Role of vascular endothelial growth factor on erythropoietin-related endothelial cell proliferation

The vascular actions of recombinant human erythropoietin (rhEPO) are of particular relevance for fully understanding rhEPO effects. This study examines the mechanisms of action of rhEPO on endothelial cells from bovine aorta (BAEC). First, the studies demonstrated that rhEPO acts on BAEC proliferation as a comitogenic growth factor in the presence of fetal calf serum (FCS). The main experimental findings disclosed that an interaction between rhEPO and vascular endothelial growth factor (VEGF) is instrumental for the growth-promoting action of rhEPO, as shown by the blockade (92.8+/-2.2% inhibition, P < 0.01) of the rhEPO-induced BAEC proliferation by a specific anti-VEGF antibody and by the capability of VEGF for substituting FCS in the induction of rhEPO-related BAEC proliferation (increase in BAEC number in the absence of FCS: 20 U/ml rhEPO alone, 0.3+/-2.8%; 5 x 10(-11) M VEGF alone, 52.9+/-3.1%; 20 U/ml rhEPO + 5 X 10(-11) M VEGF, 117.8+/-6.9%, P < 0.01 between the two agents combined with respect to each agent alone). The existence of a positive interaction between rhEPO and VEGF was further demonstrated by observing an increased cytosolic Ca2+ ([Ca2+]i) mobilization response to VEGF (10(-11)M) in BAEC pretreated or not with 20 U/ml rhEPO (delta[Ca2+]i = 704+/-111 versus 246+/-36 nM, respectively, P < 0.01). To further examine the mechanism of the potentiation of VEGF effect by rhEPO, we analyzed the mRNA expression of the VEGF receptors KDR/flk-1 and flt-1. The results disclosed that BAEC pretreatment with rhEPO upregulated the expression of both KDR/flk-1 and flt-1, therefore providing a structural basis for the aforementioned positive interactions between VEGF and rhEPO. Furthermore, inhibition by genistein suggests that tyrosine phosphorylation was involved in the VEGF receptor upregulation. The mechanisms identified in the present study disclose an interaction at the level of mRNA expression and functional effects between a hormone with predominantly hemopoietic effects, namely, erythropoietin, and an angiogenic factor, namely, VEGF. This relationship between rhEPO and VEGF might be of particular importance in neovascularization processes and in patients receiving rhEPO as a treatment.     

Retinoids downregulate vascular endothelial growth factor/vascular permeability factor production by normal human keratinocytes

PURPOSE: We describe the clinical presentation, angiographic findings, and clinical outcome in a group of patients with pseudoaneurysms treated by a new endovascular technique using Guglielmi electrolytically detachable platinum coils (GDCs). METHODS: We retrospectively reviewed the angiographic and clinical findings in a series of 11 patients with pseudoaneurysms occurring in a variety of locations: seven in the cavernous carotid artery, one in the petrous carotid artery, two in the anterior cerebral artery, and one in the cervical vertebral artery. RESULTS: All aneurysms were cured with GDC embolization. The only complication was a branch occlusion, which resolved with heparinization and produced no clinical sequelae. CONCLUSION: Pseudoaneurysms can be safely and effectively treated by embolization with GDCs. Consideration needs to be given to the anatomic location of the pseudoaneurysm and the acuity of onset. Treatment efficacy may by improved if there are bony confines around the aneurysm or if therapy takes place in the subacute period, when the wall of the pseudoaneurysm has matured and stabilized.     

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。     

Pentoxifylline inhibits hypoxia-induced upregulation of tumor cell tissue factor and vascular endothelial growth factor

The aim of this study was to compare the performance of CT and MRI in the diagnosis of longitudinal stress fracture of the tibia (LSFT). A retrospective study of imaging findings was performed in 15 patients with LSFT. The CT and MR images were compared for detection of fracture line, callus, bone marrow edema, and soft tissues changes. The CT and MRI techniques allowed the detection of the fracture line in 82 and 73 % of cases, respectively. The callus was always visualized with CT or MRI. The MRI technique had a markedly higher sensitivity than CT in the detection of bone marrow edema (73 vs 18 %) and soft tissue lesions (87 vs 9 %). This may cause a misleading aggressive appearance on MRI. Computed tomography remains the best imaging modality for diagnosis of LSFT. However, MRI findings should be known to obviate the performance of CT or bone biopsy.     

