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Apolipoprotein E4 Expression Causes Gain of Toxic Function in Isogenic Human Induced Pluripotent Stem Cell-Derived Endothelial Cells

ABSTRACT: Objective: The ApoE (apolipoprotein) allele epsilon 4 is a major genetic risk factor for Alzheimer disease, cardiovascular disorders, and stroke, indicating that it significantly impacts cerebral and vascular systems. However, very little is known about how APOE genotype affects brain endothelial cells, which form a network of tight junctions to regulate communication between the brain and circulating blood factors. Approach and Results: Here, we present a novel model of endothelial dysfunction using isogenic human induced pluripotent stem cell-derived cells harboring different alleles of the APOE gene, specifically ApoE 3/3, 3/4, and 4/4. We show for the first time that ApoE4 expression by endothelial cells is sufficient to cause a toxic gain of cellular dysfunction. Using RNAseq, we found significant effects of ApoE4 on signaling pathways involved in blood coagulation and barrier function. These changes were associated with altered cell function, including increased binding of platelets to ECs with the 3/4 or 4/4 genotype. ApoE4-positive cells exhibited a proinflammatory state and prothrombotic state, evidenced by higher secretion of Aβ (amyloid-β) 40 and 42, increased release of cytokines, and overexpression of the platelet-binding protein VWF (vonWillebrand factor). Immunohistochemistry of human brain Alzheimer disease brains also showed increased VWF expression with the ApoE4/4 genotype. Finally, pharmacological inhibition of inflammation in ECs by celastrol rescued overexpression of VWF in cells expressing ApoE4. Conclusions: These cells provide novel insight into ApoE4-mediated endothelial dysfunction and provide a new platform to test potential therapies for vascular disorders.

ORGANISM(S): Homo sapiens

PROVIDER: GSE111366 | GEO | 2020/05/24

REPOSITORIES: GEO

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Project description:Human APOE genotype is intricately involved in the homeostasis of the central nervous system, which is particularly evident from its association with neurodegenerative disease. Compared to APOE3, APOE4 is associated with greater cognitive decline in aging, poorer outcomes following stroke and traumatic brain injury, and is a major genetic risk factor for Alzheimer’s disease. Human APOE genotypes can differentially modulate neuronal function via both direct and indirect pathways. Of the indirect pathways, increasing evidence supports that APOE4-associated neurovascular dysfunction plays a significant role in neurodegeneration. For example, deficits in cerebral blood flow, neuronal-vascular coupling and blood-brain barrier integrity are observed with APOE4 both during aging and in Alzheimer’s disease patients; data that has been recapitulated in mouse models. Therefore, the identification of cellular mechanistic pathways that contribute to APOE-modulated vascular function is important for fundamental and disease-focused research. Highly specialized brain endothelial cells (BECs) are essential for virtually all neurovascular functions and, therefore, the influence of apoE on the cerebrovasculature likely involves alterations in brain endothelial cells function, directly or via supporting cells. ApoE synthesis has been demonstrated in multiple cell types in both the brain (e.g. astrocytes, pericytes and neurons) and periphery (e.g. hepatocytes and macrophages). Current research has largely focused on the effects of apoE produced by supporting cell types, particularly astrocytes and pericytes, on BEC function. However, less explored is whether BECs produce apoE to modulate their function. Data from limited studies on apoE production by BECs and APOE genotype-specific functional differences are conflicting. Evaluation of whether the APOE genotype of BECs modulates their function is important, as this could represent a novel mechanisms that contributes to altered cerebrovascular function in aging and AD. The goal of this study was to evaluate the role of human APOE genotype in modulating the phenotype and/or function of brain endothelial cells in vitro. To this end, we utilized primary brain endothelial cells isolated from APOE-targeted replacement mice that express human APOE3 or APOE4 under the endogenous mouse APOE promoter. BECs are highly specialized and functionally complex to maintain homeostasis of the central nervous system and APOE genotype could theoretically alter levels of any number of molecules and associated functions. Thus, without a priori knowledge we initially evaluated the extent that APOE modulated the transcriptomic profile of brain endothelial cells.

Project description:Cerebrovascular dysfunction is a hallmark feature of Alzheimer's disease (AD). One of the greatest risk factor for AD is the apolipoprotein E4 (E4) allele. APOE4 genotype has been shown to negatively impact on perivascular amyloid clearance, however, its direct influence along with the other APOE variants (APOE2 and APOE3) on the molecular integrity of the cerebrovasculature has not been largely explored. To address this, we employed a 10-plex tandem isobaric mass tag approach in combination with an ultra-high pressure liquid chromatography MS/MS (Q-Exactive) method, to interrogate unbiased proteomic changes in cerebrovessels from AD and healthy control brains on different APOE genotype backgrounds. We first interrogated changes between healthy control homozygote E2/E2, E3/E3 and E4/E4 cases to identify underlying genotype specific effects on cerebrovessels. EIF2 signaling, regulation of eIF4 and 70S6K signaling and mTOR signaling were the top significantly altered pathways in E4/E4 compared to E3/E3 cases. EIF2, nitric oxide and sirtuin signaling pathways were the top significantly altered pathways in E4/E4 compared to E2/E2 cases. We used a Two-way ANOVA analysis to identify AD-dependent changes and their interactions with APOE genotype. The highest number of significantly regulated proteins from this interaction was observed in E3/E4 (192) and E4/E4 (189) cases. As above, EIF2, mTOR signaling and eIF4 and 70S6K signaling were the top three significantly altered pathways in E4 allele carriers (i.e. E3/E4 and E4/E4 genotypes). Of all the cerebrovascular cell specific markers identified in our proteomic analyses, endothelial cell, astrocyte, and smooth muscle cell specific protein markers were significantly altered in E4/E4 cases, while endothelial cells and astrocyte specific protein markers were altered in E3/E4 cases. These proteomic changes provide novel insights to explain the longstanding link between APOE4 and cerebrovascular dysfunction. The early and converging APOE4 dependent changes we identified could provide novel targets in the cerebrovasculature for developing disease modifying strategies to mitigate the effects of APOE4 genotype on increasing the risk for, and the path towards AD pathogenesis.

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Apolipoprotein E4 Expression Causes Gain of Toxic Function in Isogenic Human Induced Pluripotent Stem Cell-Derived Endothelial Cells

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