The matricellular extracellular matrix protein thrombospondin-1 (TSP1) stimulates focal adhesion disassembly through a sequence (known as the hep I peptide) in its heparin-binding domain. This mediates signaling through a receptor co-complex involving calreticulin and low-density lipoprotein (LDL) receptor-related protein (LRP). We postulate that this transition to an intermediate adhesive state enhances cellular responses to dynamic environmental conditions. Since cell adhesion dynamics affect cell motility, we asked whether TSP1/hep I-induced intermediate adhesion alters cell migration. Using both transwell and Dunn chamber assays, we demonstrate that TSP1 and hep I gradients stimulate endothelial cell chemotaxis. Treatment with focal adhesion-labilizing concentrations of TSP1/hep I in the absence of a gradient enhances endothelial cell random migration, or chemokinesis, associated with an increase in cells migrating, migration speed, and total cellular displacement. Calreticulin-null and LRP-null fibroblasts do not migrate in response to TSP1/hep I, nor do endothelial cells treated with the LRP inhibitor receptor-associated protein (RAP). Furthermore, TSP1/hep I-induced focal adhesion disassembly is associated with reduced chemotaxis to basic fibroblast growth factor (bFGF) but enhanced chemotaxis to acidic (a)FGF, suggesting differential modulation of growth factor-induced migration. Thus, TSP1/hep I stimulation of intermediate adhesion regulates the migratory phenotype of endothelial cells and fibroblasts, suggesting a role for TSP1 in remodeling responses.
Osteopontin is an Arg-Gly-Asp (RGD)–containing acidic glycoprotein postulated to mediate cellular adhesion and migration in a growing number of normal and pathological conditions through interaction with integrin molecules. In this report, we have investigated the potential contributions of osteopontin and one of its receptors, the αvβ3 integrin, to endothelial regenerative processes by using both in vivo and in vitro models. In vivo, uninjured rat arterial endothelium had undetectable levels of osteopontin and β3-integrin mRNA by in situ hybridization. After balloon catheter denudation, osteopontin mRNA levels correlated temporally and spatially with active endothelial proliferation and migration, with the highest levels observed at the wound edge between 8 hours and 2 weeks after injury, declining to uninjured levels at 6 weeks, when regeneration was complete. Osteopontin protein levels, as determined by immunocytochemistry, paralleled the time course of mRNA expression. Likewise, β3-integrin mRNA and protein levels were substantially elevated in regenerating endothelial cells but were not detectable in uninjured or healed endothelium. In vitro, rat smooth muscle cell–derived and bacterial expressed mouse recombinant osteopontins both stimulated the adhesion and directed migration of bovine aortic endothelial cells through interactions with the αvβ3 receptor. Structural mutants of osteopontin confirmed the importance of the RGD domain for both adhesion and migration of endothelial cells through αvβ3. These data suggest important roles for osteopontin and β3 integrin in regenerating endothelium.
NADPH oxidase has been shown to play an important role in cardiovascular biology. The goal of the present study was to determine whether NADPH oxidase activity is important for endothelial cell growth and migration. In proliferation assays, growth factor- or serum-induced DNA synthesis in three different types of human endothelial cells was abrogated by inhibitors of NADPH oxidase, but not by inhibitors of xanthine oxidase or nitric oxide synthase. Moreover, vascular endothelial growth factor-induced migration of human endothelial cells was suppressed in the presence of NADPH oxidase inhibitors. These results support a potential role for NADPH oxidase in mediating angiogenesis.
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Endothelial cell (EC) migration is a critical event during multiple physiological and pathological processes. ECs move in the plane of the endothelium to heal superficially injured blood vessels but migrate in three dimensions during angiogenesis. We herein investigate differences in these modes of movement focusing on caveolae and their defining protein caveolin-1. Using a novel approach for morphological analysis of transmigrating cells, we show that ECs exhibit a polarized distribution of caveolin-1 when traversing a filter pore. Strikingly, in these cells caveolin-1 seems to be released from caveolar structures in the cell rear and to relocalize at the cell front in a cytoplasmic form. In contrast, during planar movement caveolin-1 is concentrated at the rear of ECs, colocalizing with caveolae. The phosphorylatable Tyr14 residue of caveolin-1 is required for polarization of the protein during transmigration but does not alter polarization during planar movement. Palmitoylation of caveolin-1 is not essential for redistribution of the protein during either mode of movement. Thus, ECs migrating in three dimensions uniquely exhibit dissociation of caveolin-1 from caveolae and phosphorylation-dependent relocalization to the cell front.
To test the hypothesis that the phosphatidylinositol 3-kinase (PI3 kinase)/protein kinase Akt signaling pathway is involved in nitric oxide (NO)-induced endothelial cell migration and angiogenesis, we treated human and bovine endothelial cells with NO donors, S-nitroso-l-glutathione (GSNO) and S-nitroso-N-penicillamine (SNAP). Both GSNO and SNAP increased Akt phosphorylation and activity, which were blocked by cotreatment with the PI3 kinase inhibitor wortmannin. The mechanism was due to the activation of soluble guanylyl cyclase because 8-bromo-cyclic GMP activated PI3 kinase and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ) blocked NO-induced PI3 kinase activity. Indeed, transfection with adenovirus containing endothelial cell NO synthase (eNOS) or protein kinase G (PKG) increased endothelial cell migration, which was inhibited by cotransfection with a dominant-negative mutant of PI3 kinase (dnPI3 kinase). In a rat model of hind limb ischemia, adenovirus-mediated delivery of human eNOS cDNA in adductor muscles resulted in time-dependent expression of recombinant eNOS, which was accompanied by significant increases in regional blood perfusion and capillary density. Coinjection of adenovirus carrying dnPI3 kinase abolished neovascularization in ischemic hind limb induced by eNOS gene transfer. These findings indicate that NO promotes endothelial cell migration and neovascularization via cGMP-dependent activation of PI3 kinase and suggest that this pathway is important in mediating NO-induced angiogenesis.
We have recently identified a novel protein, named angiomotin, by its ability to bind the angiogenesis inhibitor angiostatin in the yeast two-hybrid system. Angiomotin belongs to a family with two other members, AmotL-1 and -2 characterized by coiled-coil and C-terminal PDZ binding domains. Here we show that the putative PDZ binding motif of angiomotin serves as a protein recognition site and that deletion of three amino acids in this site results in inhibition of chemotaxis. Furthermore, endothelial cells expressing mutant angiomotin failed to migrate and form tubes in an in vitro tube formation assay. To study the effect of angiomotin on embryonic angiogenesis, we generated transgenic mice expressing wild-type angiomotin and the C-terminal deletion mutant driven by the endothelial cell-specific receptor tyrosine kinase (TIE) promoter. Expression of mutant angiomotin in endothelial cells inhibited migration into the neuroectoderm and intersomitic regions resulting in death at embryonic day 9.5. In contrast, mice expressing wild-type angiomotin developed normally and were fertile. These results suggest that the putative PDZ binding motif of angiomotin plays a critical role in regulating the responsiveness of endothelial cells to chemotactic cues.