Abstract
Endothelin (ET) synthesis is enhanced at sites of ischemia or in injured vessels. The purpose of this study was to explore the possibility of autocrine stimulation of endothelial cell migration by members of the endothelin family. Experiments with microvascular endothelial cell transmigration in a Boyden chemotactic apparatus showed that endothelins 1 and 3, as well as a selective agonist of ETB receptor IRL-1620, equipotently stimulated migration. Endothelial cell migration was unaffected by the blockade of ETA receptor, but it was inhibited by ETB receptor antagonism. Based on our previous demonstration of signaling from the occupied ETB receptor to constitutive nitric oxide (NO) synthase (Tsukahara, H., Ende, H., Magazine, H. I., Bahou, W. F., and Goligorsky, M. S. (1994) J. Biol. Chem. 269, 21778-21785), we next examined the contribution of ET-stimulated NO production to endothelial cell migration. In three independent cellular systems, 1) migration and wound healing by microvascular endothelial cells, 2) wound healing by Chinese hamster ovary cells stably expressing ETB receptor with or without endothelial NO synthase, and 3) application of antisense oligodeoxynucleotides targeting endothelial NO synthase in human umbilical vein endothelial cells, an absolute requirement for the functional NO synthase in cell migration has been demonstrated. These findings establish the permissive role of NO synthesis in endothelin-stimulated migration of endothelial cells.
Highlights
Formation of new blood vessels, driven by the morphogenetic program and/or by the functional demand for increased blood supply, is initiated by the budding of endothelial cells off the microcirculatory bed
Tissue hypoxia represents a physiologic stimulus for angiogenesis, and several autocrine angiogenic mediators produced by endothelial cells have been recognized [1,2,3]
Since we and others have previously demonstrated that occupancy of ETB receptors in endothelial cells is accompanied by the activation of constitutive endothelial NO synthase [9, 10], we hypothesized that the observed motogenic effects of members of the ET family may be mediated by the release of NO
Summary
Formation of new blood vessels, driven by the morphogenetic program and/or by the functional demand for increased blood supply, is initiated by the budding of endothelial cells off the microcirculatory bed. Tissue hypoxia represents a physiologic stimulus for angiogenesis, and several autocrine angiogenic mediators produced by endothelial cells have been recognized [1,2,3]. Leibovich et al [13] have shown that production of angiogenic activity by activated monocytes (assayed by chemotaxis of endothelial cells and corneal angiogenesis) is absolutely dependent on L-arginine and NO synthase. These observations are in concert with findings reported by Ziche et al [12] who detected the potentiation by sodium nitroprusside of the angiogenic effect of substance P in the rabbit cornea. We demonstrated that endogenous NO production by the endothelial cells is a prerequisite for the motogenic and angiogenic effects of this factor. In the present study, we used three different approaches (migration and wound healing by endothelial cells, wound healing by Chinese hamster ovary cells expressing ETB receptor with or without eNOS, and application of antisense oligodeoxynucleotides targeting eNOS) to provide evidence that the effect of ET-1 on cell migration is mediated via ETB receptor and requires functional enzymatic machinery for NO generation
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