The levels of circulating vascular endothelial growth factor and soluble Flt-1 in pregnancies complicated by preeclampsia

To evaluate the role of vascular endothelial growth factor (VEGF) in the pathogenesis of preeclampsia, we measured total VEGF, free VEGF and soluble Flt-1 (sFlt-1) concentrations and determined their relationships. Maternal serum samples were collected from 20 patients with preeclampsia and 20 normotensive women with uncomplicated pregnancies matched with the patients with preeclampsia for gestational age and parity. The serum concentrations of total VEGF (2.39+/-0.75 vs. 0.28+/-0.14) and sFlt-1 (934.5+/-235.5 vs. 298.0+/-161.2) were significantly increased in the patients with preeclampsia compared to the women with uncomplicated pregnancies. However the serum concentration of free VEGF (21.5+/-6.3 vs. 134.0+/-16.3) was lower in patients with preeclampsia. There was a positive correlation between the serum concentrations of total VEGF and sFlt-1 with systolic and diastolic blood pressure, respectively. There was a negative correlation between the serum concentration of free VEGF and systolic and diastolic blood pressure. There was a strong negative correlation between free VEGF and sFlt-1 concentrations. In conclusion, we found VEGF and sFlt-1 were related to the pathogenesis of preeclampsia. Although reduced concentrations of free VEGF might interfere with endothelial cell function and survival, further studies are required to clarify its specific role in the pathogenesis of preeclampsia.     

VEGF/Flk-1 interaction, a requirement for malignant ascites recurrence.

Vascular endothelial growth factor (VEGF) plays an important role in the production of ascitic fluid associated with malignant tumor growth. In an experimental model for malignant ascites formation, mice were inoculated intraperitoneally with syngeneic mouse sarcoma tumor cells. Ascites development was not prevented by administering tumor necrosis factor (TNF) simultaneously with the tumor cell inoculation. When the malignant ascites was first drained and renewal of ascites was monitored, however, a TNF dose-dependent inhibition of ascitic fluid accumulation was observed. Northern blot analyses indicated transient downregulation by TNF on the expression of VEGF mRNA in tumor cells. Monoclonal antibody, (mAb) DC101 generated against the mouse VEGF receptor Flk-1 prevented the recurrence of malignant ascites in mice similar to TNF inhibition. In addition, exogenous soluble human Flt-1 used as an inhibitor of endogenous VEGF binding also inhibited ascites recurrence. These data demonstrate that the observed inhibitory effect of TNF on reestablishment of malignant ascites can be achieved equally by inhibition of the interaction of VEGF with its receptor Flk-1.     

Time-Dependent Alterations of VEGF and Its Signaling Molecules in Acute Lung Injury in a Rat Model of Sepsis

Molecular mechanisms of sepsis-associated acute lung injury (ALI) are poorly defined. Since vascular endothelial growth factor (VEGF) is a potent vascular permeability and mitogenic factor, it might contribute to the development of ALI in sepsis. Thus, using lipopolysaccharide (LPS)-induced (15 mg/kg, intraperitoneal) endotoxemic rat model, we studied the timeline (1, 3, 6, and 10 h) of pulmonary VEGF expression and its signaling machinery. Levels of pulmonary VEGF and its angiogenic-mediating receptor, Flk-1, were downregulated by LPS in a time-dependent manner; levels of plasma VEGF and its permeability-mediating receptor, Flt-1, in contrast, was upregulated with time. In addition, blockade of Flt-1 could improve the downregulated pulmonary VEGF level and attenuate the elevated plasma and pulmonary levels of TNF-α, followed by improvement of arterial oxygenation and wet-to-dry weight ratio of the lung. Expression of signaling, pro- and or apoptotic factors after LPS administration were as follows: phosphorylated Akt, a downstream molecule was downregulated time dependently; endothelial nitric oxide synthase levels were significantly reduced; pro-apoptotic markers caspase 3 and Bax were upregulated whereas levels of Bcl-2 were downregulated. The present findings show that VEGF may play a role through the expression of Flt-1 in LPS-induced ALI. Moreover, downregulation of VEGF signaling cascade may account for LPS-induced apoptosis and impaired physiological angiogenesis in lung tissues, which in turn may contribute to the development of ALI induced by LPS.     

