Cyclic stretch of brain microvascular endothelial cells and regulation of amyloid processing and expression: evidence for contribution of vascular pulsatility in Alzheimer's disease

Thesis by publication.Bibliography: pages 222-238.1. Introduction -- 2. Literature review -- 3. Optimisation of mechanical stretch of endothelial cells for protein and RNA quantification -- 4. Cyclic stretch as a novel modulator of APP and associated inflammatory markers in human cerebral endothelial cells -- 5. Signalling pathways that mediate cerebral endothelial responses to cyclic stretch -- 6. Effect of endothelial cell passaging and cyclic stretch on APP expression, amyloid secretion, and NO signalling -- 7. Effect of cyclic stretch in cerebral endothelial cells pre-exposed to Aβ -- 8. Efflect of cyclic stretch and glycosphingolipid inhibition on cerebral endothelial cells -- 9. A preliminary study of the effect of a high salt diet on APP processing, eNOS signalling and aortic stiffness -- 10. Conclusions, limitations and future directions -- Appendices.Alzheimer's disease (AD) is characterised by amyloid-β (Aβ) plaques arising from amyloid precursor protein (APP) processed by β secretase-1 (BACE-1). Increasing evidence suggests a role of vascular factors in AD, namely hypertension, elevated pulse pressure and arterial stiffness, all associated with increased vascular pulsatility imposing mechanical stretch on the endothelium. This thesis addresses the role of cyclic stretch on human cerebral microvascular endothelial cells (HCMECs) in expression and processing of APP. It also investigates effect of high salt diet on APP processing in rat brains, given associations of high salt diet and cognitive impairment. In vitro studies involved cultured HCMECs subjected to 0%, 5%, 10% or 15% stretch (18 hours, 1 Hz) and analysis of protein and RNA expression, nitric oxide and Aβ levels. In vivo study included treatment of rats with high (8% NaCl, HS) or a low (0.26% NaCl, control) for 10-13 weeks and examination of brain tissue. Established for the first time was that APP expression and Aβ secretion are altered in response to HCMECs stretch, and that this response is differentially mediated in early and late passage HCMECs. In late passage HCMECs, APP and BACE-1 expression increased 2-3-fold with 10 and 15% stretch compared to 0%, with proportional increases in Aβ42/Aβ40 with % stretch (R2=0.21). In early passage HCMECs stretched at 15%, APP expression, BACE-1, Aβ42 levels were decreased 2-3-fold compared to late passage HCMECs. Glycosphingolipid inhibition prior to cyclic stretching at 15% increased APP expression and Aβ42 secretion 1-fold. In vivo findings provide preliminary evidence of altered APP processing in HS rats compared to controls parallel with increases in markers of arterial stiffness. Overall results suggest a role of arterial stiffness and vascular pulsatility strengthening the evidence of vascular contributions to AD. Future studies identifying associated molecular mechanisms will provide novel therapeutic targets for AD.1 online resource (xx, 238 pages : illustrations

High blood pressure and cyclic stretch after cerebral amyloid deposition and endothelial function

Theoretical thesis.Bibliography: leaves 50-61.1. Introduction -- 2. Material and methods -- 3. Results -- 4. Discussion -- 5. Summary and conclusions.Background: Amyloid β (Aβ) deposition is a hallmark of Alzheimer’s disease (AD). Increased pulsatility, endothelial dysfunction (ED) and inflammation, indicators of vascular stiffness, are associated with AD. Additionally, vascular stiffness is linked to hypertension, a risk factor for AD. Aim: This study aimed to determine effects of high blood pressure (BP) on cerebral Aβ deposition in rodent models, spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rats (WKY) and investigate effects of cyclic stretch (CS) on expression of amyloid precursor protein (APP), endothelial nitric oxide synthase (eNOS) and intercellular cell adhesion molecule-1 (ICAM-1) in human cerebral microvascular endothelial cells (hCMEC). Methods: Hippocampal (HC) and frontal cortex (FC) regions of SHR and WKY rats were analysed using western blotting to determine effect of BP on cerebral Aβ deposition. hCMEC were subjected to 5%, 10% or 20 % CS compared to control (0% CS) to evaluate pulsatility, ED and inflammation using western blotting and/or RTqPCR. Results: Aβ oligomerisation increased in SHR compared to WKY in HC (P<0.01) and FC (P<0.001). APP mRNA expression increased at 5%, decreased at 20% CS; eNOS decreased at both (P<0.0001). APP and ICAM-1 protein expression dose-dependently increased at 5% and 10% CS (P<0.01) and decreased at 20% CS. eNOS protein levels decreased at all CS (P<0.0001). Conclusions: Results suggest that high BP and CS respectively alter the processing and expression of cerebral APP. Prolonged CS may induce ED by increasing ICAM-1, thereby mitigating eNOS expression. Findings mechanistically support the association of elevated pulsatility and arterial stiffness with AD.Mode of access: World wide web1 online resource (xi, 61 leaves illustrations (some colour

Pulsatile stretch as a novel modulator of amyloid precursor protein processing and associated inflammatory markers in human cerebral endothelial cells

Abstract Amyloid β (Aβ) deposition is a hallmark of Alzheimer’s disease (AD). Vascular modifications, including altered brain endothelial cell function and structural viability of the blood-brain barrier due to vascular pulsatility, are implicated in AD pathology. Pulsatility of phenomena in the cerebral vasculature are often not considered in in vitro models of the blood-brain barrier. We demonstrate, for the first time, that pulsatile stretch of brain vascular endothelial cells modulates amyloid precursor protein (APP) expression and the APP processing enzyme, β-secretase 1, eventuating increased-Aβ generation and secretion. Concurrent modulation of intercellular adhesion molecule 1 and endothelial nitric oxide synthase (eNOS) signaling (expression and phosphorylation of eNOS) in response to pulsatile stretch indicates parallel activation of endothelial inflammatory pathways. These findings mechanistically support vascular pulsatility contributing towards cerebral Aβ levels