Extracellular signal-regulated protein kinase/Jun kinase cross-talk underlies vascular endothelial cell growth factor-induced endothelial cell proliferation

Ligand binding to vascular endothelial cell growth factor (VEGF) receptors activates the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK). Possible cross-communication of ERK and JNK effecting endothelial cell (EC) actions of VEGF is poorly understood. Incubation of EC with PD 98059, a specific mitogen-activated protein kinase kinase inhibitor, or transfection with Y185F, a dominant negative ERK2, strongly inhibited VEGF-activated JNK. JNK was also activated by ERK2 expression in the absence of VEGF, inhibited 82% by co-transfection with dominant negative SEK-1, indicating upstream activation of JNK by ERK. VEGF-stimulated JNK activity was also reversed by dominant negative SEK-1. Other EC growth factors exhibited similar cross-activation of JNK through ERK. VEGF stimulated the nuclear incorporation of thymidine, reversed 89% by PD 98059 and 72% by Y185F. Dominant negative SEK-1 or JNK-1 also significantly reduced VEGF-stimulated thymidine incorporation. Expression of wild type Jip-1, which prevents JNK nuclear translocation, inhibited VEGF-induced EC proliferation by 75%. VEGF stimulated both cyclin D1 synthesis and Cdk4 kinase activity, inhibited by PD 98059 and dominant negative JNK-1. Important events for VEGF-induced G1/S progression and cell proliferation are enhanced through a novel ERK to JNK cross-activation and subsequent JNK action.     

Role of vascular endothelial cell growth factor in Ovarian Hyperstimulation Syndrome

Controlled ovarian hyperstimulation with gonadotropins is followed by Ovarian Hyperstimulation Syndrome (OHSS) in some women. An unidentified capillary permeability factor from the ovary has been implicated, and vascular endothelial cell growth/permeability factor (VEGF) is a candidate protein. Follicular fluids (FF) from 80 women who received hormonal induction for infertility were studied. FFs were grouped according to oocyte production, from group I (0-7 oocytes) through group IV (23-31 oocytes). Group IV was comprised of four women with the most severe symptoms of OHSS. Endothelial cell (EC) permeability induced by the individual FF was highly correlated to oocytes produced (r2 = 0.73, P < 0.001). Group IV FF stimulated a 63+/-4% greater permeability than FF from group I patients (P < 0. 01), reversed 98% by anti-VEGF antibody. Group IV fluids contained the VEGF165 isoform and significantly greater concentrations of VEGF as compared with group I (1,105+/-87 pg/ml vs. 353+/-28 pg/ml, P < 0. 05). Significant cytoskeletal rearrangement of F-actin into stress fibers and a destruction of ZO-1 tight junction protein alignment was caused by group IV FF, mediated in part by nitric oxide. These mechanisms, which lead to increased EC permeability, were reversed by the VEGF antibody. Our results indicate that VEGF is the FF factor responsible for increased vascular permeability, thereby contributing to the pathogenesis of OHSS.     

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.     

Association of vascular endothelial growth factor expression with intratumoral microvessel density and tumour cell proliferation in human epidermoid lung carcinoma

Vascular endothelial growth factor (VEGF) expression, vascularisation and tumour cell proliferation were analysed in 91 human epidermoid lung carcinomas using immunohistochemistry. A polyclonal anti-VEGF antibody was used for VEGF expression, a polyclonal antibody directed against human von Willebrand factor (factor VIII) to identify blood vessels and the proliferating cell nuclear antigen (PCNA) as a marker for proliferating cells. Positive staining for VEGF was obtained in 54 out of 91 cases (59%), the number of blood vessels varied from zero to 64 counts (mean 9.4) and the proportion of PCNA-positive cells varied from 1.3% to 72.1% (mean 25.2%). The mean PCNA labelling index and mean microvessel count in VEGF-positive tumours were significantly higher than those in VEGF-negative tumours (Wilcoxon rank sum test, P<0.0001; p<0.05). In addition, PCNA labelling index significantly increased with increasing VEGF expression (Jonckheere test, P<0.0001). In contrast, no association was found between PCNA labelling index and tumour vascularity (r=0.07, P=0.48). The close correlation of VEGF expression with tumour cell proliferation and microvessel density suggests that VEGF acts both as an autocrine growth factor and as stimulator for angiogenesis. However, tumour cell proliferation and microvessel growth and/or density may be regulated by separate mechanisms.     

