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.     

Vascular endothelial growth factor increases the intracellular magnesium

Vascular endothelial growth factor (VEGF) is one of the key players in the process of angiogenesis. However, its underlying mechanism remains unclear. Mg2+ is the most abundant intracellular divalent cation in the body and plays critical roles in many cell functions. We investigated the effect of VEGF on intracellular Mg2+ in human umbilical vein endothelial cells (HUVECs). VEGF-A165 increased the intracellular Mg2+ concentration ([Mg2+]i) in a dose-dependent manner, with or without extracellular Mg2+, and the increase of [Mg2+]i was blocked by pretreatment with SU1498, tyrosine kinase inhibitors (tyrphostin A-23 and genistein), phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin and LY294002) or phospholipase Cgamma (PLCgamma) inhibitor (U73122). In contrast, mitogen-activated protein kinase inhibitors (SB202190 and PD98059) had no effect on the VEGF-induced [Mg2+]i increase. These results suggest that VEGF-A165 increases the [Mg2+]i from the intracellular Mg2+ stores through the tyrosine kinase/PI3K/PLCgamma-dependent signaling pathways.     

Vascular endothelial growth factor: Therapeutic possibilities and challenges for the treatment of ischemia

Vascular endothelial growth factor (VEGF) is a notable chemokine that plays critical roles in angiogenesis and vasculogenesis. The contemporary body of literature contains a substantial amount of information regarding its chemical properties as well as its fundamental role in vascular development. Studies strongly indicate its potential use as a therapeutic agent, especially in the vascular restoration of injured and ischemic tissues. VEGF therapy could be most beneficial for diseases whose pathologies revolve around tissue inflammation and necrosis, such as myocardial infarction and stroke, as well as ischemic bowel diseases such as acute mesenteric ischemia and necrotizing enterocolitis. However, a delicate balance exists between the therapeutic benefits of VEGF and the hazards of tumor growth and neo-angiogenesis. Effective future research surrounding VEGF may allow for the development of effective therapies for ischemia which simultaneously limit its more deleterious side effects. This review will: (1) summarize the current understanding of the molecular aspects and function of VEGF, (2) review potential benefits of its use in medical therapy, (3) denote its role in tumorigenesis and inflammation when overexpressed, and (4) elucidate the qualities which make it a viable compound of study for diagnostic and therapeutic applications.     

Vascular endothelial growth factor restores erectile function through modulation of the insulin-like growth factor system and sex hormone receptors in diabetic rat

Previous studies have shown that intracavernous injection of vascular endothelial growth factor (VEGF) restored erectile function in diabetic rats. However, the mechanism of VEGF in diabetes-related erectile dysfunction (ED) has not been fully investigated. We hypothesize that intracavernous injection of VEGF may reverse diabetes-related ED through modulation of the insulin-like growth factor system and sex hormone receptors. To test this hypothesis the erectile function of treated and control rats was analyzed by measurement of intracavernous pressure (ICP) following electrostimulation of the cavernous nerves. Mean ICP was significantly lower in non-treated diabetic rats compared to controls. After VEGF injection, ICP was significantly higher than in non-treated diabetic rats. IGFBP-3 mRNA and protein expression was significantly higher in non-treated diabetic rat crura than controls, while VEGF-treated animals had control levels. ER-beta and PR mRNA and protein expression was significantly lower in non-treated diabetic rat crura. After VEGF injection, ER-beta and PR mRNA and protein expression was similar to control levels. Expression of AR and ER-alpha was the same in all groups. These findings suggest that orthotopic injection of VEGF may improve the functional recovery of diabetes-related ED through modulation of the insulin-like growth factor system and sex hormone receptors. To our knowledge, this is the first study demonstrating that VEGF treatment restores erectile function through restoration of the insulin-like growth factor system and sex hormone receptor genes at the mRNA and protein levels in diabetic rat crura. These results may be important in understanding the pathogenesis of diabetes-related ED and also in providing better strategies for management of this disease.     

