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.     

Expression of vascular endothelial growth factor in human gallbladder lesions

Pregnancy-induced hypertension has been suggested to be mediated by several mechanisms, including reduced nitric oxide (NO) synthesis. In this study, the effects of chronic treatment with the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) on blood pressure and the underlying changes in vascular reactivity were investigated in virgin and late-pregnancy Sprague-Dawley rats. The systolic blood pressure was 120+/-2, 124+/-5, 116+/-4, and 171+/-5 mm Hg in untreated virgin, virgin treated with L-NAME, untreated pregnant, and pregnant treated with L-NAME rats, respectively. The rats were killed, and the thoracic aorta was cut into strips for measurement of active stress in response to alpha1-adrenergic stimulation with phenylephrine and membrane depolarization by high KCl. In pregnant rats, the maximal active stress to phenylephrine (0.31+/-0.03 x 10(4) N/m2) and the high-KCl-induced active stress (0.55+/-0.09 x 10(4) N/m2) were smaller than those in virgin rats. By contrast, in the L-NAME-treated pregnant rats, the maximal phenylephrine-induced active stress (0.76+/-0.1 x 10(4) N/m2) was greater than that in virgin rats (0.52+/-0.1 x 10(4) N/m2), whereas the high-KCl-induced active stress (1.08+/-0.14 x 10(4) N/m2) was indistinguishable from that in virgin rats (1.03+/-0.14 x 10(4) N/m2). Treatment with L-NAME did not affect the phenylephrine-releasable Ca2+ stores in pregnant rats and had minimal effect on active stress in virgin rats. Thus, reduction of NO synthesis during late pregnancy is associated with a significant increase in blood pressure and vascular responsiveness to alpha-adrenergic stimulation, which can possibly be explained in part by enhanced Ca2+ entry from extracellular space. However, other mechanisms such as increased myofilament force sensitivity to Ca2+ and/or activation of a completely Ca2+-independent mechanism cannot be excluded.     

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.     

Vascular endothelial growth factor, placenta growth factor and their receptors in isolated human trophoblast

The expression of the angiogenic growth factors, vascular endothelial cell growth factor (VEGF) and placenta growth factor (PIGF) was demonstrated in isolated human term cytotrophoblast and in vitro differentiated syncytiotrophoblast. RNase protection assays demonstrated VEGF expression in both cytotrophoblast and syncytiotrophoblast while prominent PIGF expression was detected in both types of trophoblast by Northern blot analyses. VEGF expression increased approximately eightfold in trophoblast cultured under hypoxic conditions (1 per cent O2) yet PIGF expression decreased 73 +/- 5.5 per cent in the same trophoblast. These results suggest distinct regulatory mechanisms govern expression of VEGF and PIGF in trophoblast. Characterization of the VEGF/PIGF receptors, KDR and flt-1, revealed the presence of flt-1 mRNA in isolated cytotrophoblast and in vitro differentiated syncytiotrophoblast. KDR was not detected in the isolated trophoblast. Exogenous rhVEGF induced c-Jun N-terminal kinase (JNK) activity in the normal trophoblast indicating that the flt-1 receptors on trophoblast are functional. Trophoblast-derived VEGF/PIGF could act in a paracrine fashion to promote uterine angiogenesis and vascular permeability within the placental bed. In addition, presence of function flt-1 on normal trophoblast suggests that VEGF/PIGF functions in an autocrine manner to perform an as yet undefined role in trophoblast invasion, differentiation, and/or metabolic activity during placentation.     