Nerve growth factor effects on human and mouse melanoma cell invasion and heparanase production

The role of growth factor networks in regulating the progression of human melanocytes towards tumorigenicity and ultimately the malignant phenotype is poorly understood. In particular, the autocrine and paracrine influences that modulate cellular invasion and extracellular matrix degradative enzymes of melanoma cells remain undefined at the molecular level. We report here that nerve growth factor (NGF) can modify some metastasis-associated cellular properties of human and mouse melanoma cells. Treatment of early-passage human metastatic melanoma cells (MeWo) or their variants (3S5, 70W) with biologically active 2.5S NGF resulted in (a) delayed density-dependent inhibition of melanoma cell growth; (b) increased in vitro invasion through a reconstituted basement membrane; and (c) time- and dose-dependent induction of heparanase, a heparan-sulfate-specific endo-beta-D-glucuronidase associated with human melanoma metastasis. These effects of NGF were most marked in the 70W brain-colonizing cells (70W > MeWo > 3S5). The NGF enhancement of heparanase secretion was not species-specific, since it was also observed in murine B16 melanoma cells; the highest NGF stimulation of heparanase was found in brain-colonizing murine B16-B15b variant (B16-B15b > B16-BL6, B16-F10, B16-F1). NGF also increased the invasive capacity of the human 70W and murine B16-B15b sublines in a chemoinvasion assay performed with filters coated with purified heparan sulfate proteoglycan (HSPG). The enhancement of chemotactic response and heparanase production was detected at NGF concentrations sufficient to fully saturate both low- and high-affinity NGF receptors (NGFR), the neurotrophin receptor (p75) and the trkA gene product, respectively. The results suggest that, in addition to the effects of NGF on cellular development and differentiation within the peripheral and central nervous systems, NGF can exert changes in the invasive properties of neuroectoderm-derived melanoma cells.     

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.     

Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells

Vascular endothelial growth factor (VEGF) is a regulator of vasculogenesis and angiogenesis. To investigate the role of nitric oxide (NO) in VEGF-induced proliferation and in vitro angiogenesis, human umbilical vein endothelial cells (HUVEC) were used. VEGF stimulated the growth of HUVEC in an NO-dependent manner. In addition, VEGF promoted the NO-dependent formation of network-like structures in HUVEC cultured in three dimensional (3D) collagen gels. Exposure of cells to VEGF led to a concentration-dependent increase in cGMP levels, an indicator of NO production, that was inhibited by nitro-L-arginine methyl ester. VEGF-stimulated NO production required activation of tyrosine kinases and increases in intracellular calcium, since tyrosine kinase inhibitors and calcium chelators attenuated VEGF-induced NO release. Moreover, two chemically distinct phosphoinositide 3 kinase (PI-3K) inhibitors attenuated NO release after VEGF stimulation. In addition, HUVEC incubated with VEGF for 24 h showed an increase in the amount of endothelial NO synthase (eNOS) protein and the release of NO. In summary, both short- and long-term exposure of human EC to VEGF stimulates the release of biologically active NO. While long-term exposure increases eNOS protein levels, short-term stimulation with VEGF promotes NO release through mechanisms involving tyrosine and PI-3K kinases, suggesting that NO mediates aspects of VEGF signaling required for EC proliferation and organization in vitro.     

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.     

The effect of linomide on the migration and the proliferation of capillary endothelial cells elicited by vascular endothelial growth factor

In order to assess the mechanism of action of the quinoline-3-carboxyamide linomide as an antiangiogenic drug, the effect of linomide was studied in vitro on postcapillary endothelial cells exposed to vascular endothelial growth factor (VEGF). Linomide did not block the spontaneous replication of endothelial cells, but significantly suppressed endothelial cell growth and migration elicited by VEGF. It is concluded that linomide appears to be an effective tool to inhibit VEGF-dependent angiogenesis.     

