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THE ROLE OF HYPERHOMOCYSTEINEMIA, HYPERTENSION, AND CEREBRAL HYPOPERFUSION IN AMYLOID BETA-INDUCED CEREBRAL ENDOTHELIAL CELL DYSFUNCTION IN ALZHEIMER'S DISEASE AND CEREBRAL AMYLOID ANGIOPATHY
Carey, Ashley Marie
Carey, Ashley Marie
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2024-12
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Biomedical Neuroscience
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http://dx.doi.org/10.34944/dspace/10896
Abstract
Recent evidence suggests that cerebrovascular dysfunction may precede and contribute to amyloid beta-(Aβ)-mediated pathology in Alzheimer’s Disease (AD), particularly promoting endothelial cell (EC) damage and stress, causing the cerebral blood flow (CBF) impairments, cerebral hypoperfusion (CH), and blood brain barrier (BBB) permeability that are pathologically characteristic in AD. Studies have emerged suggesting a link between cardiovascular diseases (CVDs) and AD pathology, showing that cerebrovascular/cardiovascular risk factors (CVRFs), including hyperhomocysteinemia (Hhcy) and hypertension (HTN), and the cerebral consequences of these CVRFs, such as CH, contribute to AD pathology and risk. Despite this, the underlying molecular mechanisms for these associations remain unclear. Previously, our lab has demonstrated that exposure of human cerebral microvascular ECs (HCMECs) to Aβ40-Q22 (vasculotropic Dutch mutant) or Aβ42 resulted in potentiated increases in apoptosis and decreases in barrier integrity and angiogenic capabilities. Previous evidence also reveals that Hhcy, HTN, and hypoperfusion exhibit similar dysfunction within peripheral blood vessels. Therefore, we tested the hypothesis that Hhcy, HTN, and CH exacerbate Aβ-induced cerebral EC apoptosis, BBB dysfunction, and angiogenesis impairment and Cerebral Amyloid Angiopathy (CAA) pathology in vitro and in vivo.
Human cerebral microvascular ECs (HCMECs) were challenged with AβQ22 and/or homocysteine (Hcy) or AβQ22/Aβ42, glucose deprivation (GD), or a combination of both under normoxia or hypoxia. Conditions involving oxygen and glucose deprivation (OGD) act to mimic tissue hypoperfusion in vitro. Apoptotic mediator expression, caspase activation, cytochrome c (CytC) release, and DNA fragmentation were measured to assess apoptosis. BBB- modulating protein expression, trans-endothelial electrical resistance (TEER), and inflammatory mediators were measured to assess EC barrier integrity. Angiogenesis inhibition and activation (pVEGFR2 Y1175 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. For the in vivo portion of this study, wild-type (WT) and Tg2576 AD mice (Swedish APP mutation) were fed with a Hhcy-inducing diet, HTN-inducing water (L-NAME), or both starting at 5 months. Mice were sacrificed at 13-14 months and tissue was harvested. Apoptotic mediator expression and vascular caspase-3 activation were measured. BBB-associated tight junction (TJ) protein expression, GFAP/IBA1 expression and morphology (neuroinflammation), and microhemorrhages were measured to assess BBB integrity. Angiogenic mediator expression and CD31+ microvessel density were utilized to determine cerebral angiogenic capability. Additionally, AD pathology and cognitive impairment were assessed in these mice.
We found that combined challenge of ECs with AβQ22 and Hcy potentiated increases in TRAIL death receptor (DR)-related apoptotic mediator expression, caspase activation, CytC release, and DNA fragmentation and potentiated decreases in anti-apoptotic mediator expression at differential timepoints. Treatment of ECs with AβQ22 and Hcy additively decreased TEER and, at certain timepoints, potentiated decreases in junction protein expression. Additionally, this combined treatment exacerbated increases in the expression of inflammatory mediators and phosphorylated BBB proteins. Combined treatment of ECs with AβQ22 and Hcy also additively decreased angiogenesis progression, angiogenesis-stimulating cytokine expression, wound healing capability, and actin polymerization levels. From our experiments involving OGD, we found that GD and hypoxia differentially potentiate Aβ-induced activation of cell death mechanisms within HCMECs, with GD potentiating apoptosis levels and hypoxia potentiating necrosis levels. Combined challenge of HCMECs with Aβ and OGD revealed exacerbated decreases in TEER, associated to potentiated dysregulation of BBB-TJ proteins, increases in MMP2 expression, and increases in proinflammatory mediator expression and monocyte migration. Furthermore, treatment of HCMECs with Aβ and OGD demonstrated potentiated decreases in angiogenic and wound healing capabilities. Our in vivo data has shown that Tg2576 mice with CVRFs demonstrate exacerbated increase in pro-apoptotic protein expression and caspase-3 activation within AD vulnerable brain regions. Additionally, Tg2576 mice with CVRFs displayed signs of exacerbated BBB dysfunction, evidenced by decreases in TJ protein expression, increases in active MMP2 expression, increases in neuroinflammation (astrogliosis, increased ICAM1), and increased presence of microhemorrhages and/or microthrombi. Tg2576 mice with CVRFs also demonstrated exacerbated increases in VEGF-A expression and a potentiated decrease in phosphorylated VEGFR2 Y1175, suggesting aberrant VEGF signaling and worsened angiogenic impairment. Finally, Tg2576 CVRF mice trended towards potentiated increases in cerebral soluble Aβ40 and fibrillar Aβ42 and as well as worsened cognitive outcomes on the Barnes Maze memory task.
To conclude, this study has revealed specific molecular mechanisms through which amyloidosis and CVRFs additively act to produce cerebrovascular damage and dysfunction and potentiate AD pathology within Tg2576 AD mice, specifically increasing cerebrovascular apoptosis, BBB permeability, and angiogenesis impairments. Identifying common molecular effects that Aβ and CVRFs exert on the cerebral vasculature will reveal novel potential druggable targets and biomarkers that could facilitate earlier disease detection, aid in the prevention of the initial stages of vascular damage within AD, and therapeutically slow or halt disease progression in common mixed AD and vascular dementias.
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