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

Abstract

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...

Protein Phosphotyrosine Phosphatase 1B (PTP1B) in Calpain-dependent Feedback Regulation of Vascular Endothelial Growth Factor Receptor (VEGFR2) in Endothelial Cells

“…Angiogenic assay of mouse aortic discs was carried out as previously described (33). Briefly, aortas from siRNA-injected mice were harvested and cleaned of connective tissues.…”

Axon guidance factor netrin-1 and its receptors regulate angiogenesis after cerebral ischemia

“…Newly-formed vessels induced by netrin-1 contain an intact EC monolayer surrounded by multiple cell layers. Nguyen et al [55] demonstrated that netrin-1 induces angiogenesis by increasing endothelial nitric oxide production via a DCC-dependent ERK1/2-eNOS feed-forward mechanism in aortic ECs. All these studies suggest that netrin-1 is a pro-angiogenic factor, albeit with some discrepancies (Table 1).…”

“…Stimulate EC Proliferation, migration, and TF [53] Independent of UNC5B, DCC, neogenin/--Increase endothelial NO production [55] DCC Promote vascular development [52] UNC5B/-----Facilitate focal angiogenesis in adult [54] ------Anti-angiogenic effect Block EC migration [27] UNC5B…”

Netrin‐1 Promotes Inflammation Resolution to Achieve Endothelialization of Small‐Diameter Tissue Engineering Blood Vessels by Improving Endothelial Progenitor Cells Function In Situ

“…Previous studies have found that netrin‐1 is present in the ECs of natural blood vessels and can promote angiogenesis through uncoordinated‐5 homolog B (UNC5B) and deleted in colorectal cancer (DCC) receptors 18, 19, 20. Additionally, netrin‐1 prevents ischemia and exerts anti‐inflammatory effects by binding to the adenosine receptor A2b 21…”