Vascular endothelial growth factor-B (VEGFB) stimulates neurogenesis: evidence from knockout mice and growth factor administration

Vascular endothelial growth factor-B (VEGFB) is an angiogenic and neuroprotective protein that reduces hypoxic and ischemic neuronal injury. To determine if VEGFB also regulates neurogenesis in the adult brain, we studied the effects of VEGFB administration in vitro and in vivo, as well as the effect of VEGFB gene knockout (KO) in mice, on bromodeoxyuridine (BrdU) incorporation and expression of immature neuronal markers in the subgranular zone (SGZ) of the hippocampal dentate gyrus and the forebrain subventricular zone (SVZ). Intracerebroventricular VEGFB administration increased BrdU incorporation into cells of neuronal lineage both in vitro and in vivo, and VEGFB-KO mice showed impaired neurogenesis, consistent with a neurogenesis-promoting effect of VEGFB. In addition, intraventricular administration of VEGFB restored neurogenesis to wild-type levels in VEGFB-KO mice. These results suggest a role for VEGFB in the regulation of adult neurogenesis, which could have therapeutic implications for diseases associated with central neuronal loss.     

Vascular endothelial growth factor and substrate mechanics regulate in vitro tubulogenesis of endothelial progenitor cells

Endothelial progenitor cells (EPCs) in the circulatory system have been suggested to maintain vascular homeostasis and contribute to adult vascular regeneration and repair. These processes require that EPCs break down the extracellular matrix (ECM), migrate, differentiate and undergo tube morphogenesis. Evidently, the ECM plays a critical role by providing biochemical and biophysical cues that regulate cellular behaviour. Using a chemically and mechanically tunable hydrogel to study tube morphogenesis in vitro, we show that vascular endothelial growth factor (VEGF) and substrate mechanics co‐regulate tubulogenesis of EPCs. High levels of VEGF are required to initiate tube morphogenesis and activate matrix metalloproteinases (MMPs), which enable EPC migration. Under these conditions, the elasticity of the substrate affects the progression of tube morphogenesis. With decreases in substrate stiffness, we observe decreased MMP expression while increased cellular elongation, with intracellular vacuole extension and coalescence to open lumen compartments. RNAi studies demonstrate that membrane type 1‐MMP (MT1‐MMP) is required to enable the movement of EPCs on the matrix and that EPCs sense matrix stiffness through signalling cascades leading to the activation of the RhoGTPase Cdc42. Collectively, these results suggest that coupled responses for VEGF stimulation and modulation of substrate stiffness are required to regulate tube morphogenesis of EPCs.     

Vascular endothelial growth factor is involved in neoangiogenesis in Hirudo medicinalis (Annelida,Hirudinea)

Vascular endothelial growth factor (VEGF) is fundamental in vertebrates for correct development of blood vessels. However, there are only few data about the presence of VEGF in invertebrates. In this study the role of VEGF in neovessel formation is investigated in Hirudo medicinalis. The leech is able to respond to administration of human VEGF by formation of new vessels. The response of H. medicinalis to this growth factor is explained by the presence of two specific VEGF-like receptors (Flt-1/VEGFR-1 and Flk-1/VEGFR-2) as demonstrated by immunohistochemistry and biochemical analysis. The VEGF-like produced by this annelid following surgical stimulation determines not only blood vessel formation, proliferation of vascular endothelial cells but also an increase of cytoplasmic calcium levels. The administration of specific VEGF receptor antibodies can inhibit angiogenesis in leeches previously stimulated with VEGF.     

Immunolocalization of vascular endothelial growth factor in rat condylar cartilage during postnatal development

It is well known that angiogenesis is essential for the replacement of cartilage by bone during skeletal growth and regeneration. To address angiogenesis of endochondral ossification in the condyle, we examined the appearance of vascular endothelial growth factor (VEGF) and its receptor Flt-1 in condylar cartilage of the growing rat. The early expression of VEGF at various sites during condylar cartilage development indicates that VEGF plays a role in the regulation of angiogenesis at each site of bone formation. From the findings of Flt-1 immunoreactivity, the VEGF produced by the chondrocytes of the hypertrophic zone should contribute to the promotion of endothelial cell proliferation and to stimulate migration and activation of osteoclasts in condylar cartilage, resulting in the invasion of these cells into the mineralized zone.Junko Aoyama and Eiji Tanaka contributed equally to this work     

