THE EFFECT OF APOLIPOPROTEIN E4 ON HUMAN PERICYTE FUNCTION USING INDUCED PLURIPOTENT STEM CELLS

Abstract

Pericytes, brain microvascular endothelial cells, and astrocytes form the neurovascular unit (NVU). Breakdown of the NVU and dysfunction of the blood-brain-barrier (BBB) are early biomarkers of dementia, including Alzheimer’s disease (AD). The strongest genetic risk factor for AD, Apolipoprotein E4 (ApoE4), has particular importance in BBB dysfunction compared to the ApoE3 variant. Pericyte dysfunction occurs in AD, resulting in enhanced capillary contraction and cell death. However, the role of pericytes in health and disease remains poorly understood, partially due to a lack of adequate models that can recapitulate the complexity of the multi-cellular NVU. The aim of this work was to investigate the effect of different ApoE genotypes (ApoE3 and ApoE4) on the function of human induced pluripotent stem cell (iPSC)-derived pericytes in vitro. iPSCs from donors diagnosed with (ApoE4 carrier, UKA iPSC line) and without (ApoE3 carrier, BNA iPSC line) AD and respective isogenic controls (UKA3 and BNA4) were differentiated into pericytes. Success of differentiation was assessed by pericyte marker expression and support of endothelial cell barrier function. Differences between ApoE3- and ApoE4-pericytes were evaluated by RNA sequencing, angiogenesis assays, Fluo4-AM calcium tracing, and collagen hydrogel contraction studies. The secretion of pro-inflammatory mediators after exposure to the environmental pollutant PM2.5 was measured by an antibody binding assay. iPSC-derived ApoE3- and ApoE4-pericytes labelled positively for NG2, PDGFR-β, CD13, CD146, α-SMA, Calponin-1, Desmin, and SM22. RNA sequencing revealed higher gene expression of α-SMA in ApoE4-pericytes compared to ApoE3-pericytes. BNA4-pericytes expressed more extracellular matrix components. All ApoE-pericytes supported endothelial cell tube formation, but BNA4-pericytes to a lesser extent. Differences in calcium release after treatment with two known vasoconstrictors Endothelin-1 (ET-1) and U46619 (U4) were donor-dependent. Collagen hydrogel contraction studies revealed increased contractile ability of ET-1-treated BNA4-pericytes compared to BNA-pericytes. Contraction was successfully inhibited by blocking the ET-1 receptor. Similarly, contraction of U4-treated UKA3-pericytes was inhibited by blocking the U4 receptor. Finally, IL-6 secretion of UKA3-pericytes increased after exposure to PM2.5 and BNA4-pericytes secreted less IL-8 than BNA-pericytes. These results suggest that these in vitro models can be used to evaluate key functions of iPSC-pericytes, and highlighted genetic and functional differences related to ApoE genotype.

Date of Award	31 Dec 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Tao Wang (Supervisor), Nigel Hooper (Supervisor) & Marco Domingos (Supervisor)