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Item Hypoxic Regulation of Angiotensin-Converting Enzyme 2 and Mas Receptor in Hematopoietic Stem/Progenitor Cells: A Translational Study(North Dakota State University, 2019) Joshi, Shrinidh AshokkumarVascular disease is the leading cause of mortality and morbidity in the western world, and account for the 1 of every 3 death’s in the US, but a cure for vascular disease is yet to be realized. Hematopoietic stem progenitor cells (HSPCs) are mobilized from bone marrow and have the innate propensity to accelerate vascular repair by reendothelialization and revascularization of ischemic areas. The vasoreparative ability of HSPCs is largely due to their capacity to home to the areas of hypoxia and their sensitivity to hypoxia plays a critical role in the vasoreparative functions of these cells. The discovery of vasoreparative potential of HSPCs resulted in a breakthrough approach of cell-based therapies for the treatment of ischemic vascular diseases. However, success of this approach is essentially dependent on the number of cells that could be collected from an individual. Therefore, novel mechanism-based strategies are needed to enhance the outcomes of autologous cell-based therapies in poor mobilizers and older adults. Recent evidence of a potential role of the vasoprotective axis of the renin angiotensin system (RAS) in HSPCs functions offers a breakthrough. Angiotensin-(1-7), the primary mediator of the protective functions which acts on Mas receptor (MasR), is generated by angiotensin converting enzyme-2 (ACE2). In this study, we tested the effects of hypoxia on stimulation of vasoreparative potential of HSPCs and in upregulation of ACE2 and MasR. Importantly, we delineated the molecular mechanism of hypoxic exposure in regulation of ACE2 and MasR in a HIF1α- dependent manner and hypoxic exposure induced shedding of the membrane bound ACE2 in HSPCs. We used luciferase, a reporter assay, cell-based assays, gene/protein expression studies and pharmacological strategies in human and mouse HSPCs to test our hypotheses. To verify the biological significance of hypoxia, we performed in vivo studies in mice and humans, which recapitulated the in vitro observations on vascular protective axis of RAS in HSPCs. Collectively, these studies provided mechanistic insights into hypoxic regulation of vascular protective axis of RAS in HSPCs and also provided compelling evidence for the clinical use of hypoxia as a promising approach for enhancing the vasoreparative outcomes of cell-based therapies.Item Understanding the Role of Receptor for Advanced Glycation Endproducts (RAGE) in Pancreatic Cancer and Melanoma(North Dakota State University, 2021) Taneja, SakshiIn this project we study the role of RAGE in the melanoma and pancreatic cancer progression. Based on published studies, we hypothesized that RAGE localization in melanoma varies with different cellular architectures. To test this hypothesis, we utilized an in vitro spheroid model and a lung colonization mice model to compare the RAGE localization in 3D architecture vs 2D monolayer culture. RAGE was found at the cell surface in WM115 and B16F10 spheroids, whereas RAGE is mostly distributed intracellularly in WM266. We also observed that RAGE is present at the surface of B16F10 melanoma cells within tumor nodules in the lungs of mice colonized with B16F10 cells. Previously, our group has demonstrated that RAGE promotes pancreatic tumor cell survival under normoxic conditions, upon gemcitabine administration. Hypoxia is also associated with increased tumor aggressiveness. Based on published reports, we hypothesized that RAGE upregulation under hypoxic conditions contributes to autophagy and migration in pancreatic cancer cells. We observed that autophagy decreases after RAGE inhibition by FPSZM1. Moreover, we observed decreased cell migration after RAGE blockage, indicating that RAGE also mediates migration under hypoxia. We also investigated Advanced Glycation Endproducts (AGEs) on proliferation and migration of pancreatic cancer cells. Based on published reports, we hypothesized that RAGE activation by AGEs contributes to the proliferation and migration in pancreatic cancer cells. We employed ribose modified BSA to activate RAGE in the murine KPC 5517 pancreatic cancer cell line. We observed that AGE-treated samples showed significant increase in migration but no change in proliferation. As RAGE is involved in the progression of melanoma and pancreatic cancer, our results will help researchers to better understand the biology of RAGE. Our research can help to design RAGE-specific antibodies and inhibitors that could target RAGE more effectively. Moreover, our findings on AGE-RAGE interactions, and on the role of RAGE in pancreatic cancer progression under hypoxia, may contribute to reduce the progression of pancreatic cancer. Our results showing that a RAGE inhibitor can reduce autophagy and migration of pancreatic tumor cells, suggest that FPS-ZM1 could be utilized as a potential therapeutic aid for the treatment of pancreatic cancer.