Browsing by Subject "Angiogenesis"
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Item Cross-talk between the skeletal muscle stem cells and endothelial cells(2018-03) Verma, MayankDuchenne muscular dystrophy (DMD) is a progressive neurodegenerative muscle disease caused by the absence of the dystrophin protein. While the muscle develops normally, it is susceptible to contraction-induced damage resulting in segmental necrosis. The damaged muscle is repaired by the resident stem cell, the satellite cell. However, after continuous rounds of regeneration/degeneration, the satellite cell pool is exhausted and the muscle fiber is replaced with fatty infiltrate and fibrosis. Although dystrophin is commonly studied in the muscle cells, its role in the vasculature has only recently been appreciated. The overall goal of research conducted in this thesis is to elucidate the role of the vascular endothelial cell, satellite cell, and their interactions in normal and DMD muscle. We have previously shown that when performed in developmental, there was increased angiogenesis and capillary density in mdx mice with the deletion of one allele for the Vascular Endothelial Growth Factor (VEGF) receptor, Flt1 gene. Interestingly, this led to an increase in muscle stem cells (satellite cells) and improved histological and contractile function. These data suggest that increasing the vasculature can increase the satellite cell pool and ameliorate the dystrophic phenotype seen in DMD model mice. However, the mechanism behind this interaction remains unclear. This thesis will attempt to fill in this gap in knowledge. In the following chapters (Figure 1), we identified VEGF receptors expressed on satellite cells and show that VEGFA binds to FLT1 to protect the cells from apoptosis. We investigated the cell-cell crosstalk between satellite cells and endothelial cells using 3-dimentional imaging. We showed that satellite cells secrete VEGFA to pattern the capillaries and in turn the endothelial cells keep the satellite cells in a quiescent state through expression of the notch ligand Delta-like protein 4 (DLL4). From a disease context, we utilized conditional Flt1 knockout mice to examine whether post-natal abolishment of Flt1 results in increased capillary density in the skeletal muscle and an improvement in the dystrophic phenotype in the mdx mice. Lastly, we utilized several strategies for recapitulating this phenomenon in a therapeutic manner. This will serve as a proof of concept to see whether FLT1 can be used as a drug target for the treatment of DMD. This information has applications beyond DMD as VEGF and its receptors are also under investigation for the treatment of peripheral artery disease, ischemic injury, as well as anti-cancer therapy. Outcomes from these studies will not only broaden our understanding of the juxtavascular niche for satellite cells but will also lead to the development of angiogenesis-targeted treatment options for DMD.Item Effect of mutant Endostatin and Kringle 5 fusion protein on Tumor angiogenesis.(2016-05) Venkatachalam, AnnapoornaAngiogenesis is important for the growth and metastasis of tumors. Endostatin is an endogenous inhibitor of angiogenesis and is shown to inhibit bFGF and VEGF induced signaling in endothelial cells. Recent evidence also indicates that it binds to αν and α5 group of integrins expressed endothelial cells and inhibits downstream signaling. Kringle 5, a potent anti-angiogenic molecule causes apoptosis of endothelial cells by associating with glucose-regulated protein 78 (GRP78). Previous studies from our laboratory have shown that a mutant (P125A) form of Endostatin (derived from collagen type XVIII) and Kringle 5 fragment of plasminogen can inhibit angiogenesis by inducing autophagy. P125A-endostatin and Kringle 5 interfere with distinct signaling pathways in endothelial cells. We hypothesized that a chimeric protein made of P125A-endostatin and Kringle 5 will have better anti-angiogenic activities. To test this hypothesis, we constructed a fusion protein consisting of P125A-endostatin and Kringle 5 (E-K5). Recombinant fusion protein was expressed in yeast and purified. E-K5 was found to inhibit proliferating endothelial cells and effectively blocked tumor induced angiogenesis. Anti-proliferative activity of E-K5 was linked to VEGF receptor and ανβ3 integrin-mediated signaling pathways. These studies establish the therapeutic potential of E-K5 as a potent anti-angiogenic molecule.Item The effects of iron deficiency on the developing brain vasculature(2016-10) Nguyen, Thu AnIron deficiency is amongst the most severe and important micronutrient deficiencies, affecting millions of people globally, especially pregnant women and young children. Particularly, iron deficiency anemia (IDA) adversely affects the cognitive performance, behavior and growth of infants, preschool and school-aged children. Recently it was demonstrated that iron deficiency (FeD) during gestational and early postnatal life, induces angiogenesis/vasculogenesis in the developing neonatal rat brain. Previously, the impacts of FeD on the developing brain were reported in postnatal rats. Although FeD and thyroid hormone disruption share similar brain development deficits, combined FeD and impaired thyroidal status did not alter the developing brain vasculature in neonatal rats. Hence, the altered vasculature is an important independent effect of FeD on the developing brain. In this study, I further demonstrated that the angiogenesis effect of FeD is evident since birth and continues into the postnatal stage. Moreover, previous research showed restoration of other aspects of FeD’s impacts on the neonatal rat brain with Fe repletion or supplementation. Therefore, I hypothesized