Transcriptional regulation of vascular endothelial growth factor gene expression in ovarian bovine granulosa cells

From rape (Brassica napus) seedlings proteins able to bind fatty acids and their CoA-esters were purified by gel filtration and cation-exchange chromatography. Among the four proteins detected, one of them (peak IV) appeared purified to homogeneity. This protein is a monomer with a molecular mass of about 9 kDa, as estimated by gel filtration and by polyacrylamide gel electrophoresis. The isoelectric point of the rape protein was higher than 10.5 as determined by chromatofocusing. The pure rape protein appeared furthermore to be able to transfer several phospholipids (phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine) between membranes. The rape protein, having a multifunctional property, was thus called acyl-binding/lipid-transfer protein (AB-LTP). In order to compare this protein to plant lipid-transfer proteins (LTPs), its structure was determined. The amino acid analysis of the rape AB-LTP revealed a high amount of alanine, an absence of histidine and tryptophan and the presence of eight cysteine residues. The N-terminal amino acid sequence of the rape protein revealed a high homology to plant LTPs. These observations led us to propose that the rape AB-LTPs belong to a category of plant proteins interacting with lipids and playing a role in the fatty acid dynamics.     

Enhanced expression of vascular endothelial growth factor in metastatic melanoma

Tumour growth is dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is a secreted endothelial cell-specific cytokine. VEGF is angiogenic in vivo and it also acts as a vascular permeability factor. VEGF is overexpressed in many skin disorders characterized by angiogenesis and increased vascular permeability. We investigated VEGF expression in 22 primary cutaneous melanomas, 33 melanoma metastases and six naevocellular naevi using immunohistochemistry. VEGF accumulated on the vascular endothelia in the normal dermis, suggesting that a constitutive low level of VEGF expression may regulate skin vessel function under normal physiological conditions. No VEGF was detected in the cells of naevocellular naevi or normal dermis. In contrast, 32% of the primary and 91% of the metastatic melanomas contained melanoma cells staining for VEGF. Expression of VEGF was more frequent in metastases than in primary melanomas (P <0.0001). Tumour-infiltrating inflammatory cells expressed VEGF in all melanomas. A high number of VEGF-expressing inflammatory cells was associated with high VEGF expression in melanoma cells (P = 0.003). Our results suggest that VEGF is up-regulated during the course of melanoma progression and dissemination and that tumour-infiltrating cells expressing VEGF may contribute to the progression of melanoma.     

青蒿琥酯对急性单核细胞白血病SHI-1细胞株血管内皮生长因子及其受体表达的影响

目的 研究青蒿琥酯对急性单核细胞白血病SHI-1细胞株血管内皮生长因子(VEGF)及其受体( VEGFR)的影响。方法酶联免疫吸附法检测非细胞毒性浓度(5、10、20 ng/ml)青蒿琥酯作用SHI-1细胞后培养上清液VEGF浓度,流式细胞术检测有或无青蒿琥酯作用时,SHI-1细胞表面VEGFR-1及VEGFR-2阳性表达率。结果培养24、48 h后,无青蒿琥酯作用的SHI-1细胞培养上清液VEGF质量浓度分别为( 980.3±2.2)、(982.4±2.3) pg/ml,VEGFR-1表达率分别为(5.40±3.11)％和(4.45±2.85)％,VEGFR-2表达率分别为(13.90.± 2.26)％和（13.95±1.96）％。5、10、20 ng/ml青蒿琥酯作用24h后,SHI-1细胞培养上清液VEGF质量浓度分别为(234.6±1.8)、(114.9±1.6)、(108.8±1.5) pg/ml,作用48 h后分别为(62.3±1.7)、(60.9±1.6)、(32.7±1.7) pg/ml,与培养相同时间无青蒿琥酯组相比,VEGF浓度明显下降（均P＜ 0.05）,且相同浓度青蒿琥酯作用24 h与48 h间差异亦有统计学意义(均P＜ 0.05)。5、10、20 ng/ml青蒿琥酯作用24 h,VEGFR-1阳性率分别为(4.30±2.21)％、(4.20±1.37)％和(3.90±1.86)％,作用48 h后分别为(3.80±2.87)％、(3.60±1.73)％和(3.00±1.82)％,相同作用时间不同浓度青蒿琥酯组间及相同浓度作用不同时间组间VEGFR-1阳性率差异均无统计学意义(均P＞ 0.05);作用24h后,SHI-1细胞VEGFR-2阳性率分别为(4.40±1.15)％、(3.10±0.68)％和(1.10±0.72)％,作用48 h后分别为(3.00±1.68)％、(2.20±0.93)％和(0.60±0.92)％,3个不同浓度青蒿琥酯作用相同时间后VEGFR-2表达率降低（均P＜ 0.05）,相同浓度作用24与48 h间差异均无统计学意义（均P＞ 0.05）。结论SHI-1细胞株高分泌VEGF,青蒿琥酯可下调VEGF分泌及VEGFR-2的表达,而对VEGFR-1表达的调节作用不显著。     

