a. Advancing age is the greatest risk factor for cardiovascular disease and with age, the prevalence of stroke and dementia increases dramatically. Degenerative vascular diseases are characterised by the accumulation of senescent vascular smooth muscle cells (VSMCs) and the deposition of fibrillar amyloid proteins into the surrounding extracellular matrix (ECM). This process of amyloidosis is driven by the secretion of extracellular vesicles (EVs) from senescent VSMCs. In health EVs carry protein cargoes that protect against amyloid deposition. In contrast, EVs secreted from senescent cells become trapped in the ECM and can actively drive amyloid formation. Excitingly it has been shown that native healthy EVs or engineered exosomes can cross the blood brain barrier and act to alleviate amyloid brain pathology. However, the mechanisms and their impact on the vasculature remain unclear highlighting the need for further mechanistic studies in model systems. In this project we investigate the role of senescence and EVs in driving ECM degeneration, amyloid deposition and cellular dysfunction and investigate the potential for EVs as therapies for amyloidosis in human disease using a novel 3D organoid system. We will take a bench to bedside approach focussing on cerebral amyloid angiopathy (CAA), a progressive, age-related, vascular amyloidosis leading to vascular dysfunction, intracerebral haemorrhage, neurodegeneration and cognitive decline.
b. Skills in cell and organoid tissue culture, advanced microscopy and EV analysis. Basic cell and molecular biology, histology and generation and bioinformatic analysis of proteomic and genomic datasets.
c. Aims: (1) the contribution of EVs to the development and remodelling of the basement membrane ECM in health and senescence and (2) whether EVs derived from healthy VSMCs can delay amyloidosis and/or rejuvenate senescent cells and the senescent ECM. In the 3rd aim we will use a novel 3D culture system that can model the flow of the perivascular drainage pathway to investigate EVs as therapeutics for amyloid clearance from the vasculature.
d. Year 1: Role of EVs in basement membrane.
Year 2: Characterise EV cargoes in health and senescence and determine their role in mediating amyloid nucleation. Quantify EV cargo deposition in CAA patients.
Year 3: Quantify amyloid formation and effect of EVs in 3D flow system.
Year 4: Test EVs in in vivo models.
e. During the rotation project we will test the role of EVs in patterning and building the VSMC basement membrane. siRNA and drugs will be used to block EV release from healthy and senescent VSMCs induced to deposit ECM. The ECM produced with and without EV blockade will be analysed by proteomics, Western blot and confocal microscopy to determine the impact of EVs on structure, composition and components of the basement membrane