Characterization of vascular permeability factor/vascular endothelial growth factor receptors on mononuclear phagocytes

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a polypeptide mediator, elaborated by certain tumors and other cell types, that exerts multiple effects on endothelium via interaction with a class of high-affinity binding sites. In this report, the interaction of VPF/VEGF with human mononuclear phagocytes (MPs) is characterized. Radioligand binding studies at 4 degrees C showed the presence of a single class of binding sites, kd approximately 300 to 500 pmol/L (approximately 20 times lower affinity than the high-affinity binding site on endothelial cells [ECs]), the occupancy of which correlated with VPF/VEGF-induced MP migration and expression of tissue factor. These binding results were paralleled by functional experiments which indicated that the same VPF/VEGF preparations were about an order of magnitude less effective in stimulating MP chemotaxis than in inducing EC proliferation. When MPs with surface-bound 125I-VPF/VEGF were warmed to 37 degrees C, endocytosis and degradation occurred. Occupancy of VPF/VEGF binding site resulted in subsequent activation of intracellular signal transduction mechanisms, as shown by an increase in MP intracellular calcium concentration. Cross-linking studies with 125I-VPF/VEGF showed a new high-molecular weight band (corresponding to putative 125I-VPF/VEGF-receptor complex), the appearance of which was blocked by excess unlabeled VPF/VEGF. Consistent with these results, immunoprecipitation of 32PO4-labeled MPs exposed to VPF/VEGF showed a single band of similar mobility, not seen in untreated controls. These results demonstrate that the interaction of VPF/VEGF with MPs, though of lower affinity than that observed with ECs, also results from interaction of the polypeptide with a specific cell-surface protein and leads to activation of intracellular transduction mechanisms.     

Expression and regulation of vascular endothelial growth factor in osteoblasts

Bone formation is linked closely to angiogenesis. Because prostaglandin E2 (PGE2) is a potent stimulator of bone formation, its effects were evaluated on vascular endothelial growth factor, a secreted endothelial cell-specific mitogen, and a potent angiogenic protein. Prostaglandin E2 increased vascular endothelial growth factor protein in conditioned media of osteoblastic RCT-3 cells within 3 hours. Prostaglandin E2 also increased the steady-state levels of vascular endothelial growth factor mRNA in a dose-dependent manner. The increased expression of vascular endothelial growth factor mRNA produced by PGE2 was rapid (maximal at 1 hour) and was enhanced by the protein synthesis inhibitor cycloheximide (5 micrograms/ml). The increase in vascular endothelial growth factor mRNA by PGE2 was inhibited strongly by pretreatment for 3 hours with dexamethasone (10(-7) M). Stimulation of vascular endothelial growth factor by PGE2 and its suppression by dexamethasone implicate the involvement of vascular endothelial growth factor in bone metabolism.     

Expression of two angiogenic factors, vascular endothelial growth factor and platelet-derived endothelial cell growth factor in human pancreatic cancer, and its relationship to angiogenesis

Twenty-three cases of ductal carcinoma in situ (DCIS), ten of which had an associated invasive component, were studied for loss of heterozygosity (LOH) of microsatellite markers on chromosome 9p and the results compared with a panel of 20 invasive breast carcinomas. In addition to the gene encoding p16, chromosome 9p is also thought to contain other putative tumour-suppressor genes. If the three panels of breast tumours showed LOH of markers in this region this would suggest that such putative genes were important in breast carcinogenesis. By studying both preinvasive and invasive breast tumours, it should also be possible to gain further information about the relationship between lesions of a different stage and to determine whether DCIS is indeed a precursor of invasive ductal carcinoma. Levels of LOH were low in the invasive-only set of tumours. Surprisingly, considerably higher levels of loss were observed in the tumours with an in situ component. Also, much heterogeneity was observed between different DCIS ducts or invasive tumour and DCIS from the same case.