Expression of vascular endothelial growth factor mRNA in non-small-cell lung carcinomas

PURPOSE: To determine the distribution of matrix metalloproteinase-1 (MMP-1) in the uveoscleral outflow pathway and other anterior segment tissues of normal human eyes. METHODS: Normal human eyes were fixed in methacarn and sectioned and immunostained using a specific polyclonal antibody to MMP-1. Immunoreactivity was visualized using diaminobenzidine. To compare the staining intensity in various tissues, the mean optical density within the ciliary body, mid-iris stroma, iris root, uveal trabecular meshwork, cornea, and sclera was determined using imaging densitometry. To determine the cellular distribution of MMP-1 in ciliary muscle, additional sections were double-immunostained using antibodies to MMP-1 and calponin. These sections were examined by confocal laser scanning microscopy. Specificity of the antibody to MMP-1 in ocular tissues was confirmed by western blot analysis with uveal tract homogenates. RESULTS: Moderate-to-strong MMP-1 immunoreactivity was observed in ciliary muscle, iris, sclera, corneal endothelium, and ciliary nonpigmented epithelium. Lighter immunoreactivity was observed in corneal epithelium, blood vessels, trabecular meshwork, Schlemm's canal, and associated collector channels. Confocal microscopy showed that ciliary muscle MMP-1 was primarily inside ciliary muscle cells. Densitometry showed that net optical density was approximately fivefold greater in ciliary muscle, iris root, and sclera than in trabecular meshwork. CONCLUSIONS: MMP-1 was prominently identified in regions of the anterior segment of normal human eyes associated with the uveoscleral outflow pathway and in the iris, corneal endothelium, and ciliary nonpigmented epithelium. These data support the hypothesis that MMP-1 activity is involved in regulating uveoscleral outflow facility.     

Localization of vascular endothelial growth factor in human retina and choroid

OBJECTIVE: To examine the distribution and relative levels of vascular endothelial growth factor (VEGF) in the nondiabetic and preproliferative diabetic human retina and choroid. METHODS: Immunohistochemical localization was performed on frozen sections from cryopreserved postmortem human tissue using a polyclonal antibody against VEGF and a streptavidin peroxidase system. Eyes from 5 subjects without diabetes and 8 subjects with diabetes were examined and analyzed using a 7-point immunohistochemical grading system. RESULTS: In subjects without diabetes, weak or no VEGF immunoreactivity was associated with retinal blood vessels. In subjects with diabetes, we found significantly increased immunoreactivity in the retinal vascular endothelium and blood vessel walls. Vascular endothelial growth factor immunoreactivity was also associated with intravascular leukocytes in subjects with and without diabetes. In the choroid of subjects without diabetes, immunoreactivity was almost exclusively associated with intravascular leukocytes, whereas in diabetic subjects, immunoreactivity was localized within choriocapillaris endothelium, choroidal neovascular endothelium, and migrating retinal pigment epithelium cells. CONCLUSIONS: The observed increase in VEGF immunoreactivity in the diabetic retina and choroid suggests that VEGF may contribute to 2 well-documented events during retinopathy: increased vascular permeability and angiogenesis.     

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.     

Expression of vascular endothelial growth factor (VEGF) in various compartments of the human hair follicle

Hair follicle vascularization appears to be closely related to the processes involved in hair cycle regulation, in which growth factors, cytokines and other bioactive molecules are involved. In particular, vascular endothelial growth factor (VEGF), essential for angiogenesis and vascular permeability, may be responsible for maintaining proper vasculature around the hair follicle during the anagen growth phase. The aim of our study was to compare the in vitro angiogenic capacity, i.e. the steady-state expression of the VEGF gene, of different cultured cell types derived from normal human hair follicles, corresponding to different follicular compartments. Human dermal papilla cells (DPC), fibrous sheath fibroblasts, dermal fibroblasts, and follicular and interfollicular keratinocytes were cultured and studied in vitro for VEGF expression at the mRNA level using RT-PCR, and for VEGF protein synthesis by radioimmunoprecipitation and immunocytochemistry. In vivo examination for VEGF expression in human terminal hair follicles was performed using immunohistochemical methods. In the present report the expression of four different VEGF molecular isoforms, differing in their angiogenic capacity, are described in different cultured follicular cell types for the first time. Cultured follicular cells strongly expressed mRNA of four VEGF molecular species identified as the 121-, 145-, 165- and 189-amino acid splice variants, the most prominent being the 121-amino acid molecule. DPC, and also other mesenchymal cells such as fibrous sheath fibroblasts and dermal fibroblasts, in vivo and in vitro strongly expressed VEGF mRNA and synthesized a 46-kDa VEGF protein, whereas follicular and interfollicular keratinocytes in vitro expressed lower levels of VEGF mRNA and proteins than mesenchymal cells. As the highest expression of VEGF was found in DPC, we suggest that DPC are mainly responsible for angiogenic processes possibly related to the human hair cycle.     