Transforming growth factor beta 1 down-regulates vascular endothelial growth factor receptor 2/flk-1 expression in vascular endothelial cells

Although the importance of the vascular endothelial growth factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in angiogenesis is well established, very little is known about the regulation of VEGFR expression in vascular endothelial cells. We have cloned partial cDNAs encoding bovine VEGFR-1 (flt) and -2 (flk-1) and used them to study VEGFR expression by bovine microvascular- and large vessel-derived endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor beta 1 (TGF-beta 1) reduced the high affinity 125I-VEGF binding capacity of both cell types in a dose-dependent manner, with a 2.0-2.7-fold decrease at 1-10 ng/ml. Cross-linking experiments revealed a decrease in 125I-VEGF binding to a cell surface monomeric protein corresponding to Flk-1 on the basis of its affinity for VEGF, molecular mass (185-190 kDa), and apparent internalization after VEGF binding. Immunoprecipitation and Western blot experiments demonstrated a decrease in Flk-1 protein expression, and TGF-beta 1 reduced flk-1 mRNA levels in a dose-dependent manner. These results imply that TGF-beta 1 is a major regulator of the VEGF/Flk-1 signal transduction pathway in endothelial cells.     

Corticosteroids inhibit the expression of the vascular endothelial growth factor gene in human vascular smooth muscle cells

The vascular endothelial growth factor (VEGF) is a specific mitogen for vascular endothelial cells and enhances vascular permeability and edemagenesis. VEGF is also a major regulator of angiogenesis and may be a key target for inhibiting angiogenesis in angiogenesis-associated diseases. Among the extensively studied angiostatic compounds are several corticosteroids when used alone or in combination with heparin. In this study we present evidence for an additional mechanism of action of hydrocortisone, cortisone and dexamethasone in inhibiting edemagenesis or angiogenesis. In cultures of aortic human vascular smooth muscle cells these corticosteroids (1 x 10(-8) to 1 x 10(-12) M) abolished the platelet-derived growth factor-induced (PDGF) expression of the VEGF gene in a dose-dependent manner. In contrast, two precursors of corticosteroids, desoxycorticosterone or pregnenolone, did not affect PDGF-induced VEGF expression. Our findings indicate that the capacity of corticosteroids to reduce edema or to prevent new blood vessel formation may be attributed, at least in part to the ability of these agents to abolish the expression of VEGF.     

Vascular endothelial growth factor expression is increased in renal cell carcinoma

PURPOSE: To compare the expression of VEGF in renal cell carcinoma (RCC) and normal kidney. MATERIALS AND METHODS: RT-PCR and Western blot analysis were performed on tumour and normal adjacent kidney collected from 31 patients (29 RCC and 2 oncocytomas) as well as proliferating vascular endothelial cells (VEC) in culture. RESULTS: Expression of 3 VEGF isoforms was detected in normal renal parenchyma and all ROC by RT-PCR, but was not apparent in proliferating VEC. In 27 RCC, Western blot analysis demonstrated 3-37 fold increases in VEGF expression when compared to normal parenchyma. Immunohistochemistry demonstrated VEGF staining of both tumour cells and adjacent vascular endothelium. Normal kidney showed no staining for VEGF. In the 2 remaining RCC and both oncocytomas VEGF was not increased. CONCLUSIONS: VEGF expression is increased in RCC and may have a paracrine effect in these tumours in stimulating angiogenesis.     

