The Effects of Hutchinson-Gilford Progeria Syndrome on Gene Expression in Vascular Cells

Abstract

Hutchinson-Gilford Progeria Syndrome (HGPS) is a fatal, accelerated-aging disease caused by a mutation in the nuclear envelope protein Lamin A. The resulting mutant protein, progerin, accumulates on the nuclear envelope, causing nuclear blebbing, altered gene expression, and other cellular defects. The primary pathology of HGPS is atherosclerosis, leading to stroke or heart attack. This dissertation first introduces the disease, its pathology and molecular mechanisms, and establishes what is currently known about how HGPS affects vasculature.Given that atherosclerosis begins with endothelial dysfunction in non-HGPS individuals, Chapter 2 of this dissertation examines whether the HGPS endothelium has an altered shear stress response, contributing to atherogenesis. I examined morphology and gene expression of HGPS and healthy iPSC-derived endothelial cells (viECs) after exposure to steady laminar shear stress (12 dynes/cm2 for 24 hours) in a parallel-plate flow channel. It was found that elongation in response to flow is impaired in HGPS viECs compared with healthy viECs. Differential expression (DE) analysis showed fewer significant differentially expressed genes and a lower magnitude of gene expression change after flow in HGPS compared with healthy viECs. Gene Set Enrichment Analysis identified differences in the gene sets altered by flow-induced DE, including Cholesterol Homeostasis, which was overrepresented in HGPS viECs. LGALS3, encoding the atherosclerosis marker galectin-3, was a main driver of this overrepresentation. RT-PCR confirmed LGALS3 as a shear-sensitive gene robustly upregulated in HGPS viECs compared with healthy viECs after flow. Treatment with an adenine base editor correcting the HGPS mutation restored LGALS3 expression to healthy levels. These observations indicate that HGPS ECs have an aberrant molecular response to atheroprotective shear stress, contributing to atherogenesis in HGPS patients. The vasculature of children with HGPS becomes occluded and stiffened, with high levels of extracellular matrix (ECM) deposition found in the atherosclerotic plaques and significant thickening of the arterial adventitia when compared with non-HGPS atherosclerotic patients. The smooth muscle cells (SMCs) in the medial layer experience a cyclic stretch as blood pumps through the arteries, which induces a genetic response. Here, I assess whether physiological cyclic stretch induces an aberrant response in HGPS SMCs, by exposing iPS-derived SMCs (viSMCs) on flexible-bottom plates to 10% cyclic stretch via the FlexCell instrument. After 8- and 24-hour increments, RNA was isolated and the expression of select ECM genes is analyzed via RT-PCR. In chapter 3, I show that an 8h period of cyclic stretch is not sufficient for significant differential expression of ECM genes in viSMCs. After 24h of stretch, HGPS viSMCs upregulate collagen 1 and collagen 3, while fibronectin expression remains sustained. This differs from healthy viSMCs in which these ECM genes are downregulated in response to stretch during this time. Additionally, this study gives insights for optimization and expansion of these stretch experiments. This body of work aims to add to the field of knowledge regarding the vascular pathology of HGPS, while outlining areas for further exploration of the disease mechanism.