Synergistic induction of endothelial tissue factor by tumor necrosis factor and vascular endothelial growth factor: functional analysis of the tumor necrosis factor receptors

Tissue factor expression on the surface of endothelial cells can be induced by tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF) in a synergistic manner. We have investigated the role of the two different TNF receptors for this synergy. Firstly, stimulation of the 60 kDa TNF receptor (TNFR60) by a mutant of TNF specific for TNFR60 induced responses comparable to wild-type TNF. In contrast, stimulation of TNFR80 by a TNFR80-specific TNF mutein did not result in enhancement of tissue factor expression even in the presence of a suboptimal TNFR60 triggering. Secondly, we tested neutralizing TNF receptor antibodies for inhibition of tissue factor synthesis induced by VEGF and TNF. A TNFR60-specific antibody inhibited tissue factor production over a broad range of TNF concentrations, indicating an essential role of TNFR60 in the TNF/VEGF synergy. In contrast, blocking of TNF binding to TNFR80 strongly inhibited TNF-induced tissue factor expression at low, but less pronounced at high, TNF concentrations. In conclusion, these data are in agreement with a model in which TNFR80 participates in the synergy between VEGF and low concentrations of soluble TNF by passing the ligand to the signalling TNFR60.     

Vascular endothelial growth factor inhibits maturation of dendritic cells induced by lipopolysaccharide,but not by proinflammatory cytokines

Purpose: Dendritic cells (DCs) play an important role in the hosts immunosurveillance against cancer. It has been shown that the function of DCs is impaired and their population decreased in a cancer-bearing host. In the present study, we investigated the mechanism of down-regulation of DCs in a cancer-bearing host. Methods: We evaluated the relationship between DC infiltration and production of vascular endothelial growth factor (VEGF) in carcinoma tissue by immunohistochemistry. Furthermore, functional and phenotypical alterations of DCs were evaluated when monocyte-derived, mature DCs were treated with VEGF in vitro. Monocyte-derived DCs were generated in a culture of monocyte with interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor, and the maturation of DCs was induced by either lipopolysaccharide (LPS) or a proinflammatory cytokine cocktail: tumor-necrosis factor , prostaglandin E₂, IL-6, and IL-1. Results: A significant inverse correlation was found between the density of DCs and the quantity of VEGF production in gastric carcinoma tissue (r=–0.39, p<0.05). In LPS-induced maturation, the ability of mature DCs to stimulate allogenic T cells and produce IL-12 (p70 heterodimer) was suppressed by the addition of VEGF in a dose-dependent manner. A lesser expression of costimulatory molecules (CD80 and CD86) was seen in DCs treated with exogenous VEGF than in DCs not treated with VEGF. The population of dead DCs (early and late apoptosis) treated with VEGF increased more than that without VEGF treatment, using the annexin V and propidium iodide evaluation in DCs matured by LPS. In contrast, in DCs matured by the proinflammatory cytokine cocktail, the down-regulation of costimulatory molecules and induction of DC apoptosis was not seen. Conclusions: These findings show that the inhibition of DC maturation by VEGF differs depending on the maturation status of the DCs.Abbreviations APC antigen-presenting cells - DC dendritic cells - ELISA enzyme-linked immunosorbent assay - FACS fluorescence-activated cell sorter - FCS fetal calf serum - FITC fluorescein isothiocyanate - GM-CSF granulocyte-macrophage colony-stimulating factor - HLA human leukocyte antigen - IL interleukin - LPS lipopolysaccharide - mAb monoclonal antibody - MHC major histocompatibility complex - PBS phosphate-buffered saline - PCNA proliferative cell nuclear antigen - PE phycoerythrin - PG prostaglandin - PI propidium iodide - TNF tumor-necrosis factor - VEGF vascular endothelial growth factor This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology in Japan.     