Expression and prognostic relevance of vascular endothelial growth factor (VEGF) and its receptor (FLT-1) in nephroblastoma

AIMS: To investigate the prognostic relevance of vascular endothelial growth factor (VEGF) and its receptor Flt-1 in nephroblastoma and whether tumour microvessel density (MVD) immunoreactivity, determined by the CD31 antigen, is related to the expression of VEGF and Flt-1. METHODS: The expression of VEGF and Flt-1 and MVD were investigated by means of immunohistochemical analysis in 62 Wilms's tumours. Patients were treated preoperatively with chemotherapy and had a mean follow up of 5.7 years. RESULTS: In general, VEGF and Flt-1 were expressed in normal kidney parenchyma and to a variable extent in the three main components of Wilms's tumour, namely: the blastemal, epithelial, and stromal cells. In tumour tissue, 52% and 47% of blastemal cells were positive for VEGF and Flt-1, respectively. A non-significant correlation was found between the expression of VEGF and Flt-1 in blastemal and epithelial cells and the clinicopathological stage. MVD was significantly higher in VEGF and Flt-1 positive tumours than in VEGF and Flt-1 negative tumours. Univariate analysis showed that the expression of VEGF and Flt-1 in blastemal cells was indicative of clinical progression and tumour specific survival. In addition, MVD expression was indicative of clinical progression. Epithelial staining was of no prognostic value. In a multivariate analysis, VEGF protein expression by blastemal cells was an independent prognostic marker for clinical progression. CONCLUSIONS: These results indicate that VEGF and Flt-1 protein expression are closely related to MVD and seem to be an important predictor for poor prognosis in treated patients with Wilms's tumour. Therefore, the expression of these molecules in primary Wilms's tumour may be useful in identifying those patients at high risk of tumour recurrence and in guiding antiangiogenic treatment.     

Expression of vascular endothelial growth factor,placental growth factor,and their receptors Flt-1 and KDR in human placenta under pathologic conditions

The vascular endothelial growth factor (VEGF) family and its receptors have multifunctional activities besides angiogenesis, and some of these molecules are induced by hypoxia/ischemia. They are known to be expressed in human placenta, but little is known about their involvement in pathologic conditions. We have investigated the expression patterns of VEGF, placental growth factor (PlGF), and their receptors fms-like tyrosine kinase (Flt-1) and kinase insert domain-containing region (KDR) in placentas with histopathological changes. Forty-two placentas from normal and complicated pregnancies delivered in the second and third trimesters were fixed with paraformaldehyde and embedded in paraffin. In situ hybridization and immunohistochemistry were performed on serial sections. In the villi with characteristic hypoxic/ischemic changes (HIC), including increased syncytial knots, infarction, or hypercapillarization, intense immunostaining for VEGF was detected in the media of blood vessels, and increased staining for KDR was demonstrated in the endothelial cells. Strong PlGF immunoreactivity was localized to the degenerative trophoblasts around the infarctions. Marked Flt-1 mRNA expression in the syncytiotrophoblast layers of HIC villi was identified, but some samples did not show ligand expression in these regions. Positive immunostaining for VEGF, PlGF, and Flt-1 was observed in infiltrated neutrophils and macrophages in the placentas with chorioamnionitis (CAM). These findings suggested that in the hypoxic/ischemic regions, VEGF and KDR expression is increased within the villous vessels by paracrine regulation, whereas the expression of PlGF and Flt-1 is enhanced in villous trophoblasts by autocrine regulation. The Flt-1 gene may also be up-regulated directly by hypoxia/ischemia independently of ligand mediation. Furthermore, the results indicated that VEGF and PlGF stimulate inflammatory cell migration by autocrine regulation via the Flt-1 receptor in the CAM placenta. Thus, various functions of VEGF family members participate in the development of pathologic changes in the placenta.     

