Role of vascular endothelial growth factor in regulation of physiological angiogenesis

Evidence accumulating over the last decade has established the fundamental role of vascular endothelial growth factor (VEGF) as a key regulator of normal and abnormal angiogenesis. The biological effects of VEGF are mediated by two tyrosine kinase receptors, Flt-1 (VEGFR-1) and KDR (VEGFR-2). The signaling and biological properties of these two receptors are strikingly different. VEGF is essential for early development of the vasculature to the extent that inactivation of even a single allele of the VEGF gene results in embryonic lethality. VEGF is also required for female reproductive functions and endochondral bone formation. Substantial evidence also implicates VEGF as an angiogenic mediator in tumors and intraocular neovascular syndromes, and numerous clinical trials are presently testing the hypothesis that inhibition of VEGF may have therapeutic value.     

Eicosanoid regulation of vascular endothelial growth factor expression and angiogenesis in microvessel endothelial cells

12(R)-Hydroxy-5,8,14-eicosatrienoic acid (HETrE) is a potent inflammatory and angiogenic eicosanoid in ocular and dermal tissues. Previous studies suggested that 12(R)-HETrE activates microvessel endothelial cells via a high affinity binding site; however, the cellular mechanisms underlying 12(R)-HETrE angiogenic activity are unexplored. Because the synthesis of 12(R)-HETrE is induced in response to hypoxic injury, we examined its interactions with vascular endothelial growth factor (VEGF) in rabbit limbal microvessel endothelial cells. Addition of 12(R)-HETrE (0.1 nm) to the cells increased VEGF mRNA levels with maximum 5-fold increase at 45 min. The increase in VEGF mRNA was followed by an increase in immunoreactive VEGF protein. 12(R)-HETrE (0.1 nm) rapidly activated the extracellular signal-regulated kinases (ERKs) ERK1 and ERK2. Moreover, preincubation of cells with PD98059, a selective inhibitor of MEK-1, inhibited 12(R)-HETrE-induced VEGF mRNA. Addition of VEGF antibody to cells grown in Matrigel-coated culture plates inhibited 12(R)-HETrE-induced capillary tube-like formation, suggesting that VEGF mediates, at least in part, the angiogenic response to 12(R)-HETrE. The results indicate that in microvessel endothelial cells, 12(R)-HETrE induces VEGF expression via activation of ERK1/2 and that VEGF mediates, at least in part, the angiogenic activity of 12(R)-HETrE. Given the fact that both VEGF and 12(R)-HETrE are produced in the cornea after hypoxic injury, their interaction may be an important determinant in the development of neovascularized tissues.     

血管内皮生长因子与一氧化氮

血管内皮生长因子（ＶＥＧＦ）是内皮细胞特异性促有丝分裂原,具有促进内皮细胞增生、迁移及增加血管通透性的作用,其强大的促进新血管形成的作用使其在梗塞性血管病的基因治疗中发挥巨大作用。但其作用机制仍不清楚。研究表明ＶＥＧＦ与一氧化氮９ＮＯ）间存在密切关系,ＮＯ是ＶＥＧＦ发挥许多重要生理作用过程中必不可少的因素。探讨ＶＥＧＦ与ＮＯ的关系有助于进一步阐明ＶＥＧＦ的作用机制。     

Effects of nitric oxide donors on vascular endothelial growth factor gene induction

Nitric oxide (NO) has been reported to modulate the vascular endothelial growth factor (VEGF) gene by accumulating hypoxia-inducible factor-1alpha (HIF-1alpha) protein, but there is a contradiction among effects of various NO donors. The effects of NO donors including S-nitroso-N-acetyl-penicillamine (SNAP), S-nitroso-glutathione (GSNO), 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC18), 3-[(+/-)-(E)-ethyl-2(')-[(E)-hydroxyimino]-5-nitro-3-hexenecarbamoyl]-pyridine (NOR4), 3-morpholinosydnonimine (SIN-1), and nitroprusside (SNP) on the VEGF reporter gene were examined. SNAP, GSNO, NOC18, and NOR4 enhanced the VEGF reporter activity under normoxia and modulated the hypoxic induction. In contrast, SNP had only an inhibitory effect. An NO scavenger attenuated the reporter activation by NO donors except NOR4, but did not ameliorate the inhibitory effect of SNP. A reducing compound dithiothreitol suppressed NO-induced activation of the VEGF reporter gene. SNAP, GSNO, and NOC18 induced the accumulation of HIF-1alpha protein, while others did not. These results suggest that SNAP, GSNO, and NOC compounds are suitable for pharmacological studies in HIF-1-mediated VEGF gene activation by NO.     

Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by activating VEGF receptor-2 (VEGFR-2). The role of its homolog, placental growth factor (PlGF), remains unknown. Both VEGF and PlGF bind to VEGF receptor-1 (VEGFR-1), but it is unknown whether VEGFR-1, which exists as a soluble or a membrane-bound type, is an inert decoy or a signaling receptor for PlGF during angiogenesis. Here, we report that embryonic angiogenesis in mice was not affected by deficiency of PlGF (Pgf-/-). VEGF-B, another ligand of VEGFR-1, did not rescue development in Pgf-/- mice. However, loss of PlGF impaired angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. Transplantation of wild-type bone marrow rescued the impaired angiogenesis and collateral growth in Pgf-/- mice, indicating that PlGF might have contributed to vessel growth in the adult by mobilizing bone-marrow-derived cells. The synergism between PlGF and VEGF was specific, as PlGF deficiency impaired the response to VEGF, but not to bFGF or histamine. VEGFR-1 was activated by PlGF, given that anti-VEGFR-1 antibodies and a Src-kinase inhibitor blocked the endothelial response to PlGF or VEGF/PlGF. By upregulating PlGF and the signaling subtype of VEGFR-1, endothelial cells amplify their responsiveness to VEGF during the 'angiogenic switch' in many pathological disorders.     

Leptin and vascular endothelial growth factor regulate angiogenesis in tooth germs

Leptin, a 16 kDa non-glycolated polypeptide of 146 amino acids produced by the ob gene, has a variety of physiological roles not only in lipid metabolism, hematopoiesis, thermogenesis and ovarian function, but also in angiogenesis. This study focuses to investigate the possibility that leptin, as an angiogenic factor, may regulate the angiogenesis during tooth development. We firstly studied the expression of leptin and vascular endothelial growth factor (VEGF) during tooth development immunohistochemically. This investigation revealed that leptin is expressed in ameloblasts, odontoblasts, dental papilla cells and stratum intermedium cells. This expression pattern was similar to that of VEGF, one of the most potent angiogenic factors. Interestingly, more leptin-positive cells were observed in the upper third portion of dental papilla, which is closest to odontoblastic layer, compared to middle and lower thirds. Moreover, in the dental papilla, more CD31 and/or CD34-positive vascular endothelial cells were observed in the vicinity of ameloblasts and odontoblasts expressing leptin and VEGF. These findings strongly suggest that ameloblasts, odontoblasts and dental papilla cells induce the angiogenesis in tooth germs by secretion of leptin as well as VEGF.     

血管内皮生长因子家族及其受体与肿瘤血管生成研究进展

血管内皮生长因子(vascular endothelial growth factor,VEGF),又名血管通透性因子(vascular permeability factor,VPF)是重要的血管生成正性调节因子,是目前抗癌治疗的研究靶点之一。现已发现的VEGF家族成员包括VEGF—A、VEGF—B、VEGF—C、VEGF—D、VEGF—E和胎盘生长因子(placenta growth factor,PLGF)。VEGF的受体有VEGFR—1(fit—1)、VEGFR-2(flk-1／KDR)、VEGFR-3(fit-4)、neuropilin(NPR1／NPR2)。该家族的成员可以选择性地增强血管和／或淋巴管内皮细胞的有丝分裂,刺激内皮细胞增殖并促进血管生成,提高血管特别是微小血管的通透性,使血浆大分子外渗沉积在血管外的基质中,促进新生毛细血管网的建立,为肿瘤细胞的生长提供营养等。作者对VEGF家族成员及其受体的理化特征、VEGF与肿瘤的关系、VEGF抑制剂的研制作一综述。     

Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor or vasculotropin, is a recently characterized endothelial-specific mitogen which is angiogenic in vivo. Here we demonstrate that VEGF is angiogenic in vitro: when added to microvascular endothelial cells grown on the surface of three-dimensional collagen gels, VEGF induces the cells to invade the underlying matrix and to form capillary-like tubules, with an optimal effect at approximately 2.2nM (100ng/ml). When compared to basic fibroblast growth factor (bFGF) at equimolar (0.5nM) concentrations, VEGF was about half as potent. The most striking effect was seen in combination with bFGF: when added simultaneously, VEGF and bFGF induced an in vitro angiogenic response which was far greater than additive, and which occurred with greater rapidity than the response to either cytokine alone. These results demonstrate that like bFGF, VEGF induces an angiogenic response via a direct effect on endothelial cells, and that by acting in concert, these two cytokines have a potent synergistic effect on the induction of angiogenesis in vitro. We suggest that the synergism between VEGF and bFGF plays an important role in the control of angiogenesis in vivo.     

Phosphorylated endothelial nitric oxide synthase mediates vascular endothelial growth factor-induced penile erection

The objective of the present study was to evaluate whether vascular endothelial growth factor (VEGF)-induced penile erection is mediated by activation of endothelial nitric oxide synthase (eNOS) through its phosphorylation. We assessed the role of constitutively activated eNOS in VEGF-induced penile erection using wild-type (WT) and eNOS-knockout (eNOS(-/-)) mice with and without vasculogenic erectile dysfunction. Adult WT and eNOS(-/-) mice were subjected to sham operation or bilateral castration to induce vasculogenic erectile dysfunction. At the time of surgery, animals were injected intracavernosally with a replication-deficient adenovirus expressing human VEGF145 (10(9) particle units) or with empty virus (Ad.Null). After 7 days, erectile function was assessed in response to cavernous nerve electrical stimulation. Total and phosphorylated protein kinase B (Akt) as well as total and phosphorylated eNOS were quantitatively assessed in mice penes using Western immunoblot and immunohistochemistry. In intact WT mice, VEGF145 significantly increased erectile responses, and in WT mice after castration, it completely recovered penile erection. However, VEGF145 failed to increase erectile responses in intact eNOS(-/-) mice and only partially recovered erectile function in castrated eNOS(-/-) mice. In addition, VEGF145 significantly increased phosphorylation of eNOS at Serine 1177 by approximately 2-fold in penes of both intact and castrated WT mice. The data provide a molecular explanation for VEGF stimulatory effect on penile erection, which involves phosphorylated eNOS (Serine 1177) mediation.     

Prognostic role of serum vascular endothelial growth factor,basic fibroblast growth factor and nitric oxide in patients with colorectal carcinoma

CCR2, and its principle ligand MCP-1/CCL2, have been well documented for their ability to induce monocyte infiltration and promote the pathogenesis of rheumatoid arthritis and atherosclerosis. In order to assess additional roles for CCR2, we inserted allogeneic implants into CCR2-/- and MCP-1-/- mice and characterized T cell responses and the regulatory role of CCR2 on MCP-1 expression. The results demonstrate a marked decrease in lymphocyte infiltration in both CCR2-/- and MCP-1-/- animals. In contrast, IL-12 and CTL function were only suppressed in CCR2-/- animals. Further, whereas MCP-1 was only transiently elevated in the inflammatory fluid of WT animals, levels were sustained within the implants (5000pg/ml; >8 days) and serum (243pg/ml) of CCR2-/- mice. Higher levels of MCP-1 were also observed in the culture supernatants of CCR2-/- macrophages as compared to WT cells despite no difference in mRNA levels. Evidence that MCP-1 levels are regulated by receptor binding and internalization was suggested by its rapid decline when added to WT macrophages at 37 degrees C but not 4 degrees C. These studies indicate that CCR2 plays an important role in regulating T cell responses and controlling the level of MCP-1 at inflammatory sites.     