Expression of vascular endothelial growth factor (VEGF) and its cognate receptors in human pheochromocytomas

Pheochromocytomas are well-vascularized tumors, suggesting that a potent angiogenic factor may be involved in the mechanism of their formation. As vascular endothelial growth factor (VEGF) is a potent mitogen for vascular endothelial cells, here we have investigated the mRNA and protein expression of VEGF and the mRNA expression of its two receptors (Flt-1 and Flk-1/KDR) in pheochromocytomas tissue. An increase in VEGF mRNA (mainly isoforms VEGF(121) and VEGF(165)) and in VEGF protein expression were observed by semi-quantitative RT-PCR and Western blot, respectively, compared to normal adrenomedullary tissue. Flk-1/KDR, and Flt-1 levels of mRNA were also increased markedly in tumors and correlated with levels of VEGF mRNA. Therefore, we speculate that upregulation of VEGF expression and its receptors might be important in the pathogenesis of pheochromocytomas.     

Aldosterone upregulates vascular endothelial growth factor expression in mouse cortical collecting duct epithelial cells through classic mineralocorticoid receptor

Accumulating evidence shows that aldosterone plays an important role in the pathogenesis of renal fibrosis but its mechanism has not been completely defined. Recently, exogenous administration of aldosterone significantly alleviated ischemic states in a model of femoral artery ligated rats, accompanied by an obvious enhancement of VEGF upregulation. We hypothesized that aldosterone may also regulate the expression of VEGF in the kidney. To confirm this, cultured cortical collecting duct epithelial cells (M-1 cell line) were incubated with aldosterone and control media, respectively. The pathway by which aldosterone regulates VEGF expression was tested by the administration of spironolactone, a specific mineralocorticoid receptor (MR) antagonist. VEGF expression was detected by immunofluorescence staining, ELISA, Western blot and RT-PCR. Aldosterone induced an elevation of VEGF excretion in a time- and dose-dependent manner. Western blotting showed a 1.4-fold elevation in cytosolic VEGF expression following aldosterone (10(-8) M) incubation for 48 h (p<0.01). After aldosterone (10(-7) M) incubation for 48 h, the mRNA level of VEGF164 and VEGF120 showed 1.8- and 1.7-fold increases, respectively (p<0.01). This upregulation was almost completely blocked by spironolactone as shown both by mRNA levels and cytosolic protein levels. In addition, the mRNA of aldosterone receptor was detected in M-1 cells. We demonstrated for the first time that aldosterone induced VEGF expression in M-1 cells, an effect mediated by classic mineralocorticoid receptor. This finding provides experimental evidence for the local non-hemodynamic action of aldosterone.     

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.     

Hypoxia-inducible factor and vascular endothelial growth factor in the neuroretina and retinal blood vessels after retinal ischemia

Retinal ischemia arises from circulatory failure. As the retinal blood vessels are key organs in circulatory failure, our aim was to study the retinal vasculature separately from the neuroretina to elucidate the role of hypoxia-inducible factor (HIF) 1α and 1β and vascular endothelial growth factor (VEGF) in retinal ischemia. Retinal ischemia was induced in porcine eyes by applying an intraocular pressure, followed by 12 h of reperfusion. HIF-1α mRNA expression was not affected by ischemia, while immunofluorescence staining was higher after ischemia in the neuroretina. HIF-1β immunoreactivity and mRNA expression were unaffected. VEGF protein levels in the vitreous humor and VEGF staining in the neuroretina were more pronounced in eyes subjected to ischemia than in the sham eyes. VEGF may be activated downstream of HIF-1 and is known to stimulate retinal neovascularization, which causes sight-threatening complications. These results emphasize the need for pharmacological treatment to block the HIF and VEGF signaling pathways in retinal ischemia.     