that the angiogenesis effects in developing Fe deficient rat brains are reversible with Fe repletion. qPCR data showed an increase in brain mRNA level of endothelial cell marker genes and angiogenesis associated genes in the developing iron deficient rat brain compared to control at birth, postnatal days 7 and 14. FeD also increases vessel branch points suggesting the FeD induced angiogenesis occurs since birth in the neonatal rat brains. After iron repletion on postnatal day 7, mRNA level of the same genes and vessel branch points had no significant increase compared to control at postnatal day 14 and returned to approximately the same level as control at postnatal day 30. These results suggest that FeD induced angiogenesis could be reversible as evident by vessel regression upon iron repletion. Brain angiogenesis is critically regulated by hypoxia-inducible factor 1 (HIF1) which is activated during hypoxia. I hypothesized that neonatal FeD results in tissue hypoxia and activates hypoxia-inducible factor 1 alpha (HIF1α) leading to induction of expression of several genes associated with angiogenesis. Western blot densitometry indicated no significant difference in HIF1α expression in Fe deficient brains compared to control at postnatal day 7 and 14. Finally, since it is unknown whether the induced vasculature is beneficial or not to the Fe deficient developing brain, we also assessed mRNA level of an endothelial integrity marker, plasmalemmal vesicle- associated protein, PLVAP or PV-1 that was expressed in a fenestrated blood brain barrier. The mRNA level of PV-1 gene was not altered in both FeD and Fe repletion groups, however, PV-1 specific roles as a BBB integrity marker in the process of vessel sprouting remains to be determined. Overall, this study on the independent effects of FeD on the developing brain vasculature demonstrated reversibility of the induced blood vessels outgrowth with Fe repletion at postnatal day 7, however, it is unclear if hypoxic signaling through the HIF1 pathway via HIF1α activation contributes to brain angiogenesis due to FeD.Item The Functional Role Of Mir-210 In Hypoxia-Induced Angiogenesis(2017-02) Schnettler, EricaHypoxia induced microRNA-210 is implicated in ischemic disorders and in tumor progression. In the present study, we have used a knock out mouse model to investigate how miR-210 regulates angiogenesis. Our studies show that miR-210 sensitizes endothelial cells to bFGF-mediated signaling by targeting FGFRL1 which lacks a receptor kinase domain. FGFRL1 was found to be a negative regulator of bFGF induced pro-angiogenic signaling. In the absence of miR-210, FGFRL1 levels are increased in endothelial cells and as a consequence dampened hypoxia-induced vessel sprouting. miR-210 KO mice showed reduced angiogenesis of FGF-containing matrigel plugs. Furthermore, tumor angiogenesis was attenuated in miR-210 KO animals. These data suggest that miR-210 targets FGFRL1 and sensitizes endothelial cells to bFGF and regulates tumor angiogenesis.Item Non-canonical pathways of HIF1-alpha regulation in ovarian cancer: implications for tumor angiogenesis and metastasis.(2011-10) Joshi, Hemant PrakashThe high rates of mortality associated with epithelial ovarian cancer (EOC) are a direct consequence of its metastatic nature. Activation of angiogenesis is a significant factor in generation of metastases and is contingent upon the cellular response to hypoxia within the tumor microenvironment. Hypoxia-inducible factor 1 (HIF1) is a transcription factor composed of HIF1α and HIF1β subunits and is the master regulator of the hypoxic response. Hypoxia and HIF1 are therefore critical mediators of tumor angiogenesis and metastasis. Regulation of HIF1 is primarily at the level of protein. In normoxia, the HIF1α subunit is hydroxylated via an oxygen- and iron-dependent mechanism and targeted for destruction. In hypoxia, low oxygen levels preclude hydroxylation and HIF1α is stabilized, allowing for its association with constitutively expressed HIF1β to form bioactive HIF1. We have identified two novel mechanisms of HIF1α regulation that are oxygen-responsive in EOC cells (EOCCs). The first involves dynamins, a class of proteins involved in endocytic processes such as transferrin/iron uptake. Exposing EOCCs to hypoxic conditions results in lower levels of dynamin 2. Impairment of dynamin 2 activity in normoxia causes accumulation of HIF1α protein due to a rapid decrease in intracellular iron levels and HIF1α polyubiquitination. Treatment with a form of iron that is not dependent on dynamins for endocytosis reverses this effect. Conversely, overexpression of dynamin 2 in hypoxia results in suppression of HIF1α protein levels. A second novel mechanism of HIF1α control involves microRNAs (miRNAs), ~22 nucleotide, non-coding RNA molecules that repress translation of target mRNAs by binding their 3' untranslated regions (UTRs). Using microarray and qPCR analysis, we found that exposing EOCCs to hypoxia reduced levels of miR-199a-1, a miRNA that is located in an intron within the dynamin 2 gene and is predicted in silico to target the HIF1α 3' UTR. We further demonstrated that miR-199a-1 directly targets the HIF1α 3' UTR and overexpression of miR-199a-1 suppresses HIF1α protein levels and HIF1-driven gene expression. Moreover, cells stably overexpressing miR-199a-1 exhibit marked defects in migratory ability. We corroborated these findings in vivo by overexpressing miR-199a-1 in a mouse model of metastatic EOC and found significant reductions in tumor vessel density and tumor burden. Together, these findings provide insight into non-canonical, dynamin-dependent and miRNA-based mechanisms of HIF1 regulation that may have important implications in the progression of EOC.