Vascular endothelial growth factor and nitric oxide in rat liver regeneration

In this work we investigated the role of nitric oxide (NO) in the angiogenesis mediated by vascular endothelial growth factor (VEGF) during rat liver regeneration after two-thirds partial hepatectomy. Sham operated (Sh) and partially hepatectomized (PH) male Wistar rats were randomized in three experimental groups: control (treated with vehicle); pre-treated with sodium nitroprusside (SNP: 0.25 mg/kg body weight, i.v. at a rate of 1 ml/h) and pre-treated with the preferential iNOS inhibitor, aminoguanidine (AG, 100 mg/kg body weight, i.p.). Animals were killed at 5, 24 and 72 h after surgery. At 5 h post-surgery, NO production was estimated by EPR (Sh-Control: 37.65+/-10.70; PH-Control: 88.13+/-1.60(); Sh-SNP: 90.35+/-3.11(); PH-SNP: 119.5+/-12.10()(#); Sh-AG: 33.27+/-5.23, PH-AG: 36.80+/-3.40(#)) (p<0.05 vs Sh-Control; (#)p<0.05 vs PH-Control). At 24 h after PH, VEGF levels showed no difference between PH-Control and PH-SNP animals. However, after 72 h, VEGF protein levels in PH-SNP animals were found to be increased (above 300%) with respect to PH-Control. On the other hand, aminoguanidine (AG) pre-treatment blocked the rise of inhibition of NO generation and decreased VEGF expression. Our results demonstrated that NO plays a role in modulating VEGF protein expression after hepatectomy in rats.     

Interleukin-6 increases vascular endothelial growth factor and angiogenesis in gastric carcinoma

Interleukin-6 (IL-6) is a proinflammatory cytokine associated with the disease status of gastric carcinoma (GC). Vascular endothelial growth factor (VEGF) is a potent tumor angiogenic factor in GC. In this study, we attempted to clarify whether IL-6 can regulate VEGF and angiogenesis in GC. GC samples from 54 surgical specimens were subjected to immunohistochemical examination of IL-6, VEGF, and tumor microvessels, and results showed that IL-6 was positively correlated with VEGF expression and tumor vasculature. We determined VEGF expression in four GC cell lines by ELISA, revealing that GC cells can produce significant amount of VEGF with increasing dose and duration of IL-6 stimulation. Next, a luciferase reporter gene assay was employed to determine the signaling pathway driving the VEGF promoter by IL-6, which showed that the JAK/STAT pathway is involved in the stimulation of VEGF gene expression. The effects of IL-6 on angiogenesis in vitro and in vivo were evaluated by HUVEC studies and the Matrigel plug assay, respectively. Results showed that IL-6 effectively promoted HUVEC proliferation and tube formation in vitro and Matrigel plug vascularization in vivo, primarily by inducing VEGF in GC. This study provides evidence that the multifunctional cytokine, IL-6, may induce VEGF expression which increases angiogenesis in gastric carcinogenesis.     

Endogenous production of nitric oxide by vascular endothelial growth factor down-regulates proliferation of choriocarcinoma cells

The trophoblast-like choriocarcinoma cell line BeWo expresses a receptor for vascular endothelial growth factor (VEGF) and proliferates in response to VEGF. Nitric oxide (NO) seems to play a key role in the VEGF-induced proliferation of endothelial cells but the NO mechanistic regulation of VEGF-stimulated trophoblast proliferation is presently unclear. We assessed the effect of exogenous VEGF on BeWo cell proliferation by [3H]thymidine incorporation. The VEGF-induced proliferation of BeWo cells was significantly increased by the NO synthase (NOS) inhibitor, N(omega)-nitro-l-arginine methyl ester (L-NAME), but was inhibited by the NO donor, sodium nitroprusside. Treatment of the cells with 10 ng/ml of VEGF increased not only eNOS expression but also NO production. The extracellular signal-regulated kinase (Erk) of the mitogen-activated protein kinase (MAPK) family was activated by VEGF as demonstrated by the phosphorylation of Erk in Western blots. The effects of VEGF on NO production and the expression of endothelial NOS (eNOS) were attenuated by treating BeWo cells with the selective inhibitor of MAPK kinase, PD98059. VEGF-stimulated proliferation of BeWo cells was inhibited by the tyrosine kinase inhibitor genistein but increased by PD98059. Other kinase inhibitors, including LY294002 (phosphoinositide 3-kinase inhibitor) and SB203580 (P38 MAPK inhibitor), had no effect on the proliferation of the cells and NO production. These results indicate that endogenous NO production down-regulates the VEGF-stimulated proliferation of BeWo cells and that the activation of Erk plays an important role in this mechanism.     

