Dephosphorylation of endothelial nitric oxide synthase contributes to the anti‐angiogenic effects of endostatin

147

152

Netrin-1 is critical for axonal pathfinding which shares similarities with formation of vascular network. Here we report that netrin-1 induction of angiogenesis is mediated by an increase in endothelial nitric oxide (NO ⅐ ) production, which occurs via a DCC-dependent, ERK1͞2-eNOS feed-forward mechanism. Exposure of mature aortic endothelial cells to netrin-1 resulted in a potent, dose-dependent increase in NO ⅐ production, detected by electron spin resonance. Scavenging NO ⅐ with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) abolished netrin-1 stimulated angiogenesis. Netrin-1-stimulated NO ⅐ production or angiogenesis was inhibited by DCC antibody, DCC small interfering RNA (siRNA), specific inhibitors (PD98059, U0126), or siRNAs for MEK1͞2. PTIO attenuated ERK1͞2 phosphorylation, indicating a feed-forward mechanism. Netrin-1 induced a time-dependent phosphorylation of eNOS s1179, s116 and a rapid dephosphorylation of eNOSt497. Only eNOS s1179 was sensitive to U0126 or PTIO. These data characterized a mechanism whereby netrin-1 promotes angiogenesis, which may broadly relate to cardiovascular, neuronal and cancer physiology.nitric oxide N etrin-1 is one of the first discovered axon-guiding molecules that are critical for neuronal development (1, 2). It is a secreted protein that is released to circulation after being produced by a variety of cells (3). Upon activation of its attraction-selective receptor deleted in colorectal cancer (DCC) or neogenin, netrin-1 induces axonal outgrowth and crossover through the midline (2, 4); this is at least partially mediated by activation of the mitogen activated protein kinase ERK1͞2 (5).Formation of vascular network shares many similarities with neuronal pathfinding (6). A number of mitogens including VEGF and EGF have been shown to regulate endothelial cell growth and proliferation. Interestingly, netrin-1 is structurally homologous to the endothelial mitogens. It has an N-terminal type IV laminin repeat, followed by three cystein-rich EGF modules and a positively charged C-terminal domain. A recent study demonstrated that netrin-1 stimulates growth of umbilical vein endothelial cells and vascular smooth muscle (7). However, in the presence of the repulsive receptor UNC5B in developing capillaries, netrin-1 induces endothelial filopodial retraction (8). Our current study uniquely studied proliferation and migration of adult mature aortic endothelial cells, and endothelial outgrowth from adult mouse aortic discs to examine the angiogenic effects of netrin-1. Compared to umbilical vein or developing vessels, aorta shares the closest physiology with large adult coronary arteries where therapeutic angiogenesis is beneficial for patients with ischemic coronary artery diseases (9, 10). More importantly, the signaling mechanisms underlying netrin-1 modulation of angiogenesis were identified.Using the highly specific and sensitive electron spin resonance technology for direct and characteristic measurement of nitric oxide gas radical (NO ⅐ ), we examined a potent...

Section: Discussionsupporting

confidence: 88%

Netrin-1 induces angiogenesis via a DCC-dependent ERK1/2-eNOS feed-forward mechanism

Nguyen

Cai

2006

147

152

“…Endostatin and TNP470 both regulate eNOS (20,21), which is upstream of the two TSP1-sensitive steps that we identified in this signaling cascade. Our evidence that TSP1 inhibits at least two targets downstream of eNOS implicates the eNOS-sGC-cGMP pathway as a convergence point for proangiogenic and antiangiogenic signaling.…”

Section: Discussionmentioning

confidence: 83%

“…TNP470 inhibits myristoylation of eNOS and redistributes eNOS from the plasma membrane (20). Endostatin increases activity of the phosphatase PP2A, which dephosphorylates eNOS at Ser-1177 and decreases NO synthesis (21).…”

mentioning

confidence: 99%

Thrombospondin-1 inhibits endothelial cell responses to nitric oxide in a cGMP-dependent manner

