Cell Biology & Molecular Genetics
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Item Investigation of the molecular mechanisms of vascular endothelial dysfunction in Hutchinson-Gilford progeria syndrome through in vitro 2D and 3D models(2021) Gete, Yantenew; Cao, Kan; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder with features of accelerated aging. Predominantly, HGPS is caused by a de novo point mutation in the LMNA gene (c.1824C > T; p.G608G) resulting in progerin, a toxic lamin A protein variant. Children with HGPS typically die from coronary artery diseases or strokes at an average age of 14.6 years. Endothelial dysfunction is a known driver of cardiovascular pathogenesis; however, it is currently unknown how progerin antagonizes endothelial function in HGPS. In this study, I used human iPSC-derived endothelial cell (iPSC-EC) models that cultured under both static and fluidic culture conditions. HGPS iPSC-ECs show reduced endothelial nitric oxide synthase (eNOS) expression and activity compared to healthy controls and concomitant decreases in intracellular nitric oxide (NO) level, which result in deficits in capillary-like microvascular network formation. In addition, expression of matrix metalloproteinase 9 (MMP-9) was reduced in HGPS iPSC-ECs while expression of tissue inhibitor metalloproteinases 1 and 2 (TIMP1 and TIMP2) were upregulated relative to healthy controls. Moreover, I used an adenine base editor (ABE7.10max-VRQR) to correct the pathogenic c.1824C > T allele in HGPS iPSC-ECs. Remarkably, ABE7.10max-VRQR correction of the HGPS mutation significantly reduced progerin expression to a basal level, rescued nuclear blebbing, increased intracellular NO level, normalized TIMPs , and restored angiogenic competence in HGPS iPSC-ECs. Furthermore, to elucidate the effects of progerin on endothelial cells and vascular remodeling, in collaboration with Dr. Truskey’s lab at Duke university, we developed tissue-engineered blood vessels (TEBVs) using iPSC-ECs and smooth muscle cells (iPSC-SMCs) from normal and HGPS patients. Relative to normal TEBVs, HGPS TEBVs showed reduced function and exhibited markers of cardiovascular disease associated with endothelium, including a reduction in both vasoconstriction and vasodilation with increased inflammation markers, VCAM-1 and E-selectin protein. Hence, the TEBV model has identified a role of the endothelium in HGPS. Together, the results of the study provide molecular insights of endothelial dysfunction in HGPS and suggest that ABE7.10max-VRQR could be a promising therapeutic approach for correcting HGPS-related cardiovascular phenotypes.Item INVESTIGATION OF DEFECTIVE CELL SIGNALING CASCADE INVOLVED IN THE OSTEOGENESIS IN HUTCHINSON-GILFORD PROGERIA SYNDROME(2018) Choi, Ji Young; Cao, Kan; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Human bone homeostasis is maintained through constant bone remodeling, which balances bone formation by osteoblasts and bone resorption by osteoclasts. Patients with Hutchinson-Gilford progeria syndrome (HGPS) have low bone mass that manifests in a high risk of fractures and an atypical skeletal geometry, suggesting impaired bone remodeling. HGPS is a premature aging disease caused by truncated lamin A that is permanently farnesylated. The mutant lamin A is referred as progerin. Several previous clinical reports discussed abnormal skeletal development of the children with HGPS, but the molecular mechanistic study on defective osteogenesis of HGPS stem cells need to be further elucidated. The major aim of my dissertation research is to investigate dysfunction in stem cell differentiation due to aberrant cell signaling in osteoprogenitor cells that express progerin. To achieve this aim, the study demonstrates both in vitro and in vivo models of HGPS to support defective mechanism of the canonical WNT/β-catenin pathway, seemingly at the level of efficiency of nuclear import of β-catenin and impaired osteoblast differentiation. Restoring β-catenin activity