Characterization of multiple signaling pathways of insulin in the regulation of vascular endothelial growth factor expression in vascular cells and angiogenesis

The effects of insulin on vascular endothelial growth factor (VEGF) expression in cultured vascular cells and in angiogenesis were characterized. Insulin increased VEGF mRNA levels in mouse aortic smooth muscle cells from 10(-9) to 10(-7) m with an initial peak of 3.7-fold increases at 1 h and a second peak of 2.8-fold after 12 h. The first peak of VEGF expression was inhibited by LY294002, an inhibitor of phosphatidylinositol (PI) 3-kinase, and by the overexpression of dominant negative forms of p85 subunit of PI 3-kinase or Akt. Inhibitors of MEK kinase, PD98059, or overexpression of dominant negative forms of Ras was ineffective. In contrast, the chronic effect of insulin on VEGF expression was partially inhibited by both LY294002 or PD98059 as well as by the overexpression of dominant negatives of PI 3-kinase or Ras. The importance of PI 3-kinase-Akt pathway on VEGF expression was confirmed in mouse aortic smooth muscle cells isolated from insulin receptor substrate -1 knockout (IRS-1-/-) mice that showed parallel reductions of 46-49% in insulin-stimulated VEGF expression and PI 3-kinase-Akt activation. Insulin-induced activation of PI 3-kinase-Akt on hypoxia-induced VEGF expression and neovascularization was reduced by 40% in the retina of neonatal hypoxia model using IRS-1-/- mice. Thus, unlike other cells, insulin can regulate VEGF expression by both IRS-1/PI 3-kinase-Akt cascade and Ras-MAPK pathways in aortic smooth muscle cells. The in vivo results provide direct evidence that insulin can modulate hypoxia-induced angiogenesis via reduction in VEGF expression in vivo.     

The neurotransmitter dopamine inhibits angiogenesis induced by vascular permeability factor/vascular endothelial growth factor

Angiogenesis has an essential role in many important pathological and physiological settings. It has been shown that vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), a potent cytokine expressed by most malignant tumors, has critical roles in vasculogenesis and both physiological and pathological angiogenesis. We report here that at non-toxic levels, the neurotransmitter dopamine strongly and selectively inhibited the vascular permeabilizing and angiogenic activities of VPF/VEGF. Dopamine acted through D2 dopamine receptors to induce endocytosis of VEGF receptor 2, which is critical for promoting angiogenesis, thereby preventing VPF/VEGF binding, receptor phosphorylation and subsequent signaling steps. The action of dopamine was specific for VPF/VEGF and did not affect other mediators of microvascular permeability or endothelial-cell proliferation or migration. These results reveal a new link between the nervous system and angiogenesis and indicate that dopamine and other D2 receptors, already in clinical use for other purposes, might have value in anti-angiogenesis therapy.     

A synthetic glycosaminoglycan mimetic binds vascular endothelial growth factor and modulates angiogenesis

In a previous study, we showed that in situ injection of glycosaminoglycan mimetics called RGTAs (ReGeneraTing Agents) enhanced neovascularization after skeletal muscular ischemia (Desgranges, P., Barbaud, C., Caruelle, J. P., Barritault, D., and Gautron, J. (1999) FASEB J. 13, 761-766). In the present study, we showed that the RGTA OTR4120 modulated angiogenesis in the chicken embryo chorioallantoic membrane assay, in a dose-dependent manner. We therefore investigated the effect of OTR4120 on one of the most specific angiogenesis-regulating heparin-binding growth factors, vascular endothelial growth factor 165 (VEGF165). OTR4120 showed high affinity binding to VEGF165 (Kd = 2.2 nm), as compared with heparin (Kd = 15 nm), and potentiated the affinity of VEGF165 for VEGF receptor-1 and -2 and for neuropilin-1. In vitro, OTR4120 potentiated VEGF165-induced proliferation and migration of human umbilical vein endothelial cells. In the in vivo Matrigel plug angiogenesis assay, OTR4120 in a concentration as low as 3 ng/ml caused a 6-fold increase in VEGF165-induced angiogenesis. Immunohistochemical staining showed a larger number of well differentiated VEGFR-2-expressing-cells in Matrigel sections of OTR4120-treated plug than in control sections. These findings indicate that OTR4120 enhances the VEGF165-induced angiogenesis and therefore may hold promise for treating disorders characterized by deficient angiogenesis.