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.     

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.     

Expression of the angiogenic factors, basic fibroblast growth factor and vascular endothelial growth factor, in the ovary

In adult tissues, vascular growth (angiogenesis) occurs normally during tissue repair, such as in the healing of wounds and fractures. Inappropriate vascular growth is associated with various pathological conditions. These conditions include tumor growth, retinopathies, hemangiomas, fibroses, and rheumatoid arthritis in the case of rampant vascular growth and nonhealing wounds and fractures in the case of inadequate vascular growth. The female reproductive organs exhibit dramatic, periodic growth and regression, accompanied by equally dramatic changes in their rates of blood flow. Thus, it is not surprising that they are some of the few adult tissues in which angiogenesis occurs as a normal process. Ovarian follicles and corpora lutea contain and produce angiogenic factors. These angiogenic factors bind heparin and seem to belong to the fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) families of proteins. Based on our studies of the pattern of expression of FGF and its major receptors in bovine, ovine, and porcine corpora lutea, we have suggested that FGF may influence not only luteal cell proliferation but also cell death, thereby regulating cell turnover in the luteal vascular and nonvascular compartments. In addition, we recently have shown that luteal expression of VEGF is greatest during the early luteal phase, coincident with luteal vascularization. Moreover, VEGF is present exclusively in luteal connective tissue and perivascular (arteriolar smooth muscle and capillary pericyte) cells. In fact, the first thecal-derived cells to invade the granulosa-derived regions immediately after ovulation seem to be VEGF-containing pericytes. We have therefore hypothesized that ovarian pericytes play a key role in vascularization of developing follicles and corpora lutea. Further understanding of the specific physiological roles of these factors in follicular and luteal growth, development, and function will ultimately lead to improved methods of regulating fertility.     

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.     

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.     

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.     

Levels of vascular endothelial growth factor, hepatocyte growth factor/scatter factor and basic fibroblast growth factor in human gliomas and their relation to angiogenesis

Angiogenesis is a possible target in the treatment of human gliomas. To evaluate the role of 3 growth factors, vascular endothelial growth factor (VEGF), hepatocyte growth factor/scatter factor (HGF/SF) and basic fibroblast growth factor (bFGF), in the angiogenic cascade, we determined their levels in extracts of 71 gliomas by enzyme-linked immunosorbent assay (ELISA). The levels of bFGF were only marginally different between gliomas of World Health Organization (WHO) grade II (low grade) and grades III and IV (high grade). In contrast, the mean concentrations of VEGF were 11-fold higher in high-grade tumors and those of HGF/SF 7-fold, respectively. Both were highly significantly correlated with microvessel density (p < 0.001) as determined by immunostaining for factor VIII-related antigen. In addition, VEGF and HGF/SF appeared to be independent predictive parameters for glioma microvessel density as determined by multiple regression analysis. We measured the capacity of all 3 factors to induce endothelial tube formation in a collagen gel. In this assay, bFGF was found to be an essential cofactor with which VEGF as well as HGF/SF were able to synergize independently. According to the concentrations of angiogenic factors, extracts from high-grade tumors were significantly more potent in the tube formation assay than the low-grade extracts (p = 0.02). Adding neutralizing antibodies to bFGF, VEGF and HGF/SF together with the extracts, tube formation was inhibited by up to 98%, 62% and 54%, respectively. Our findings suggest that bFGF is an essential cofactor for angiogenesis in gliomas, but in itself is insufficient as it is present already in the sparsely vascularized low-grade tumors. Upon induction of angiogenesis in high-grade tumors, bFGF may synergize with rising levels of not only VEGF but possibly also with HGF/SF, which appears here to be an independent angiogenic factor.