Isenberg

Ridnour

Perruccio

et al. 2005

240

261

Redox signaling plays an important role in the positive regulation of angiogenesis by vascular endothelial growth factor, but its role in signal transduction by angiogenesis inhibitors is less clear. Using muscle explants in 3D culture, we found that explants from mice lacking the angiogenesis inhibitor thrombospondin-1 (TSP1) exhibit exaggerated angiogenic responses to an exogenous NO donor, which could be reversed by providing exogenous TSP1. To define the basis for inhibition by TSP1, we examined the effects of TSP1 on several proangiogenic responses of endothelial cells to NO. NO has a biphasic effect on endothelial cell proliferation. The positive effect at low doses of NO is sensitive to inhibition of cGMP signaling and picomolar concentrations of TSP1. NO stimulates both directed (chemotactic) and random (chemokinetic) motility of endothelial cells in a cGMP-dependent manner. TSP1 potently inhibits chemotaxis stimulated by NO. Low doses of NO also stimulate adhesion of endothelial cells on type I collagen in a cGMP-dependent manner. TSP1 potently inhibits this response both upstream and downstream of cGMP. NO-stimulated endothelial cell responses are inhibited by recombinant type 1 repeats of TSP1 and a CD36 agonist antibody but not by the N-terminal portion of TSP1, suggesting that CD36 or a related receptor mediates these effects. These results demonstrate a potent antagonism between TSP1 and proangiogenic signaling downstream of NO. Further elucidation of this inhibitory signaling pathway may identify new molecular targets to regulate pathological angiogenesis.angiogenesis ͉ cell adhesion ͉ chemotaxis ͉ proliferation ͉ CD36 R edox signaling plays a central role in both developmental and pathological angiogenesis (1). Low to moderate concentrations of NO act on endothelial cells to stimulate angiogenesis by activating soluble guanylyl cyclase (sGC), thereby increasing cGMP, which activates cGMP-dependent protein kinases (2) and other cGMP-dependent pathways (3, 4). These responses lead to downstream activation of the Ras-Raf-extracellular-regulated kinase (ERK) (5), vasodilator-stimulated phosphoprotein (6), and phosphatidylinositol 3-kinase-Akt pathways (7). At specific doses, NO donors induce endothelial cell chemotaxis (7,8), permeability (9), and proliferation in vitro (10, 11) and angiogenesis in vivo (12).Consistent with these observations, endogenous NO synthesis in endothelial cells is induced by some angiogenic factors, including vascular endothelial growth factor (VEGF) (13). VEGF receptor signaling activates Akt in a phosphatidylinositol 3-kinase-and PKC-dependent manner, which phosphorylates endothelial NO synthase (eNOS) on Ser-1177 (14, 15). This modification increases eNOS activity and NO synthesis. eNOS activation plays a crucial role in VEGF-induced angiogenesis and vascular permeability (16,17). Conversely, NO induces VEGF transcription by inducing synthesis and stabilization of . These data suggest a positive feedback loop between NO and VEGF signaling. However, inhibitory effects of ...

“…Vascular endothelial growth factor (VEGF) is a critical cytokine involved in angiogenesis, and nitric oxide (NO) is a downstream effector (2,3). VEGF increases NO levels in endothelial cells (ECs) by activating endothelial NO synthase (eNOS͞NOS3) (4)(5)(6)(7). Recently, the role of eNOS in EC migration has been demonstrated in vivo and in vitro (8,9) but the precise mechanism by which NO regulates migration is unknown.…”

mentioning

confidence: 99%

Matrix metalloproteinase 13 mediates nitric oxide activation of endothelial cell migration

López-Rivera

Lizarbe

Martínez‐Moreno

et al. 2005

To explore the mechanisms by which NO elicits endothelial cell (EC) migration we used murine and bovine aortic ECs in an in vitro wound-healing model. We found that exogenous or endogenous NO stimulated EC migration. Moreover, migration was significantly delayed in ECs derived from endothelial NO synthase-deficient mice compared with WT murine aortic EC. To assess the contribution of matrix metalloproteinase (MMP)-13 to NO-mediated EC migration, we used RNA interference to silence MMP-13 expression in ECs. Migration was delayed in cells in which MMP-13 was silenced. In untreated cells MMP-13 was localized to caveolae, forming a complex with caveolin-1. Stimulation with NO disrupted this complex and significantly increased extracellular MMP-13 abundance, leading to collagen breakdown. Our findings show that MMP-13 is an important effector of NO-activated endothelial migration.angiogenesis ͉ endothelium ͉ vasular remodeling W ound healing is an orchestrated cascade of enzymatic activities that converge toward damage repair. Wound healing involves inflammation and angiogenesis and is tightly regulated by cytokines (1). Vascular endothelial growth factor (VEGF) is a critical cytokine involved in angiogenesis, and nitric oxide (NO) is a downstream effector (2, 3). VEGF increases NO levels in endothelial cells (ECs) by activating endothelial NO synthase (eNOS͞NOS3) (4-7). Recently, the role of eNOS in EC migration has been demonstrated in vivo and in vitro (8, 9) but the precise mechanism by which NO regulates migration is unknown.ECs migrate as a result of an injury and during angiogenesis from preexisting vessels. The process is tightly regulated by matrix turnover, in which matrix metalloproteinases (MMPs) play a pivotal role (10-12). We have previously reported that NO induces MMP-13 expression and activity in bovine aortic ECs (BAECs) (13,14).MMPs are extracellular matrix-degrading endopeptidases. MMP expression and activity can be found in physiological and pathological situations, such as tissue development, atherosclerosis, ovarian function, arthritis, osteoarthritis, cancer, angiogenesis, and wound healing (15). MMP-13 was initially discovered in mammalian cell carcinomas and is also expressed by several cell types, including endothelium (13,(16)(17)(18).Here, we present evidence that MMP-13 is a downstream effector of NO-activated EC movement. We have developed a wound-healing model in BAECs and aortic cells from eNOS WT and eNOS-deficient mice. We show that aortic ECs lacking MMP-13 experience delayed migration, and aortic ECs from eNOS null mice present delayed cell migration and a significant decrease in MMP-13 expression. In addition, we present data showing that MMP-13 exists in association with caveolin-1 in resting cells, and that this complex is disrupted in the presence of NO, leading to the secretion of MMP-13 to the extracellular media. We postulate that NO induces EC movement in part via the disruption of the MMP-13͞caveolin-1 complex, which in turn releases the secretion of active MMP-13 to the ...