Browsing by Subject "Progeria"
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Item Open Access An Induced Pluripotent Stem Cell-derived Tissue Engineered Blood Vessel Model of Hutchinson-Gilford Progeria Syndrome for Disease Modeling and Drug Testing(2018) Atchison, Leigh JoanHutchison-Gilford Progeria Syndrome (HGPS) is a rare, accelerated aging disorder caused by nuclear accumulation of progerin, an altered form of the Lamin A gene. The primary causes of death are stroke and cardiovascular disease at an average age of 14 years. It is known that loss or malfunction of smooth muscle cells (SMCs) in the vasculature leads to cardiovascular defects, however, the exact mechanisms are still not understood. The contribution of other vascular cell types, such as endothelial cells, is still not known due to the current limitations of studying such a rare disorder. Due to limitations of 2D cell culture, mouse models, and the limited HGPS patient pool, there is a need to develop improved models of HGPS to better understand the development of the disease and discover novel therapeutics.
To address these limitations, we produced a functional, three-dimensional tissue model of HGPS that replicates an arteriole-scale tissue engineered blood vessel (TEBV) using induced pluripotent stem cell (iPSC)-derived cell sources from HGPS patients. To isolate the specific effects of HGPS SMCs, we initially used human cord blood-derived endothelial progenitor cells (hCB-EPCs) from a separate, healthy donor and iPSC-derived SMCs (iSMCs). TEBVs fabricated from HGPS patient iSMCs and hCB-EPCs (HGPS iSMC TEBVs) showed disease attributes such as reduced vasoactivity, increased medial wall thickness, increased calcification, excessive extracellular matrix protein deposition, and cell apoptosis relative to TEBVs fabricated from primary mesenchymal stem cells (MSCs) and hCB-EPCs or normal patient iSMCs with hCB-EPCs. Treatment of HGPS iSMC TEBVs for one week with the rapamycin analog Everolimus (RAD001), increased HGPS iSMC TEBV vasoactivity and iSMC differentiation in TEBVs.
To improve the sensitivity of our HGPS TEBV model and study the effects of endothelial cells on the HGPS cardiovascular phenotype, we adopted a modified differentiation protocol to produce iPSC-derived vascular smooth muscle cells (viSMCs) and endothelial cells (viECs) from normal and Progeria patient iPSC lines to create iPSC-derived vascular TEBVs (viTEBVs). Normal viSMCs and viECs showed structural and functional characteristics of vascular SMCs and ECs in 2D culture, while HGPS viSMCs and viECs showed various disease characteristics and reduced function compared to healthy controls. Normal viTEBVs had comparable structure and vasoactivity to MSC TEBVs, while HGPS viTEBVs showed reduced vasoactivity, increased vessel wall thickness, calcification, apoptosis and excess ECM deposition. In addition, HGPS viTEBVs showed markers of cardiovascular disease associated with the endothelium such as decreased response to acetylcholine, increased inflammation, and altered expression of flow-associated genes.
The treatment of viTEBVs with multiple Progeria therapeutics was evaluated to determine the potential of the HGPS viTEBV model to serve as a platform for drug efficacy and toxicity testing as well as to further elucidate the mechanisms behind each drugs mode of action. Treatment of viTEBVs with therapeutic levels of the farnesyl-transferase inhibitor (FTI), Lonafarnib, or Everolimus improved different aspects of HGPS viTEBV structure and function. Treatment with Everolimus alone increased response to phenylephrine, improved SMC differentiation and cleared progerin through autophagy. Lonafarnib improved acetylcholine response, decreased ECM deposition, decreased calcification and improved nitric oxide production. Most significantly, combined therapeutic treatment with both drugs showed an additive effect by improving overall vasoactivity, increasing cell density, increasing viSMC and viEC differentiation, and decreasing calcification and apoptosis in treated HGPS viTEBVs. On the other hand, toxic doses of both drugs combined resulted in significantly diminished HGPS viTEBV function through increased cell death. In summary, this work shows the ability of a tissue engineered vascular model to serve as an in vitro personalized medicine platform to study HGPS and potentially other rare diseases of the vasculature using iPSC-derived cell sources. It has also further identified a potential role of the endothelium in HGPS. Finally, this HGPS viTEBV model has proven effective as a drug testing platform to determine therapeutic and toxic doses of proposed therapeutics based on their specific therapeutic effects on HGPS viTEBV structure and function.
Item Open Access Tissue Engineered Blood Vessels to Study Endothelial Dysfunction in Hutchinson-Gilford Progeria Syndrome(2022) Abutaleb, Nadia OsamaHutchinson-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.