rescues osteoblast differentiation and significantly improves bone mass. In particular, HGPS patient-derived induced pluripotent stem cells (iPSCs)-osteoprogenitors and primary mesenchymal stem cells (MSCs) expressing the HGPS mutant progerin display defects in osteoblast differentiation, characterized by deficits in alkaline phosphatase activity and mineralizing capacity. Mechanistic investigation reveals that canonical WNT/β-catenin pathway, a major signaling cascade involved in skeletal homeostasis, is impaired by progerin, causing a reduction in nuclear active β-catenin protein levels and reciprocal aberrant cytoplasmic accumulation which causes reduced transcriptional activity for osteogenesis. Non-farnesylation of progerin in MSCs attains higher level of active β-catenin protein expression and consequently increasing the signaling, enhancing mineralization capacity and ameliorating the defective osteogenesis. Moreover, in vivo analysis of the Zmpste24-/- HGPS mouse model demonstrates that treatment with a sclerostin-neutralizing antibody (SclAb), which targets an antagonist of canonical WNT/β-catenin signaling pathway, fully rescues the low bone mass phenotype to wild-type levels. This study implicates β-catenin signaling cascade as a therapeutic target for restoring defective skeletal microarchitecture in HGPS. Given the fundamental nature of WNT/β-catenin signaling to stem cell renewal and lineage allocation, the findings from this dissertation may provide broader inferences for the treatment options in HGPS.Item INVESTIGATION OF SMOOTH MUSCLE CELL DEATH AND GENOME INSTABILITY IN HUTCHINSON-GILFORD PROGERIA SYNDROME(2016) Zhang, Haoyue; Cao, Kan; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hutchinson–Gilford progeria syndrome (HGPS) is a severe human premature aging disorder caused by a lamin A mutant named progerin. Death occurs at a mean age of 13 y from cardiovascular problems. Previous studies revealed loss of vascular smooth muscle cells (SMCs) from large arteries in HGPS patient and mouse models, suggesting a causal connection between SMC loss and cardiovascular malfunction. The primary aim of this dissertation is to elucidate the molecular mechanisms underlying the massive SMC loss in HGPS. To study this, I develop an in vitro differentiation protocol to generate HGPS SMCs from induced pluripotent stem cells (iPSCs). My results indicate that HGPS SMCs exhibit a profound cell death phenotype, potentially recapitulating the in vivo SMC loss. Mechanistically, I find that HGPS SMCs bear deficient homologous recombination (HR). In addition, progerin accumulation strongly suppresses PARP1 and consequently triggers an activation of the error-prone non-homologous end joining (NHEJ) response during S/G2 phase. As a result, HGPS SMCs exhibit prolonged mitosis and mitotic catastrophe. Mis-regulated DNA damage response (DDR) is proposed to induce genome instability and various cellular phenotypes in HGPS, including HGPS SMC cell death. To better understand HGPS DDR misregulation, I examine HR and NHEJ in HGPS fibroblasts at different cell cycle phases. My analysis indicates that HR is deficient in S/G2 phase, whereas NHEJ, the dominant G0/G1 phase DDR pathway, is impaired in G0/G1 phase but active in S/G2 phase HGPS fibroblasts. The mis-regulation of HR and NHEJ may jeopardize genome integrity in both G0/G1 and S/G2 phase HGPS cells. Mechanistic study reveals that H2AX, a crucial upstream DDR signal, is reduced in G0/G1 but normal in S/G2 phase HGPS cells, implicating a potential cause of the cell cycle-dependent NHEJ mis-regulation. Furthermore, this reduction is correlated with impaired ATM activation and loss of H3K9me3 in HGPS. Restoration of H3K9me3 by methylene blue treatment can stimulate ATM activity, improve H2AX signaling and rescue NHEJ in G0/G1 phase HGPS cells. This dissertation not only is the first mechanistic study on HGPS SMC loss but also provides a molecular basis and therapeutic approach for the HGPS DDR deficiencies.