Tissue Engineered Blood Vessels to Study Endothelial Dysfunction in Hutchinson-Gilford Progeria Syndrome

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2022

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Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare, fatal genetic disease that causes progressive atherosclerosis and accelerated aging in children resulting in fatality at an average of 14.6 years of age. With a limited pool of HGPS patients, clinical trials face unique challenges and require reliable preclinical testing. Preclinical studies to date have relied on 2D cell culture using HGPS fibroblasts which does not accurately model the 3D physiological microenvironment and limits the scope many studies to only the fibroblast response, which may not translate to a vascular benefit. Further, only two HGPS murine models develop atherosclerosis and these exhibit symptoms that are not present in HGPS patients. An ideal model would incorporate human cells in a microenvironment mimicking in vivo conditions and replicate key aspects HGPS vascular pathology. Such a model would contribute significantly to the ability to study HGPS mechanisms and test therapies. One significant area of research that requires further study is the contribution of endothelial cells to HGPS pathology. The endothelium plays a critical atheroprotective role in maintaining vascular homeostasis. When it is damaged, dysfunctional endothelium becomes inflammatory and proatherogenic, contributing significantly to atherosclerosis and cardiovascular disease. Progressive atherosclerosis is the most severe symptom of HGPS and is the common underlying cause of mortality in HGPS patients, yet only a few studies have investigated the HGPS endothelial phenotype. The goal of our work was to develop and characterize a tissue engineered model of HGPS vasculature that could be used to study endothelial dysfunction in HGPS and test therapies to alleviate HGPS vascular pathology.In Aim 1, we developed and characterized a tissue engineered blood vessel (TEBV) model of HGPS using vascular smooth muscle cells (viSMCs) and endothelial cells (viECs) differentiated from induced pluripotent stem cells (iPSCs) from two HGPS patients. HGPS viSMCs and viECs exhibited manifestations consistent with typical symptoms of HGPS including progerin expression, abnormal nuclear morphology, and premature senescence. HGPS viECs exhibited cell responses consistent with endothelial dysfunction including impaired tube formation, elevated reactive oxygen species (ROS) levels, reduced proliferation, and increased levels of DNA damage compared with healthy viECs. HGPS viECs also displayed a blunted response to shear stress including limited flow-sensitive gene expression and reduced nitric oxide production. TEBVs fabricated with HGPS cells exhibited reduced vasoactivity compared with healthy TEBVs and replicated HGPS vascular pathology including SMC loss, excess extracellular matrix (ECM) protein deposition, inflammation, and vascular calcification. In Aim 2, we tested the effects of HGPS therapeutics the farnesyltransferase inhibitor Lonafarnib and mTOR inhibitor Everolimus, currently in Phase I/II clinical trial, on endothelial dysfunction and HGPS TEBVs phenotype. Everolimus decreased reactive oxygen species levels, increased proliferation, and reduced DNA damage in HGPS vascular cells. Lonafarnib improved flow-sensitive gene expression of HGPS viECs exposed to physiological shear stress. Both Lonafarnib and Everolimus were able to restore nitric oxide production to healthy levels in HGPS viECs exposed to physiological shear stress. While Everolimus improved vasoconstriction, Lonafarnib increased vasodilation, ECM deposition, inflammation, and calcification in HGPS TEBVs. Combination treatment with Lonafarnib and Everolimus maintained the benefits of each monotherapy and also resulted in additional benefits such as improved endothelial marker expression and reduced apoptosis. The 3D TEBV model was critical to reveal the benefit of Lonafarnib and Everolimus combination treatment which was more limited in 2D studies but became clear in the TEBVs. In Aim 3, we tested a recently developed targeted gene therapy for HGPS known as adenine base editing (ABE). ABE corrects the genetic heterozygous point mutation that causes HGPS with high efficiency and minimal off-target effects. ABE ameliorated all the dysfunctional endothelial phenotypes that we tested in HGPS viECs including elevated ROS levels, reduced proliferation, and increased DNA damage. Most critically for endothelial function, ABE restored nitric oxide production and flow-sensitive gene expression to healthy levels in HGPS viECs. In TEBVs, ABE restored healthy levels of vasoconstriction and vasodilation, improved SMC retention, increased proliferation, and inhibited excess ECM protein expression. In summary, this work contributes data supporting the hypothesis that progerin induces endothelial dysfunction in HGPS endothelial cells which could contribute to the vascular pathology observed in HGPS. This work also tests current and novel therapies in the first 3D tissue engineered model of HGPS, validating the model as a valuable platform for preclinical testing that can supplement and improve information gathering from current 2D and animal models.

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Abutaleb, Nadia Osama (2022). Tissue Engineered Blood Vessels to Study Endothelial Dysfunction in Hutchinson-Gilford Progeria Syndrome. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/25301.

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