Regulation of retinal endothelial cells and pericytes by VEGF, TGF-beta1, and SPARC

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Regulation of retinal endothelial cells and pericytes by VEGF, TGF-beta1, and SPARC

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Title: Regulation of retinal endothelial cells and pericytes by VEGF, TGF-beta1, and SPARC
Author: Yan, Qi, 1962-
Abstract: Ocular diseases characterized by retinal neovascularization are among the principal causes of visual loss worldwide. In an effort to understand the components of the retinal angiogenesis and its regulation by various molecules, I developed a procedure to isolate retinal microvascular endothelial cells (EC) and pericytes from the primate and bovine species. This was important because retinal capillary EC are different from EC derived from other vascular beds. Retinal EC expressed von Willebrand factor, acetylated low-density lipoprotein, CD31, and did not contain smooth muscle a-actin. Monkey retinal EC could be maintained to passage 3, whereas bovine retinal EC could be maintained to passage 8. Subconfluent cultures of monkey retinal EC secreted extracellular matrix proteins that included fibronectin, laminin, collagen types I and IV, and SPARC. VEGF and bFGF both stimulated retinal EC replication, which was also elevated under hypoxia. The synergism of VEGF and bFGF on retinal EC proliferation and cell invasion was observed in a 3-dimensional assay but not in 2-dimensional dish culture.Unlike VEGF and bFGF, the role of transforming growth factor (TGF)-$\beta1$ in angiogenesis has been controversial. The effects of TGF-$\beta1$ on retinal microvascular cells are not fully defined. I have found that TGF-$\beta1$ inhibited the proliferation of both retinal EC and pericytes in a concentration-dependent manner. Moreover, TGF-$\beta1$ induced specifically apoptotic cell death in retinal EC but not in pericytes. Apoptosis of retinal EC mediated by TGF-$\beta1$ was associated with a decreased level of the cyclin-dependent kinase inhibitor p21$\rm\sp{waf1/cip1},$ compared with that observed in the apoptosis-resistant cells. In contrast, the translation product of the tumor suppressor gene p53 was increased in the TGF-$\beta1$-treated apoptotic cells, suggesting p53 mediated retinal EC apoptosis is independent of p21$\rm\sp{waf1/cip1}.$ Thus, I have proposed that p21$\rm\sp{waf1/cip1}$ and p53 function in distinct pathways that are protective or permissive, respectively, for the apoptotic signals mediated by TGF-$\beta1.$SPARC is expressed in a variety of tissues during embryogenesis and remodeling, and is believed to regulate vascular morphogenesis and cellular differentiation. Although usually limited in normal adult tissues, SPARC is expressed at significant levels in the adult bovine retina, whereas newborn calf retina showed a similar expression pattern with a greatly reduced level. Strong reactivity with anti-SPARC antibody was found in the soma of ganglion cells and their axons. SPARC was also present in retinal astrocytes but not in Mueller cells, capillaries of the inner retina but not large vessels. To determine the significance of SPARC expression in retina, I test two hypotheses. (1) SPARC has counteradhesive properties that can cause cell rounding, and would thus induce retinal EC apoptosis in vitro; (2) SPARC is one of the endogenous inhibitors that either maintain the quiescence of retinal EC under normal physiological conditions or inhibit retinal neovascularization. My conclusions are that SPARC or its peptides did not induce retinal EC apoptosis in vitro under the experimental conditions used; SPARC and its peptides were potent inhibitors of retinal EC replication, as they effectively inhibited EC proliferation induced by retinal extract. Future study will focus on testing the role of SPARC in retinal neovascularization through the use of wild-type vs. SPARC-null mice in an animal model of the retinopathy, and on delineating mechanisms whereby SPARC regulates retinal pericyte function.
Description: Thesis (Ph. D.)--University of Washington, 1998

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