Analyses of lymphatic vessel network development in the mouse mesentery : a dissertation /
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| Format: | Thesis Book |
| Language: | English |
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[College Station, Tex.] :
[Texas A&M University System Health Science Center],
[2009]
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| Subjects: |
| Abstract: | ABSTRACT: Regulatory mechanisms for angiogenesis are relatively well established compared to lymphangiogenesis. There is evidence that a combination of vascular endothelial growth factors VEGF-A/C with hypoxia or collagen matrix promotes the development of lymphatic-like structures, along with blood vessels in mouse embryoid bodies (EB). In this study we tested the hypothesis that hypoxia combined with prolonged VEGF-A/C treatment would induce early lymphangiogenesis in addition to angiogenesis in mouse EBs, while under similar conditions specific extracellular matrix (ECM) proteins would promote the development of lymphatic-like structures over angiogenesis. EBs were subjected to four conditions and were maintained under normoxia and hypoxia (21% and 2.6% O[2], respectively) with or without VEGF-A/C. Transcriptional microarray and immunofluorescence analyses of normoxic and hypoxic EBs, showed very low expression of the early lymphatic endothelial cell (LEC) markers, LYVE-1 and Prox1 at early time points. Double immunofluorescence using MEA32 and Prox1/LYVE-1 demonstrated that combined hypoxia and VEGF-A/C treatment promoted the formation of blood vessel-like structures in EBs at E22.5, whereas only unorganized Prox1[superscript+]/LYVE-1[superscript+] LECs were detected in these EBs. We then hypothesized that EB-derived LECs grown on collagen-I would promote a more migratory and proliferative phenotype, while those grown on laminin would promote differentiation of EBs. Our data revealed that in EBs, at E36.5, LECs attached better to collagen-I, resulting in an organized network of lymphatic-like structures as compared to EBs grown on laminin at the same time point. Also, at E36.5, EBs grown on collagen-I favored the formation of lymphatic-like structures, while blood vessel-like structures were repressed. Collectively, our data demonstrate that hypoxia combined with growth factors promotes angiogenesis, whereas the combination of these conditions with specific ECM proteins facors lymphangiogenic processes in mouse EBs. In addition, these processes are very carefully timed and orchestrated in embryonic development. During vascular development, after the formation of endothelium-lined vessels, the next phase is maturation and remodeling. This phase involves two critical events, valve formation and muscle cell recruitment. The mechanisms regulating lymphatic maturation are still being unraveled. In this dissertation, we employed mouse whole-mount mesenteric immunohistochemistry techniques to investigate the spatial and temporal patterns of eNOS expression and muscle cell investiture, as they relate to the maturation and remodeling of lymphatic networks. We hypothesized that valve formation in collecting lymph vessels would precede muscle cell investiture. Our findings demonstrate that the earliest investiture of smooth muscle actin positive (SMA[superscript+]) muscle cells occurs a day after valve formation, at E17.5 and coincides with the onset of eNOS expression. We also hypothesized that hydrodynamic forces such as shear stress derived by lymph flow or 'function' would serve as an impetus for moderating 'form' or maturation of lymphatic networks during development. Results from E17.5 data analyses demonstrated that at valve sites, eNOS expression, possibly an indication of lymph flow-derived shear stress, was confined intimately with recruited SMA[superscript+] muscle cells in both a temporal and a spatial manner. Our findings also demonstrate that eNOS-associated segments at E17.5 and E18.5 begin to become specified into a collecting lymphatic phenotype, by the down-regulation of LYVE-1 along the vessel segment, and the up-regulation of Podoplanin and Prox1 at the valve sites. In addition, we established a qualitative and quantitative approach to characterize the remodeling of complex disorganized embryonic lymphatic networks into relatively more organized adult vessel networks. Furthermore the data provides evidence that the remodeling of lymphatic networks begins during late embryonic time points, but continues after birth and during postnatal week 1. |
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| Item Description: | Vita. "Major Subject: Medical Sciences". "Submitted to the Office of Research and Graduate Studies of The Texas A&M University System Health Science Center in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 2009." Approved as to style and content by: Mariappan Muthuchamy, David C. Zawieja, Emily Wilson, James L. Lessard, Harris J. Granger. |
| Physical Description: | xiii, 190 leaves : illustrations ; 28 cm. |
| Bibliography: | Includes bibliographical references (leaves 174-185). |