutch mutant), promote death receptor 4 and 5 (DR4/DR5)-mediated apoptosis in human cECs, barrier permeability and angiogenic impairment. Previous studies show that Hhcy also induces EC dysfunction, but it remains unknown whether Aβ and homocysteine function through common molecular mechanisms. We tested the hypotheses that Hhcy exacerbates Aβ-induced cEC DR4/5-mediated apoptosis, barrier dysfunction, and angiogenesis defects. This study was the first to demonstrate that Hhcy specifically potentiates Aβ40-E22Q-mediated activation of the DR4/5-mediated extrinsic apoptotic pathway in cECs, including DR4/5 expression, caspase 8/9/3 activation, cytochrome-c release and DNA fragmentation. Additionally, we revealed that Hhcy intensifies the deregulation of the same cEC junction proteins mediated by Aβ, precipitating BBB permeability. Furthermore, Hhcy and Aβ40-E22Q, impairing VEGF-A/VEGFR2 signaling and VEGFR2 endosomal trafficking, additively decrease cEC angiogenic capabilities. Overall, these results show that the presence of the CV risk factor Hhcy exacerbates Aβ-induced cEC apoptosis, barrier dysfunction, and angiogenic impairment. This study reveals specific mechanisms through which amyloidosis and Hhcy jointly operate to produce brain EC dysfunction and death, highlighting new potential molecular targets against vascular pathology in comorbid AD/CAA and Hhcy conditions.

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