Indiana University-Purdue University Indianapolis (IUPUI) / Elaborate vascularization of the retina is crucial for the development and functioning of the eye. The proper patterning of astrocytes is a key event preceding retinal angiogenesis by providing guidance cues for endothelial cells, yet how this is regulated still remains obscure. The dual function of proteoglycans in both extracellular matrix (ECM) composition and cell signal transduction suggests their potential in the regulation of astrocyte migration. The current study demonstrated that non-cell-autonomous regulation by neuroretina cell surface proteoglycan is crucial for PDGF-A regulated astrocyte migration. Ablation of glycosaminoglycan side chains of proteoglycans in neuroretina led to impaired astrocyte migration, incomplete retinal angiogenesis, and hyaloid vessel persistence. This is followed by severe photoreceptor degeneration as a result of reactive gliosis, which cannot be rescued by constitutively activated Kras signaling. Notably, inner limiting membrane (ILM), the basement membrane of the retina, was breached in proteoglycan-deficient retinae prior to the formation of astrocytic network. Herein we propose that cell surface proteoglycans are essential for the initial assembly of ILM, and this cannot be compensated by secreted ECM proteoglycans. In support of this, after removal of ILM in retinal explant by Collagenase digestion, establishment of a new ILM can be achieved by incubation with exogenous laminin-supplemented Matrigel. This basement membrane reconstitution failed, however, in proteoglycan-deficient retinae or in wild type samples digested with a combination of Heparinase and ChABC in addition to Collagenase. Taken together, our study reveals a novel function of neuroretinal cell surface proteoglycans in the initial assembly of basement membrane which subsequently serves as a permissive substratum necessary for astrocyte migration.

Indiana University-Purdue University Indianapolis (IUPUI) / The transcription factor and tumor suppressor protein p53 critically regulates cell survival or death in response to cellular stress. p53 can activate genes involved in a wide variety of processes, including apoptosis, cell cycle arrest, angiogenesis, metabolism, and senescence. Mutations in p53 are common in cancer and alter its interactions with other proteins, but there are other mechanisms and posttranslational modifications that can alter these interactions as well. In some tumors, such as renal cell carcinoma, p53 is commonly inactive even though mutations to TP53 are rare. This suggests that there are other biochemical mechanisms of inhibition, which we explore in this study. Mutations in the DNA-binding domain of p53 result in conformational changes that enable p53 to interact with and inhibit its family member p73, thereby promoting cell survival instead of apoptosis. In contrast, it has been reported that wild-type p53 does not bind to p73. We found that JNK-mediated phosphorylation of Thr81 in the proline-rich domain (PRD) of p53 enabled wild-type p53 to form a complex with p73. The dimerization of wild-type p53 with p73 facilitated the expression of apoptotic target genes such as PUMA and BAX, as well as the induction of apoptosis. In addition to the apoptotic function of p53, the tumor suppressor also plays a major role in the inhibition of angiogenesis. Here we also report a new mechanism where the Mdm2 oncoprotein can indirectly inactive p53 through the regulation of the tumor suppressor VHL. In response to hypoxia, VHL can bind p53, which results in activation of several anti-angiogenic targets of p53 such as THBS1 and COL18A1. Mdm2 regulates the VHL-p53 interaction by conjugating nedd8 to VHL within a region that is important for the VHL-p53 interaction, blocking the induction of anti-angiogenic genes and resulting in a proangiogenic phenotype. Due to its positive regulation of major proangiogenic proteins and its negative regulation of potent inhibitors of angiogenesis, we propose that the oncoprotein Mdm2 is the angiogenic switch. These findings refine our understanding of p53 interactions and activation, specifically for p53-p73 induced cell death and p53-VHL inhibition of angiogenesis. / 2020-08-05

Indiana University-Purdue University Indianapolis (IUPUI) / Fracture repair is costly and difficult to treat. One of the main causations of nonunion is a lack of essential blood supply. The needed blood is supplied by the growth of new blood vessels, a process known as angiogenesis, that invade the damaged tissue early in the healing process. We proposed using bone tissue engineering as an effective therapy. This therapy uses stem cells to aid in tissue regeneration. Endothelial progenitor cells (EPCs) were selected due to their ability to form tube-like networks in vitro. EPCs were isolated from murine bone marrow and lung tissue. We tested EPC’s tube forming, proliferative, and wound migration ability in vitro. To test their ability in vivo we created a femoral fracture in young and old mice. EPCs were seeded to the fracture site upon a collagen scaffold. The in vitro studies displayed that the bone marrow and lung-derived endothelial cells presented EPC traits. In the mouse fracture model bone marrow, endothelial cells did not significantly improve the healing process. In the future, we want to improve our cell extraction and purification method, as well as test a new stem cell delivery biomaterial. We also want to select and use a growth factor (GF) that can help to promote bone regeneration in tandem with the EPCs.

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Individuals with diabetes mellitus often develop complications that traditionally have been separated into microvascular pathologies, such as retinopathy, nephropathy and neuropathy, or macrovascular pathologies, including cardiovascular disease. Increasing evidence suggests that these micro- and macro-vascular complications may be linked. Our objective is to determine if direct effects of hyperglycemia on a microvascular bed that supplies cells in large arteries, the vasa vasorum, promotes diabetes-associated accelerated atherosclerosis. Normoglycemic apolipoprotein-E deficient (ApoE-/-) mice showed continuous atherosclerosis progression throughout the study that was directly correlated to increased vasa vasorum density with time. Hyperglycemic ApoE-/- Ins2+/Akita mice and streptozotocin-injected (STZ) ApoE-/- mice also demonstrated progressive plaque growth over time, but had accelerated atherosclerosis at 15 weeks of age compared to normoglycemic controls. The increased atherosclerosis in hyperglycemic mice correlated with impaired angiogenesis at 10 and 15 weeks of age. These mice showed increased expression for a marker of hypoxia in the atherosclerotic lesions yet decreased expression of vascular endothelial growth factor (VEGF), suggesting disruption of hypoxia-mediated angiogenesis. Cell culture experiments suggested that alternative splicing of an antiangiogenic form of VEGF in macrophages as well as post-translational modifications of macrophages and smooth muscle cells may contribute to reduced VEGF expression and decreased vasa vasorum neovascularization. After 25 weeks of age, vasa vasorum expansion plateaued in normoglycemic mice but continued to increase in hyperglycemic ApoE-/- STZ-injected mice. The increase in vasa vasorum neovascularization correlates to increases in plasma cholesterol. We have shown that hyperglycemia alters the microvascular structure of the vasa vasorum in two distinct mouse models of diabetes. Initially, elevations in glucose correlate to a significant reduction in lesion vascularization that results in increased lesional hypoxia that may promote the development and progression of atherosclerosis. At later time points there appears to be a burst of neovascularization that correlate with increases in cholesterol. / Thesis / Master of Science (MSc) / People with diabetes have elevated glucose levels that affect the vessels that distribute blood in our body. This puts them at higher risk of developing cardiovascular disease and having heart attacks and strokes. One set of vessels, known as the vasa vasorum, delivers blood to the walls of larger vessels. The primary goals of this study are 1) to determine if diabetes affects the vasa vasorum and, 2) to determine if changes to the vasa vasorum increase a person’s risk of developing cardiovascular disease. The results of this study show that diabetes in mice decreases the number of vasa vasorum vessels. The decrease in vasa vasorum blood vessels appears to influence the larger blood vessels they supply which promotes an environment that is more prone to cardiovascular disease. This information could be used in the future to develop drugs that target the vasa vasorum and possibly decrease cardiovascular events.

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