Effect Of Nerve Growth Factor On Angiogenesis Of Murine Aorta Ring And Apoptosis Of Human Umbilical Vein Endothelial Cells

Background: Angiogenesis (new blood vessel formation from preexisting vessels) is closely correlated with many physiological processes and pathological changes and is co-regulated by the activators and inhibitors of endothelial cell migration and proliferation, protein hydrolization and cell differentiation. Recent studies show that nerve growth factor (NGF), a member of the neurotrophin family, promotes both proliferation and migration of vascular endothelial cells (VEC) in vitro and angiogenesis in vivo. But whether the angiogenic effect of NGF is direct or through increasing the secretion of vascular endothelial growth factor (VEGF) is still controversial.VEC apoptosis has long been considered a mechanism that counterbalances the effect of cell proliferation/survival during angiogenesis. However, recent evidence both in vitro and in vivo suggests that VEC apoptosis not only initiates vessel regression, but contributes to the removal of redundant cells throughout the vasculature and causes lumen formation, although the detailed biological mechanism remains obscure. Most angiogenic growth factors not only stimulate VEC proliferation and migration but concomitantly inhibit VEC apoptosis. However,whether NGF induces or inhibits VEC apoptosis and promotes lumen formation remains unclear.At present, NGF is thought to play its biological role by binding to the two distinct receptors: Tyrosine receptor Kinase A (TrkA) and p75 neurotrophin receptor (p75NTR). Which receptor does NGF play the above roles remains to be investigated. Objective: To explore the effect of NGF on mouse aorta ring angiogenesis, human umbilical vein endothelial cells (HUVEC) apoptosis and the formation of lumen-like structures (LLS) by HUVEC on Matrigel in vitro.Methods:To explore the effect of NGF on murine aorta ring angiogenesis, murine aorta rings were cultured in 3D Matrigel in serum-free medium in the presence of various concentrations of NGF for different time period. The identification of sprouted vascular endothelial cells was confirmed by immunofluorescent staining. The configuration of sprouting was observed and photographed under phase contrast microscope and the average sprouting area per high power field was measured by Image Pro software and analyzed statistically with SPSS13.0 software. NGF neutralizing antibody pretreatment was used to investigate the effect of NGF on angiogenesis and vascular endothelial growth factor (VEGF) receptor 2 (R2) antagonist SU5416 was used to observe whether the angiogenic role of NGF depends on VEGF. Tyrosine receptor kinase A (TrkA) antagonist K252a was used to investigate whether NGF exerts its angiogenic effect through its receptor TrkA. The effect of NGF on HUVEC apoptosis was evaluated by viable cell counts, the percentage of annexin V+ cells detected by FACS, and immunofluorescent staining for cleaved caspase 3. The expression level of the two NGF receptors TrkA and p75NTR was evaluated by immunofluorescence staining and Western blot. In order to identify the receptors that mediates NGF-induced apoptosis in HUVEC, K252a and anti-p75NTR neutralizing antibody were used to prevent binding of the two receptors to their ligand NGF. The configuration of LLS on Matrigel was observed under phase contrast microscope and the cellular composition of LLS was investigated using immunofluorescence for cleaved caspase 3 and DAPI and Phalloidin staining and observed under laser confocal. The area of the"lumens"was measured by Image Pro software and analyzed statistically with SPSS11.1 software.Results: (1) Effect of NGF on angiogenesis of murine aorta ring cultured in vitro:①0～100ng/ml exogenous NGF promoted sprouting from murine aorta rings cultured in Matrigel in dose-dependent manner. Sprouted cells expressed CD31. Anti-NGF neutralizing antibody markedly blocked NGF-induced sprouting angiogenesis.②SU5416 significantly reduced VEGF-induced angiogenesis but had no significant effect on NGF-induced sprouting.③K252a significantly inhibited NGF-induced angiogenesis. (2) Effect of NGF on HUVEC apoptosis and LLS formation by HUVEC in vitro.①LLS consist substantially of apoptotic cells.②NGF significantly increases cell apoptosis and LLS formation on Matrigel.③Anti-NGF neutralizing antibody significantly blocked spontaneous and NGF-induced LLS formation.④NGF treatment has no significant effect on the expression level of its two receptors, TrkA and p75NTR.⑤Blockade of both TrkA and p75NTR, but not that of either receptor alone significantly decreased NGF-induced cell apoptosis and LLS formation.Conclusions: NGF promotes angiogenesis of murine aorta ring through activating TrkA receptor; both TrkA and p75NTR mediate NGF-induced cell apoptosis which can promote LLS formation by HUVEC in vitro.