Brain edema in meningiomas is associated with increased vascular endothelial growth factor expression

OBJECTIVE: Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), an endothelial cell-specific cytokine, induces proliferation of endothelial cells and increases vascular permeability dramatically. All gliomas secrete significant amounts of VEGF, whereas meningiomas are variable in expression. Thus, we sought to determine whether the extent of VPF/VEGF expression in meningiomas correlated with differences in brain edema associated with these tumors. METHODS: Meningioma tissue samples from 37 patients (15 men, average age 65 +/- 13 yr; 22 women, average age 60 +/- 10 yr) who underwent surgery at or were referred to the University of Alabama Hospital were examined retrospectively for the extent of expression of immunoreactive VPF/VEGF. Additionally, peritumoral edema was assessed on a blinded basis radiographically from preoperative magnetic resonance imaging scans. Selected specimens were examined by in situ hybridization to document the source of VPF/VEGF. RESULTS: The predominant meningioma subclassifications were transitional (57%) or meningothelial (27%) subtypes. VPF/VEGF immunoreactivity ranged from 0 to 3.5, with a median value of 2 on a subjective 5-point scale; magnetic resonance imaging-assessed edema ranged in extent from 0 to 4 (subjective 5-point scale), with a median value of 2.5. The correlation of determination (R2) of magnetic resonance imaging-assessed tumor edema rating and VPF/VEGF staining intensity rating was 0.6087 (r = 0.78; P = 0.0001). In situ hybridization localized VPF/VEGF messenger ribonucleic acid in meningioma cells and not in normal parenchymal brain cells. CONCLUSION: These data suggest that meningioma-associated edema may be a result of the capacity of meningioma cells to produce VPF/VEGF locally, leading to increased tumor neovascularization and enhanced vascular permeability.     

The role of vascular endothelial growth factor and interleukins in the pathogenesis of severe ovarian hyperstimulation syndrome

Recent studies have suggested that the proliferation of malignant gliomas may result from activation of protein kinase C (PKC)-mediated pathways; conversely, inhibition of PKC may provide a strategy for blocking tumor growth. In the current studies, we examined the effect of a novel PKC inhibitor, calphostin C, which is a selective, highly potent, photo-activatable inhibitor of the PKC regulatory domain, on the proliferation and viability of three established and three low-passage malignant glioma cell lines, four low-passage low-grade glioma cell lines, and in adult human and neonatal rat non-neoplastic astrocyte cell lines in vitro. Under light-treated conditions, calphostin C consistently inhibited cell proliferation in each of the tumor cell lines and in the neonatal rat astrocyte cell line with a 50% effective concentration of 30 to 50 ng/ml (40 to 60 nm), which was comparable to the previously reported median inhibitory concentration (IC50) for PKC inhibition by calphostin C. Complete elimination of proliferation was achieved at concentrations of 50 to 100 ng/ml (60 to 125 nM). Cell viability decreased sharply with calphostin C concentrations of 100 to 300 ng/ml (125 to 380 nM). In contrast, under light-shielded conditions, calphostin C had a comparatively modest effect on cell proliferation and viability, with a median effective concentration of approximately 300 ng/ml. No significant inhibition of proliferation was noted in the non-neoplastic adult astrocyte cell line under either light-treated or light-shielded conditions. These findings provide further evidence that PKC may play an essential role in mediating the proliferation of both benign and malignant glioma cells in vitro and may also contribute to the proliferation of non-neoplastic immature astrocytes. Light-sensitive inhibition of proliferation and viability by agents such as calphostin C may provide a novel strategy for applying photodynamic therapy to the treatment of neoplastic glial cells.     

