Fibrin induction of tissue factor expression in human vascular endothelial cells

BACKGROUND: For the present study, we hypothesized that fibrin is an inducer of tissue factor (TF) expression in vascular endothelial cells in vitro and in vivo. METHODS AND RESULTS: To test the in vitro aspect of this hypothesis, human umbilical vein endothelial cells (HUVECs) were cocultured with physiologically relevant concentrations of fibrin (0.03 to 1.0 mg fibrin/mL) for various times (0.5 to 24 hours), and TF expression was compared with that in unstimulated HUVECs (media control). Results demonstrated that fibrin induced a time- and dose-dependent increase in TF antigen expression, functional TF procoagulant activity, and TF mRNA in HUVECs. CONCLUSIONS: These studies demonstrate that fibrin can directly regulate TF expression in HUVECs 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.     

Promoters of epithelialization induce expression of vascular endothelial growth factor in human gastric epithelial cells in primary culture

Three routes of heroin use were identified--injecting, 'chasing the dragon' and snorting. Whilst injecting and 'chasing the dragon' accounted for virtually all current heroin use, snorting had been the first route of use for nearly a fifth of heroin users in both the treatment and community samples. Overlap of lifetime experience of the different routes was widespread, with the majority of heroin users in both the treatment and community samples having used heroin by more than one route. Although less than half of the treatment sample had used heroin by injection on the first occasion, more than 90% had injected at least once, and over 80% had at some time used by 'chasing the dragon'. Only a quarter of the community sample had first used heroin by injecting, and yet, by the time of interview, two-thirds of the sample had injected. The majority of durable changes in route of heroin use were towards injecting and 'chasing the dragon', with transitions to snorting being extremely rare. For both the samples, transitions to injecting were twice as frequent as transitions to 'chasing'. Snorting appeared to be an unstable route of use, with almost all who initially snorted their heroin now using by injecting or 'chasing the dragon'.     

Expression of vascular endothelial growth factor in human gastric carcinomas

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a secreted protein which may play a pivotal role in tumor-associated microvascular angiogenesis and hyperpermeability. The expression of mRNA for VEGF was examined in eight gastric carcinoma cell lines and 30 gastric carcinoma tissues as well as corresponding normal mucosa. All the cell lines expressed VEGF mRNA at various levels that correlated well with the amounts of VEGF secreted into the condition medium. The expression of VEGF mRNA by TMK-1 cells was increased by the treatment of epidermal growth factor (EGF) or interleukin-1alpha (IL-1alpha), whereas it was decreased by the treatment of interferon-beta (IFN-beta). In gastric carcinoma tissues, the level of VEGF mRNA in primary tumors was higher than that in the corresponding normal mucosas in six (46%) of 13 well-differentiated adenocarcinomas and in two (12%) of 17 poorly differentiated adenocarcinomas, respectively. Vessel counts in well-differentiated adenocarcinomas had a tendency to be higher than those in poorly differentiated adenocarcinomas. In well-differentiated adenocarcinomas, the levels of VEGF mRNA expression tended to be higher in carcinomas of advanced stage than in early stage carcinomas. Both in situ mRNA hybridization and immunohistochemistry demonstrated the presence of VEGF expression within the tumor cells. These results suggest that VEGF may confer angiogenesis and progression of human gastric carcinomas, especially of the well-differentiated type.     

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.     

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.     

Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells

Inadequate presentation of tumor antigens by host professional antigen-presenting cells (APCs), including dendritic cells (DCs), is one potential mechanism for the escape of tumors from the host immune system. Here, we show that human cancer cell lines release a soluble factor or factors that dramatically affect DC maturation from precursors without affecting the function of relatively mature DCs. One factor responsible for these effects was identified as vascular endothelial growth factor (VEGF). Thus, VEGF may play a broader role in the pathogenesis of cancer than was previously thought, and therapeutic blockade of VEGF action may improve prospects for immunotherapy as well as inhibit tumor neovasculature.     

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。     

Serum and tissue vascular endothelial growth factor levels in hydatidiform mole

Using a specific and sensitive enzyme immunoassay for vascular endothelial growth factor (VEGF), we measured the circulating levels of VEGF in patients with hydatidiform mole as well as in maternal serum during normal pregnancy. VEGF levels in maternal serum were elevated at 7 weeks and then fell to a plateau. Serum VEGF levels were increased in patients with hydatidiform mole above the normal pregnant levels, while no differences were seen related to the development of persistent trophoblastic disease. By semi-quantitative RT-PCR in molar tissue for VEGF and placenta growth factor, a member of VEGF family, neither of the mRNA levels have no relation to the development of persistent trophoblastic disease. These observations suggest serum VEGF levels will be of value as a new circulating marker of hydatidiform mole.     

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.     

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.     

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.     

Dual pathways of tubular morphogenesis of vascular endothelial cells by human glioma cells: vascular endothelial growth factor/basic fibroblast growth factor and interleukin-8

In this study, we examined whether human glioma cells are angiogenic in a model using human microvascular endothelial cells, and also which factor is responsible for the glioma-dependent angiogenesis. Tubular morphogenesis in type I collagen gel by human microvascular endothelial cells was stimulated in the presence of 10 and 100 ng/ml of vascular endothelial growth factor (VEGF), 10 ng/ml basic fibroblast growth factor (bFGF) and 10 ng/ml of interleukin-8 (IL-8). Tube formation of the microvascular endothelial cells was assayed in the glioma cell lines IN157 and IN301, co-cultured using the double chamber method. IN301 cells had much higher levels of VEGF, bFGF and transforming growth factor-beta mRNA than IN157 cells, whereas the two had similar levels of transforming growth factor-alpha mRNA. By contrast, IN157 cells had much higher levels of IL-8 mRNA than IN301 cells. IN301-dependent tubular morphogenesis was inhibited by anti-VEGF or anti-bFGF antibody, and the inhibition was almost complete when anti-VEGF and anti-bFGF antibodies were present. On the other hand, IN157-dependent tubular morphogenesis was inhibited by anti-IL-8 antibody, but not by anti-VEGF or anti-bFGF antibodies. These findings demonstrated dual paracrine controls of tumor angiogenesis by human glioma cells. One is mediated through VEGF and/or bFGF, and the other, through IL-8.