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.     

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。     

Expression of vascular endothelial growth factor and its receptors in rats with protein-overload nephrosis

BACKGROUND: Based on the fact that vascular endothelial growth factor (VEGF) increases vascular permeability, it is speculated that VEGF might be involved in the development of proteinuria, although this remains unconfirmed. The production and site of action of VEGF remains unclear in nephrotic renal diseases. METHODS: Non-radioactive in situ hybridization was performed to examine the expression of VEGF mRNA and its receptors, flt-1 and KDR/flk-1, in a rat model of nephrosis induced by intraperitoneal injection of bovine serum albumin (BSA). Saline injected rats were served as control animals. RESULTS: Neither morphological changes nor deposition of immunoglobulin or complement were observed in our model. Proteinuria developed, reaching a maximum level in rats injected with BSA for 3 days, followed by persistent proteinuria until day 14. The expression of mRNA for VEGF and the two receptors was markedly upregulated in glomeruli of BSA-induced nephritis compared with the control group. VEGF mRNA was localized in glomerular cells, including cells in mesangium, visceral and parietal epithelial cells. In contrast, flt-1 mRNA and KDR/flk-1 mRNA were expressed on glomerular endothelial cells and cells in mesangium. The ratio of glomerular cells positive for VEGF mRNA and its receptors mRNA increased proportionately with the severity of proteinuria. Immunohistochemistry for ED-1 and proliferating cell nuclear antigen showed no significant increase in infiltrating macrophage or cellular proliferation. Conclusions: Our results suggest that altered glomerular expression of VEGF and its receptors is not associated with proliferation of endothelial cells, but rather with proteinuria in BSA-induced nephritis in rats. VEGF may play a different role in different renal diseases.     

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.     

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.     

Tissue distribution and biotransformation of zopolrestat, an aldose reductase inhibitor, in rats

Zopolrestat (Alond) is a new drug that is being evaluated as an aldose reductase inhibitor for the treatment of diabetic complications. 14C-labeled zopolrestat was orally administered to rats for a tissue distribution study and a bile duct cannulation metabolism study. Tissue samples from the distribution study were analyzed by complete oxidation and liquid scintillation counting. Urine and bile samples from the bile duct cannulation study were analyzed by microbore HPLC, with simultaneous radioactivity monitoring and atmospheric pressure ionization tandem mass spectrometry. The mass balance in the distribution study demonstrated that the greatest exposure (AUC0-infinity) occurred in the liver, followed by the ileum and large intestine. The time of maximal plasma concentrations for nearly all tissues was 4 hr after the dose, and the half-life of radioactivity in most tissues (8-10 hr) was similar to the half-life in plasma. For the bile duct-cannulated rat study, most of the radioactivity was recovered in the bile, indicating that biliary excretion is a major route of elimination of zopolrestat and its metabolites in rats. Numerous oxidative metabolites, as well as phase II conjugates, were identified in the bile and urine samples. Acyl glucuronides of zopolrestat and unchanged drug accounted for >85% of biliary radioactivity, whereas unchanged drug and degradation products of glutathione conjugates were identified as the major urinary metabolites.     

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.     

Increased vascular endothelial growth factor production in the lungs of rats with hypoxia-induced pulmonary hypertension

Vascular endothelial growth factor (VEGF) is a potent mitogenic and permeability factor targeting predominantly endothelial cells. At least two tyrosine kinase receptors, Flk-1 and Flt-1, mediate its action and are mostly expressed by endothelial cells. VEGF and VEGF receptor expression are upregulated by hypoxia in vivo and the role of VEGF in hypoxia-induced angiogenesis has been extensively studied in a variety of disease entities. Although VEGF and its receptors are abundantly expressed in the lung, their role in hypoxic pulmonary hypertension and the accompanying vascular remodeling are incompletely understood. We report in this in vivo study that hypoxia increases mRNA levels for both VEGF and Flk-1 in the rat lung. The kinetics of the hypoxic response differ between receptor and ligand: Flk-1 mRNA showed a biphasic response to hypoxia with a significant, but transient, rise in mRNA levels observed after 9-15 h of hypoxic exposure and the highest levels noted after 3 wk. In contrast, VEGF mRNA levels did not show a significant increase with acute hypoxia, but increased progressively after 1-3 wk of hypoxia. By in situ hybridization, VEGF mRNA was localized predominantly in alveolar epithelial cells with increased signal in the lungs of hypoxic animals compared with controls. Immunohistochemical staining with anti-VEGF antibodies localized VEGF peptide throughout the lung parenchyma and was increased in hypoxic compared with normoxic animals. Furthermore, hypoxic animals had significantly higher circulating VEGF concentrations compared with normoxic controls. Lung vascular permeability as measured by extravasation of Evans Blue dye was not significantly different between normoxic and hypoxic animals, although a tendency for increased permeability was seen in the hypoxic animals. These findings suggest a possible role for VEGF in the pulmonary response to hypoxia.     