Expression of vascular endothelial growth factor (VEGF) and its receptors is regulated in eyes with intra-ocular tumours

The immunolocalization and gene expression of vascular endothelial growth factor (VEGF) and its cognate tyrosine kinase receptors, Flt-1 and KDR, has been studied in ocular melanomas and retinoblastomas using in situ hybridization and immunohistochemistry. Tumour-related alterations in VEGF/VEGF-receptor expression have also been examined in separate and uninvolved iris, retina and choroid of the same eyes. Although VEGF immunoreactivity in the normal retina was virtually absent, low-level VEGF expression was evident in the ganglion cell-bodies, Müller cells and in a distinct population of amacrine cells. VEGF gene expression was absent in the iris and choroid of normal eyes. In tumour-bearing eyes, high levels of VEGF protein and gene expression were observed within the vascularized regions of the tumours, while the adjacent retina and choroid showed increased VEGF levels when compared with normals. Flt-1 and KDR gene expression and immunolocalization occurred in VEGF-expressing ganglion, Müller and amacrine cells in normal eyes. Within the intra-ocular tumours, VEGF-receptor gene expression and protein was evident in the endothelial cells and also in cells close to the vessels, while in the adjacent retina, Flt-1 and KDR levels were elevated over normal, especially in the blood vessels. Flt-1 and KDR were both observed at elevated levels in the choroid and iris blood vessels. This study suggests that VEGF, Flt-1 and KDR are expressed by neural, glial and vascular elements within normal human retina. Intra-ocular tumours demonstrate a high level of VEGF and VEGF-receptor expression; within uninvolved, spatially separate retina, choroid and iris in the same eyes, expression is also elevated, especially within the vasculature. Retinal vascular endothelia may respond to high intra-ocular levels of VEGF by increasing expression of their VEGF receptors, a phenomenon which could have relevance to neoplasm-related ocular neovascularization.     

Vascular endothelial growth factor and its receptor, Flt-1, in smokers and non-smokers

Raised levels of plasma vascular endothelial growth factor (VEGF) are found in some cancers, diabetes, and certain other conditions, but levels of its receptor, soluble Flt-1 (sFlt-1), in these diseases have yet to be reported. We hypothesised that smoking would influence levels of these molecules. Consequently, we measured VEGF and sFlt-1 by enzyme-linked immunosorbent assay (ELISA) in plasma from 92 non-smokers and 35 smokers. No difference in VEGF was seen between the groups but, despite considerable overlap, sFlt-1 was significantly lower in smokers (P = 0.027). VEGF and sFlt-1 correlated strongly with each other (P < 0.001). Although VEGF may arise from a number of cell types, including endothelial cells, the primary source of sFlt-1 is thought to be the endothelium; however, neither VEGF nor sFlt-1 correlated with levels of the endothelial cell activation/damage marker soluble thrombomodulin. Our data point to changes in levels of the VEGF receptor, sFlt-1--but not VEGF itself--in smokers, which appears to be unrelated to endothelial cell function.     

The pivotal role of VEGF on glomerular macrophage infiltration in advanced diabetic nephropathy

A growing body of evidence implicates inflammation in the development of diabetic nephropathy. We recently reported that diabetic endothelial nitric oxide synthase knockout (eNOS KO) mice develop advanced glomerular lesions resembling human diabetic nephropathy. Vascular endothelial growth factor (VEGF) is a major factor in diabetic nephropathy, and is known to be chemotactic for macrophages. Herein, we examined the association of VEGF with macrophage infiltration in experimental diabetic nephropathy. Glomerular macrophage infiltration was markedly increased in diabetic eNOS KO mice compared to diabetic C57BL/6 mice, and correlated with glomerular injury, such as mesangiolysis, glomerular microaneurysm and nodular lesions of glomerular sclerosis. An elevation of podocyte VEGF expression correlated with infiltration of Flt-1-positive macrophage in injured glomeruli in diabetic eNOS KO mice, suggesting that VEGF could contribute to macrophage migration. Neither renal nNOS nor iNOS expression was altered in both C57BL/6 and eNOS KO mice. To determine if lack of NO could affect VEGF activation of macrophages, we examined if exogenous NO can block macrophage migration induced by VEGF in in vitro studies. Exogenous NO blocked macrophage migration and hypertrophy in response to VEGF. NO mediated these effects in part by downregulating Flt-1 expression on the macrophage. In summary, NO negatively regulates VEGF-induced macrophage migration by inhibiting Flt-1 expression. The VEGF-endothelial NO uncoupling pathway might partially explain how VEGF causes glomerular disease in diabetes.     