Immunolocalization of vascular endothelial growth factor on intramuscular ectopic osteoinduction by bone morphogenetic protein-2

When recombinant human bone morphogenetic protein-2 (rhBMP-2) is implanted in soft tissues, bony tissue is induced during the course of endochondral ossification. The relationship between endochondral ossification and vascularization is important in bone formation, and vascular endothelial growth factor (VEGF) is considered to play an important role in this process. In this study, the immunohistological localization of VEGF was investigated in rhBMP-2-induced ectopic endochondral ossification in the calf muscle of rats. In addition, the characteristics of anti-VEGF antibody-reactive cells were histologically investigated using electron microscopy to examine the cause of endochondral ossification induced by recombinant human bone morphogenetic protein-2. The role of VEGF in rhBMP-2-induced osteoinduction and vascular induction was studied by observing the relationship between the localizations of anti-VEGF antibody-reactive cells and vascularization. During the process of rhBMP-2-induced ectopic endochondral ossification, fibroblast-like cells, which were located at the margin of the implant and reactive to BMP-2 at 5 days, were positive for VEGF immunostaining. Hypertrophic chondrocytes appeared 9 days and osteoblasts appeared 14 to 21 days after implantation, and all these cells were reactive with anti-VEGF antibody. Bony trabeculae subsequently appeared in the muscle, and new blood vessels were formed alongside the trabeculae. When VEGF was added to rhBMP, more new blood vessels and bone were formed in the induced bone. These findings suggested that rhBMP-2 induced the differentiation of undifferentiated mesenchymal cells to chondrocytes and osteoblasts, and these differentiated cells expressed VEGF, creating an advantageous environment for vascularization in bony tissue.     

Nicotine-induced vascular endothelial growth factor release via the EGFR-ERK pathway in rat vascular smooth muscle cells

Cigarette smoke has been firmly established as an independent risk factor for atherosclerosis and other vascular diseases. The proliferation and migration of vascular smooth muscle cells (VSMC) induced by growth factors have been proposed to play an important role in the progression of atherosclerosis. In the present study, we investigated the effects of nicotine, which is one of the important constituents of cigarette smoke, on vascular endothelial growth factor (VEGF) release, in rat VSMC. The stimulation of cells with nicotine resulted in a time- and concentration-dependent release of VEGF. Hexamethonium, an antagonist of nicotinic acetylcholine receptor (nAChR), inhibited nicotine-induced VEGF release. We next investigated the mechanisms by which nicotine induces VEGF release in the cells. The nicotine-induced VEGF release was inhibited by treatment with U0126, a selective inhibitor of MEK, which attenuated the nicotine-induced ERK phosphorylation. Nicotine induced a transient phosphorylation of ERK. Furthermore, AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) kinase, inhibited nicotine-induced ERK phosphorylation and VEGF release. These data suggest that nicotine releases VEGF through nAChR in VSMC. Moreover, VEGF release induced by nicotine is mediated by an EGFR-ERK pathway in VSMC. VEGF may contribute to the risk of cardiovascular diseases in cigarette smokers.     

Protease-activated receptor-2 regulates vascular endothelial growth factor expression in MDA-MB-231 cells via MAPK pathways

Protease-activated receptor 2 (PAR2) is a G-protein coupled receptor that is cleaved and activated by serine proteases including the coagulation protease factor VIIa (FVIIa). There is evidence that PAR2 function contributes to angiogenesis, but the mechanisms involved are poorly defined. Here we show that PAR2 activation in human breast cancer cells leads to the upregulation of vascular endothelial growth factor (VEGF). Activation of PAR2 with agonist peptide (AP), trypsin or FVIIa results in a robust increase of VEGF message and protein. Incubation of cells with PAR1-AP, PAR3-AP, PAR4-AP, or thrombin has only a modest effect on VEGF production. Cleavage blocking antibodies show that FVIIa-mediated VEGF production is PAR2 mediated. Mitogen-activated protein kinase (MAPK) pathway inhibitors U0126 and SB203580 inhibit PAR2-mediated VEGF production. Incubation of cells with PAR2-AP leads to significant extracellular regulated kinase1/2 (ERK1/2) and p38 MAPK phosphorylation and activation. Collectively, these data suggest that PAR2 signaling through MAPK pathways leads to the production of proangiogenic VEGF in breast cancer cells.