Circulating vascular endothelial growth factor in patients with colorectal cancer

BACKGROUND: Venom immunotherapy (VIT) has proven to be safe and effective in wasp venom anaphylaxis. However, there are no good parameters to indicate when to stop venom immunotherapy. OBJECTIVE: To evaluate the relationship of the lymphocyte transformation test (LTT) to history and specific IgE determination, and to address the time course of lymphocyte transformation responses to wasp (Vespula) venom during VIT and the possible utility of LTT to determine the duration of therapy. METHODS: Peripheral blood mononuclear cells (PBMCs) of 18 individuals with a history of wasp sting anaphylaxis and a positive serum-venom-specific IgE, were stimulated with wasp venom before immunotherapy, at the end of a 5-day semi-rush immunotherapy and at 24 months during venom immunotherapy. Results, expressed as stimulation index (SI), were compared with the SI in seven asymptomatic stung controls. RESULTS: In controls the median (minimum-maximum) of the SI were 2.39 (0.52-3.39) before therapy and 2.39 (1.12-6.02) when repeated after 24 months. For patients the median (minimum-maximum) of the SI were 10.13 (1.19-44.88) before immunotherapy (d0), 2.73 (0.67-12.03) at the end of the build-up immunotherapy (d5) and 4.21 (0.88-14.66) at the end of 24 months of maintenance therapy (m24). The proliferation responses in vespid-allergic patients were significantly higher than in stung controls (P = 0.006) but only 13/18 patients showed a positive LTT result before the start of immunotherapy (sensitivity of the LTT 72%). When the LTT was repeated after a 5 day build-up hyposensitization course the SI significantly dropped as compared to the pre-treatment levels (P = 0.002). The SI of the LTT was negative in eight out of 18 patients at 24 months and the median values were significantly lower than before therapy (P = 0.03). CONCLUSIONS: Although, in the absence of sting challenge data it is not possible to draw conclusions about the predictive value of the LTT, our data may suggest that abolition of the LTT during VIT might indicate clinical insensitivity. Further studies, comparing the results of sting challenges, with the results of lymphocyte transformation will be necessary in order to evaluate the role of LTT in stopping immunotherapy.     

Expression of vascular endothelial growth factor and placenta growth factor in human placenta

Normal development and function of the placenta requires invasion of the maternal decidua by trophoblasts, followed by abundant and organized vascular growth. Little is known of the significance and function of the vascular endothelial growth factor (VEGF) family, which includes VEGF, VEGF-B, and VEGF-C, and of placenta growth factor (PIGF) in these processes. In this study we have analyzed the expression of VEGF and PIGF mRNAs and their protein products in placental tissue obtained from noncomplicated pregnancies. Expression of VEGF and PIGF mRNA was observed by in situ hybridization in the chorionic mesenchyme and villous trophoblasts, respectively. Immunostaining localized the VEGF and PIGF proteins in the vascular endothelium, which was defined by staining for von Willebrand factor and for the Tie receptor tyrosine kinase, an early endothelial cell marker. VEGF-B and VEGF-C mRNAs were strongly expressed in human placenta as evidenced by Northern blot analysis. These data imply that VEGF and PIGF are produced by different cells but that both target the endothelial cells of normal human term placenta.     

Correlation of vascular endothelial growth factor messenger RNA expression with peritumoral vasogenic cerebral edema in meningiomas

Matched related cord blood transplantation (CBT) has been successfully used to rescue patients undergoing myeloablative therapy. However, few data are available on the kinetics of hematological and immunological reconstitution of CBT recipients. We have investigated the hematological engraftment and immune recovery following related CBT in three patients, with acute lymphoblastic leukemia, aged 10, 9 and 7 years and with a body weight of 31, 40 and 25 kg, respectively. All patients engrafted and none experienced acute or chronic graft-versus-host disease. The time needed to achieve granulocyte recovery was 13, 26 and 29 days, respectively and platelet recovery occurred in 28, 49 and 51 days. All patients presented a marked increase of HbF, the values observed being much greater than those documented in patients given marrow transplantation and comparable with those observed in normal children in the first year of age. The recovery of T cell immunity, as well as that of natural killer subpopulations, mimicked that described in BMT recipients, a quicker return of CD8+ T cells determining the characteristic inversion of CD4/CD8 ratio. An impressive increase in the percentage and absolute number of B lymphocytes, apparently not related to viral infections, was demonstrable in all three cases. These data suggest that CBT recipients can experience a slight delay in hematological recovery when compared with patients given BMT. The reconstitution of erythropoiesis seems to recapitulate the ontogenetic pattern and the kinetics of recovery of the immune system reproduce that observed after BMT with the peculiarity of B cell expansion in peripheral blood.     

Hypoxia induces vascular endothelial growth factor gene and protein expression in cultured rat islet cells

The formation of new microvasculature by capillary sprouting at the site of islet transplantation is crucial for the long-term survival and function of the graft. Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties, may be a key factor in modulating the revascularization of islets after transplantation. In this study, we examined the gene expression of VEGF mRNA in three tumor cell lines and in isolated whole and dispersed rat islets in vitro by Northern blot hybridization in normoxic (5% CO2, 95% humidified air) and hypoxic (1% O2, 5% CO2, 94% N2) culture conditions. Increased expression of VEGF mRNA was observed in beta-TC3, RAW 264.7, and IC-21 tumor cell lines when subjected to hypoxia. With isolated whole islets in normoxic culture, a threefold increase in VEGF mRNA (P < 0.001) was seen at 48 h as compared with freshly isolated islets. This response was similar to the 3.8-fold increase observed with islets subjected to hypoxia. Dispersed rat islet cell clusters cultured on Matrigel for 24 h under hypoxic conditions showed a 3.4-fold increase (P < 0.01) in VEGF mRNA compared with those cultured in normoxia. This correlated with increased VEGF secretion as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies revealed the presence of increased expression of VEGF protein near the center of islets after 24 h of normoxic culture. Islet cell clusters on Matrigel showed intense cellular localization of VEGF in both beta-cells and non-beta-cells. These findings suggest that rat islet cells, when subjected to hypoxia during the first few days after transplantation, may act as a major source of VEGF, thereby initiating revascularization and maintaining the vascular permeability of the grafted islets.