Attenuation of the exercise-induced increase in skeletal muscle Flt-1 mRNA by nitric oxide synthase inhibition

We investigated the vascular endothelial growth factor (VEGF) receptor [fms-like-tyrosine kinase (Flt-1 and fetal liver kinase-1 (Flk-1)] response to acute exercise. In female Wistar rats, the VEGF receptor messenger RNA (mRNA) response to a single acute exercise bout was examined using semi-quantitative Northern blot from the left gastrocnemius muscles at rest and post-exercise at 0, 1, 2, 4, 8, 16, 24 and 48 h. Exercise altered both Flt-1 and Flk-1 mRNA, with significant increases in Flt-1 mRNA at 1 and 24 h. However, post-hoc analysis was unable to discern the time point where a significant increase in Flk-1 mRNA occurred. To investigate the regulation of Flt-1 mRNA by exercise we examined if nitric oxide synthase (NOS) inhibition alters the Flt-1 mRNA response. Eight groups [Condition: Rest or Exercise; Drug: Saline, 30 mg kg(-1)N(omega)-nitro-L-arginine methyl ester (L-NAME), 300 mg kg(-1) L-NAME or 300 mg kg(-1) D-NAME] were used to determine the effect of NOS inhibition on the Flt-1 mRNA response to exercise. L-NAME, a known NOS inhibitor, attenuated the exercise-induced increase in Flt-1 mRNA by approximately 50%. These findings suggest that: (1) exercise alters Flt-1 and Flk-1 gene expression; and (2) NO is important in the regulation of the Flt-1 gene response to exercise.     

Inhibitory effects of erythromycin on wear debris-induced VEGF/Flt-1 gene production and osteolysis

Objectives  A highly vascularized and inflammatory periprosthetic tissue augments the progress of aseptic loosening, a major clinical problem after total joint replacement. The purpose of this study is to investigate the effect of erythromycin (EM) on ultra high molecular weight polyethylene (UHMWPE) particle-induced VEGF/VEGF receptor 1 (Flt-1) gene production and inflammatory osteolysis in a mouse model. Methods  UHMWPE particles were introduced into established air pouches on BALB/c mice, followed by implantation of calvaria bone from syngeneic littermates. EM treatment started 2 weeks after bone implantation (5 mg/kg day, i.p. injection). Mice without drug treatment as well as mice injected with saline alone were included. Pouch tissues were harvested 2 weeks after bone implantation. Expression of VEGF, Flt-1, RANKL, IL-1, TNF and CD68 was measured by immunostain and RT-PCR, and implanted bone resorption was analyzed by micro-CT (μCT). Results  Exposure to UHMWPE induced pouch tissue inflammation, increase of VEGF/Flt-1 proteins, and increased bone resorption. EM treatment significantly improved UHMWPE particle-induced tissue inflammation, reduced VEGF/Flt-1 protein expression, and diminished the number of TRAP⁺ cells, as well as the implanted bone resorption. Conclusion  This study demonstrated that EM inhibited VEGF and Flt-1 gene expression. The molecular mechanism of EM action on VEGF/Flt-1 signaling-mediated osteoclastogenesis warrants further investigation.     