Hypoxia and vascular endothelial growth factor stimulate angiogenic integrin expression in bovine retinal microvascular endothelial cells

PURPOSE: Integrins alphavbeta3 and alphavbeta5 are cell-to-matrix adhesion molecules that have been reported to mediate vascular cell proliferation and migration. The authors investigated the regulation of expression of these angiogenic integrins by hypoxia and vascular endothelial growth factor (VEGF) in retinal microvascular endothelial cells in culture. METHODS: Cultured bovine retinal capillary endothelial cells were exposed to human recombinant VEGF under normoxic (95% air, 5% CO2) conditions to assess the effects of VEGF. Hypoxia studies were performed under lower oxygen concentration (0.5%-1.5% O2) induced by nitrogen replacement in constant 5% CO2 conditions. Integrin family mRNA and protein expression were assessed by northern blot analysis and immunoprecipitation. RESULTS: VEGF (25 ng/ml) increased integrin alphav, beta3, and 35 mRNA after 24 hours 6.1+/-0.8-fold (P < 0.001), 5.9+/-1.1-fold (P < 0.001), and 1.9+/-0.2-fold (P < 0.01), respectively. Similarly, hypoxia stimulated gene expression of integrin alphav and beta3 after 24 hours by 5.1+/-1.7-fold (P < 0.01) and 3.0+/-0.5-fold (P < 0.01), respectively, and integrin beta5 after 9 hours 1.4+/-0.2-fold (P < 0.05). This hypoxia-induced, integrin alphav mRNA elevation was inhibited significantly by anti-VEGF neutralizing antibody. Also, a conditioned medium from confluent endothelial cells maintained under hypoxic conditions for 24 hours produced a 7.1+/-1.1-fold increase (P < 0.001) in integrin alphav mRNA expression after 24 hours, which was reversed by anti-VEGF neutralizing antibody. Induction of integrin alphav by VEGF and hypoxia was confirmed in the protein level. CONCLUSIONS: These data suggest that hypoxia stimulates expression of vascular integrins alphavbeta3 and alphavbeta5 in retinal microvascular endothelial cells partially through autocrine-paracrine action of VEGF induced by the hypoxic state.     

Vascular endothelial growth factor expression in head and neck squamous cell carcinoma

BACKGROUND: Angiogenesis is an essential process required for growth and metastasis in cancer. In breast, gastric, and prostate cancer, vascular endothelial growth factor (VEGF) has been implicated in angiogenesis; however, little is known about VEGF in HNSCC. In this study, we hypothesize that VEGF is present in elevated levels in HNSCC and may therefore play a role in promoting angiogenesis. METHODS: We obtained tumor tissue from 63 HNSCC patients undergoing primary resection. All tissue samples were analyzed by immunohistochemistry (IHC) techniques for the presence and localization of VEGF; however, only 36 had sufficient amounts of tissue for quantitative analysis of VEGF by ELISA. Nine control specimens taken from patients undergoing uvulopalatopharyngoplasty were also analyzed. RESULTS: In all 63 of our patient samples we found VEGF to be present and localized to the cancer cells and endothelial cells. The poorly differentiated cancer cells stained more intensely in comparison with the well-differentiated ones. There was a 20-fold increase in the patient levels when compared with controls levels (P > or =0.05). Analysis by enzyme-linked immunosorbent assay revealed elevated mean levels of VEGF (241 +/- 326 pg/mg total protein [TP]) with a range of 2 to 1484 pg/mg TP. The control specimens had mean levels of 13 +/- 11 pg/mg TP and a range of 1 to 78 pg/mg TP. Patients who exhibited higher levels of VEGF tended to have a higher rate of disease recurrence (P < or =0.048) and shorter disease-free interval (P < or =0.05). CONCLUSIONS: The expression of VEGF in elevated levels in the HNSCC tumor microenvironment appears to be associated with more aggressive disease. Based on our results, VEGF may be an important angiogenic factor associated with cancer cells and endothelial cells in HNSCC. Further studies are needed to better define the role of VEGF in HNSCC and its role as a potential target for therapeutic intervention.     