Expression of VEGF in routinely fixed material using a new monoclonal antibody VG1

The aim of this study was to raise and characterize a monoclonal antibody reactive with VEGF (vascular endothelial growth factor) in routinely fixed specimens and to use it to investigate its tissue distribution in normal and pathological specimens. Recombinant VEGF 189 protein was used to raise a monoclonal antibody. The specificity of the antibody was confirmed using COS cells transfected with cDNA coding for VEGF 121, 165 and 189 protein and by western blotting studies. The resulting antibody VG1 was shown to react with the 121, 165 and 189 isoforms of VEGF protein in routinely processed material. In normal tissues, there was strong staining of endometrial and salivary glands and of the mucosa of the gastro-intestinal tract. In tumours, a proportion of the neoplastic cells in lung and breast cancer and in melanoma were labelled. In all tissues, whether normal or malignant, striking VEGF positivity was seen in plasma in vessels and stroma. This study has shown that antibody VG1 detects the 121, 165 and 189 VEGF isoforms in routinely fixed specimens. The results of the normal tissue and tumour labelling are in agreement with other studies using alternative methods of detection. This should be a useful and reliable reagent for studies of VEGF and angiogenesis in human pathological material.     

Complementary actions of VEGF and angiopoietin-1 on blood vessel growth and leakage

Vascular endothelial growth factor (VEGF) and Angiopoietins are families of vascular-specific growth factors that regulate blood vessel growth, maturation and function. To learn more about the effects of these factors in vivo, we have overexpressed VEGF-A or Angiopoietin-1 (Ang1) in two systems in mice, and examined the effects on blood vessel growth and function. In one set of studies, VEGF, Ang1, or both factors, were transgenically overexpressed in the skin under the keratin-14 (K14) promoter. The skin of mice overexpressing VEGF (K14-VEGF) had numerous tortuous, capillary-sized vessels which were leaky to the plasma tracer Evans blue under baseline conditions. In contrast, the skin of mice overexpressing Ang1 (K14-Ang1) had enlarged dermal vessels without a significant increase in vessel number. These enlarged vessels were less leaky than those of wild-type mice in response to inflammatory stimuli. In double transgenic mice overexpressing VEGF and Ang1, the size and number of skin vessels were both increased; however, the vessels were not leaky. In a second set of studies, VEGF or Ang1 was systemically delivered using an adenoviral approach. Intravenous injection of adenovirus encoding VEGF (Adeno-VEGF) resulted in widespread tissue oedema within 1-2 days after administration, whereas injection of Adeno-Ang1 resulted in the skin vessels becoming less leaky in response to topical inflammatory stimuli or local injection of VEGF. The decreased leakage was not accompanied by morphological changes. Thus, overexpressing VEGF appears to promote growth of new vessels accompanied by plasma leakage, whereas overexpressing Ang1 promotes the enlargement of existing vessels and a resistance to leakage. Further understanding of the interrelationship of these factors during normal development could lead to their application in the treatment of ischaemic diseases.     

The vascular endothelial growth factor (VEGF) receptor Flt-1 (VEGFR-1) modulates Flk-1 (VEGFR-2) signaling during blood vessel formation

Mice lacking the vascular endothelial growth factor (VEGF) receptor flt-1 (VEGFR-1) die from vascular overgrowth, caused primarily by aberrant endothelial cell division (Kearney JB, Ambler CA, Monaco KA, Johnson N, Rapoport RG, Bautch VL: Vascular endothelial growth factor receptor Flt-1 negatively regulates developmental blood vessel formation by modulating endothelial cell division. Blood 2002, 99:2397-2407). Because a second high-affinity VEGF receptor, flk-1, produces a positive endothelial proliferation signal, it was logical to ask whether flt-1 affects developmental blood vessel formation by modulating signaling through flk-1. Differentiated embryonic stem cell cultures lacking flt-1 (flt-1-/-) had increased flk-1 tyrosine phosphorylation, indicating that flk-1 signaling is up-regulated in the mutant background. The selective flk-1 inhibitor SU5416 partially rescued the flt-1-/- mutant phenotype, and this rescue was accompanied by a decrease in the relative amount of flk-1 tyrosine phosphorylation. Thus reduced flk-1 signal transduction can partially compensate for the lack of flt-1. The flt-1-/- mutant phenotype was also partially rescued by Flt-1/Fc, a truncated flt-1 that binds and sequesters the VEGF ligand. Taken together, these data show that down-regulation of flk-1 signaling by two different strategies partially rescues the developmental vascular overgrowth seen in the absence of flt-1, and they support a model whereby flt-1 modulates the flk-1 signal at an early point in the pathway.     

Microvessel density, vascular endothelial growth factor and its receptors Flt-1 and Flk-1/KDR in hepatocellular carcinoma.

Assessment of angiogenesis may yield important information for an effective antiangiogenic treatment for hepatocellular carcinoma (HCC) because HCC is characteristically hypervascular We examined the relationship of microvessel density (MVD), vascular endothelial growth factor (VEGF), and VEGF receptors Flt-1 and Flk-1/KDR in 50 patients with HCC and in 3 hepatoma cell lines. VEGF messenger RNA (mRNA) was overexpressed in 26 tumors (52%), and the 3 VEGF isoforms (121, 165, and 189) were present in high frequencies. Flt-1 mRNA was overexpressed in 34 tumors (68%), with levels significantly increased in HCCs compared with the nontumorous livers. Tumor Flt-1 mRNA significantly correlated with tumor VEGF mRNA levels. Within the group of tumors 8.5 cm or less in diameter, tumors with intrahepatic metastasis in the form of tumor microsatellite formation had significantly higher VEGF mRNA levels. MVD assessed by immunohistochemical analysis with CD34 antibody was inversely related to tumor size. Angiogenesis as assessed by MVD and tumor VEGF expression seems to have a more important role in tumor growth and intrahepatic metastasis in smaller HCCs. The differential up-regulation of Flt-1 suggests that it may have an important role in angiogenesis in HCC.     

A comparison of plasma versus histologic indices of angiogenic markers in breast cancer.

BACKGROUND: Over-expression of angiogenic growth factors and their receptors, and high levels of these molecules in the blood, are a common feature of cancer although the relationships between cell expression and plasma levels are unknown. We hypothesized a significant correlation between the expression and cellular distribution of vascular endothelial growth factor (VEGF), its receptor Flt-1, and the angiopoietin receptor Tie-2 with levels of these molecules in the plasma. METHODS: The tissue expression of VEGF, Flt-1, and Tie-2 were investigated by immunohistochemistry, and plasma levels assessed by enzyme-linked immunosorbent assay in 36 patients with breast cancer and 15 with benign breast disease. RESULTS: Despite expected significant differences in plasma levels of the molecules (P<0.03 to <0.001), no significant differences were found in Tie-2, VEGF, and Flt-1 tissue expression between breast cancer and benign disease controls. No significant correlations were observed between plasma levels of their tissue expression. CONCLUSIONS: Tissue expression of Tie-2, VEGF, and Flt-1 may not be an overly sensitive tool for assessing abnormalities of coagulation, platelet activation, and angiogenesis in human cancer. Plasma markers may not be representative of tumor activity, and may not come wholly from tumor cells. Instead these markers may be indicative of endothelial dysfunction in cancer patients.     

Acute resistance exercise increases skeletal muscle angiogenic growth factor expression