The von Hippel-Lindau gene product inhibits vascular permeability factor/vascular endothelial growth factor expression in renal cell carcinoma by blocking protein kinase C pathways

Mutation or loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is regularly found in sporadic renal cell carcinomas (RCC), well vascularized malignant tumors that characteristically overexpress vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). The wild-type VHL (wt-VHL) gene product acts to suppress VPF/VEGF expression, which is overexpressed when wt-VHL is inactive. The present study investigated the pathways by which VHL regulates VPF/VEGF expression. We found that inhibition of protein kinase C (PKC) represses VPF/VEGF expression in RCC cells that regularly overexpress VPF/VEGF. The wt-VHL expressed by stably transfected RCC cells forms cytoplasmic complexes with two specific PKC isoforms, zeta and delta, and prevents their translocation to the cell membrane where they otherwise would engage in signaling steps that lead to VPF/VEGF overexpression. Other experiments implicated mitogen-activated protein kinase (MAPK) phosphorylation as a downstream step in PKC regulation of VPF/VEGF expression. Taken together, these data demonstrate that wt-VHL, by neutralizing PKC isoforms zeta and delta and thereby inhibiting MAPK activation, plays an important role in preventing aberrant VPF/VEGF overexpression and the angiogenesis that results from such overexpression.     

Angiogenesis is associated with vascular endothelial growth factor expression in cervical intraepithelial neoplasia

Squamous cell carcinoma of the cervix (SCC) is preceded by a premalignant condition known as cervical intraepithelial neoplasia (CIN). The majority of cases of CIN regress spontaneously; however, methods are needed to identify those lesions likely to progress. Increased blood vessel density, signifying angiogenesis, is an independent prognostic indicator in a number of cancers, although little is known about its significance in premalignant lesions. The aim of the present study was to determine the relationship between vessel density, expression of the potent angiogenic factor vascular endothelial growth factor (VEGF) and CIN grade. Using immunohistochemistry, mean vessel density (MVD) and VEGF expression were assessed in samples from 54 patients who had undergone cone biopsy for CIN or hysterectomy for SCC and from 16 patients with no cervical pathology. There were significant increases in MVD and VEGF expression from normal cervix through CIN I to CIN III to invasive SCC, but no difference in mean vessel diameter between groups. There was a strong correlation between mean vessel density and VEGF expression, and both were associated with histological grade of CIN. The original MVDs for a small group of patients later presenting with recurrent disease were found to be equal to or greater than the mean for their histological grade. We conclude that the onset of angiogenesis is an early event in premalignant changes of the cervix due, in part, to enhanced expression of VEGF by the abnormal epithelium.     

Role of vascular endothelial growth factor in ovarian cancer: inhibition of ascites formation by immunoneutralization

Ovarian cancer is characterized by the rapid growth of solid intraperitoneal tumors and large volumes of ascitic fluid. Vascular endothelial growth factor (VEGF) augments tumor growth by inducing neovascularization and may stimulate ascites formation by increasing vascular permeability. We examined the role of VEGF in ovarian carcinoma using in vivo models in which intraperitoneal or subcutaneous tumors were induced in immunodeficient mice using the human ovarian carcinoma cell line SKOV-3. After tumor engraftment (7 to 10 days), some mice were treated with a function-blocking VEGF antibody (A4.6.1) specific for human VEGF. A4.6.1 significantly (P < 0.05) inhibited subcutaneous SKOV-3 tumor growth compared with controls. However, tumor growth resumed when A4.6.1 treatment was discontinued. In mice bearing intraperitoneal tumors (IP mice), ascites production and intraperitoneal carcinomatosis were detected 3 to 7 weeks after SKOV-3 inoculation. Importantly, A4.6.1 completely inhibited ascites production in IP mice, although it only partially inhibited intraperitoneal tumor growth. Tumor burden was variable in A4.6.1-treated IP mice; some had minimal tumor, whereas in others tumor burden was similar to that of controls. When A4.6.1 treatment was stopped, IP mice rapidly (within 2 weeks) developed ascites and became cachectic. These data suggest that in ovarian cancer, tumor-derived VEGF is obligatory for ascites formation but not for intraperitoneal tumor growth. Neutralization of VEGF activity may have clinical application in inhibiting malignant ascites formation in ovarian cancer.     

Human neutrophils secrete vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a pivotal role in mediating neovascularization as well as other endothelial cell alterations during inflammation. In this study, we demonstrate that human neutrophils are a source of VEGF. We observed that isolated blood neutrophils released VEGF in response to different stimuli and we demonstrated by immunohistochemistry that neutrophils infiltrating inflamed tissues contain VEGF. These results indicate that neutrophil-derived VEGF may be instrumental in regulating vascular responses during acute and chronic inflammation.