AIMS: Both aerobic and resistance exercise training promote skeletal muscle angiogenesis. Acute aerobic exercise increases several pro-angiogenic pathways, the best characterized being increases in vascular endothelial growth factor (VEGF). We hypothesized that acute resistance exercise also increases skeletal muscle angiogenic growth factor [VEGF and angiopoietin (Ang)] expression. METHODS: Seven young, sedentary individuals had vastus lateralis muscle biopsies and blood drawn prior to and at 0, 2 and 4 h post-resistance exercise for the measurement of VEGF; VEGF receptor [KDR, Flt-1 and neuropilin 1 (Nrp1)]; Ang1 and Ang2; and the angiopoietin receptor--Tie2 expression. Resistance exercise consisted of progressive knee extensor (KE) exercise to determine one repetition maximum (1-RM) followed by three sets of 10 repetitions (3 x 10) of KE exercise at 60-80% of 1-RM. RESULTS: Resistance exercise significantly increased skeletal muscle VEGF mRNA and protein and plasma VEGF protein at 2 and 4 h. Resistance exercise increased KDR mRNA and Tie2 mRNA at 4 h and Nrp1 mRNA at 2 and 4 h. Skeletal muscle Flt-1, Ang1, Ang2 and Ang2/Ang1 ratio mRNA were not altered by resistance exercise. CONCLUSIONS: These findings suggest that acute resistance exercise increases skeletal muscle VEGF, VEGF receptor and angiopoietin receptor expression. The increases in muscle angiogenic growth factor expression in response to acute resistance exercise are similar in timing and magnitude with responses to acute aerobic exercise and are consistent with resistance exercise promoting muscle angiogenesis.     

Vascular endothelial growth factor modulates skeletal myoblast function

Vascular endothelial growth factor (VEGF) expression is enhanced in ischemic skeletal muscle and is thought to play a key role in the angiogenic response to ischemia. However, it is still unknown whether, in addition to new blood vessel growth, VEGF modulates skeletal muscle cell function. In the present study immunohistochemical analysis showed that, in normoperfused mouse hindlimb, VEGF and its receptors Flk-1 and Flt-1 were expressed mostly in quiescent satellite cells. Unilateral hindlimb ischemia was induced by left femoral artery ligation. At day 3 and day 7 after the induction of ischemia, Flk-1 and Flt-1 were expressed in regenerating muscle fibers and VEGF expression by these fibers was markedly enhanced. Additional in vitro experiments showed that in growing medium both cultured satellite cells and myoblast cell line C2C12 expressed VEGF and its receptors. Under these conditions, Flk-1 receptor exhibited constitutive tyrosine phosphorylation that was increased by VEGF treatment. During myogenic differentiation Flk-1 and Flt-1 were down-regulated. In a modified Boyden Chamber assay, VEGF enhanced C2C12 myoblasts migration approximately fivefold. Moreover, VEGF administration to differentiating C2C12 myoblasts prevented apoptosis, while inhibition of VEGF signaling either with selective VEGF receptor inhibitors (SU1498 and CB676475) or a neutralizing Flk-1 antibody, enhanced cell death approximately 3.5-fold. Finally, adenovirus-mediated VEGF(165) gene transfer inhibited ischemia-induced apoptosis in skeletal muscle. These results support a role for VEGF in myoblast migration and survival, and suggest a novel autocrine role of VEGF in skeletal muscle repair during ischemia.     

Role of signals from the dorsal root ganglion in neuropathic pain in a rat model

Vascular endothelial growth factor (VEGF), known as an endothelial cell-specific mitogen, has been reported to be linked also to the NO/cGMP-pathway, which has been notified in the inner ear. Up to now, VEGF has not yet been described in the inner ear. We performed immunohistochemical analysis using specific antibodies to VEGF and to both known VEGF-receptors Flt-1 and KDR/Flk-1 on paraffin-sections of temporal bones from guinea pigs (n=5). Immunoreactivity of VEGF, Flt-1 and KDR/Flk-1 was detectable in a subpopulation of vestibular ganglion cells. VEGF could be found also in the endothelium of blood vessels, in fibrocytes of the lamina propria and in the neuroepithelium. Strong immuno-labelling to Flt-1 was evident in nerve fibres, vascular endothelium and in the neuroepithelium. Fibrocytes, endothelium of blood vessels, supporting cells and calyces in the sensory epithelium revealed immunoreactivity to KDR/Flk-1. These findings give evidence that VEGF, Flt-1 and KDR/Flk-1 are constitutively expressed in the vestibule.