Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody

The hyperpermeability of tumor vessels to macromolecules, compared with normal vessels, is presumably due to vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) released by neoplastic and/or host cells. In addition, VEGF/VPF is a potent angiogenic factor. Removal of this growth factor may reduce the permeability and inhibit tumor angiogenesis. To test these hypotheses, we transplanted a human glioblastoma (U87), a human colon adenocarcinoma (LS174T), and a human melanoma (P-MEL) into two locations in immunodeficient mice: the cranial window and the dorsal skinfold chamber. The mice bearing vascularized tumors were treated with a bolus (0.2 ml) of either a neutralizing antibody (A4.6.1) (492 micrograms/ml) against VEGF/VPF or PBS (control). We found that tumor vascular permeability to albumin in antibody-treated groups was lower than in the matched controls and that the effect of the antibody was time-dependent and influenced by the mode of injection. Tumor vascular permeability did not respond to i.p. injection of the antibody until 4 days posttreatment. However, the permeability was reduced within 6 h after i.v. injection of the same amount of antibody. In addition to the reduction in vascular permeability, the tumor vessels became smaller in diameter and less tortuous after antibody injections and eventually disappeared from the surface after four consecutive treatments in U87 tumors. These results demonstrate that tumor vascular permeability can be reduced by neutralization of endogenous VEGF/ VPF and suggest that angiogenesis and the maintenance of integrity of tumor vessels require the presence of VEGF/VPF in the tissue microenvironment. The latter finding reveals a new mechanism of tumor vessel regression-i.e., blocking the interactions between VEFG/VPF and endothelial cells or inhibiting VEGF/VPF synthesis in solid tumors causes dramatic reduction in vessel diameter, which may block the passage of blood elements and thus lead to vascular regression.     

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.     

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.     

In vivo (31)P magnetic resonance spectroscopy and morphometric analysis of the perfused vascular architecture of human glioma xenografts in nude mice

The relationship between the bioenergetic status of human glioma xenografts in nude mice and morphometric parameters of the perfused vascular architecture was studied using (31)P magnetic resonance spectroscopy (MRS), fluorescence microscopy and two-dimensional digital image analysis. Two tumour lines with a different vascular architecture were used for this study. Intervascular distances and non-perfused area fractions varied greatly between tumours of the same line and tumours of different lines. The inorganic phosphate-nucleoside triphosphate (P(i)/NTP) ratio increased rapidly as mean intervascular distances increased from 100 microm to 300 microm. Two morphometric parameters - the percentage of intervascular distances larger than 200 microm (ivd200) and the non-perfused area fraction at a distance larger than 100 microm from a nearest perfused vessel (area100), - were deduced from these experiments and related to the P(i)/NTP ratio of the whole tumour. It is assumed that an aerobic to anaerobic transition influences the bioenergetic status, i.e. the P(i)/NTP ratio increased linearly with the percentage of ivd200 and the area100.     

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 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.     

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.     

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.     

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.     

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.     

Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors

Vascular endothelial growth factor (VEGF) plays a key role in physiological blood vessel formation and pathological angiogenesis such as tumor growth and ischemic diseases. Hypoxia is a potent inducer of VEGF in vitro. Here we demonstrate that VEGF is induced in vivo by exposing mice to systemic hypoxia. VEGF induction was highest in brain, but also occurred in kidney, testis, lung, heart, and liver. In situ hybridization analysis revealed that a distinct subset of cells within a given organ, such as glial cells and neurons in brain, tubular cells in kidney, and Sertoli cells in testis, responded to the hypoxic stimulus with an increase in VEGF expression. Surprisingly, however, other cells at sites of constitutive VEGF expression in normal adult tissues, such as epithelial cells in the choroid plexus and kidney glomeruli, decreased VEGF expression in response to the hypoxic stimulus. Furthermore, in addition to VEGF itself, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-2, was induced by hypoxia in endothelial cells of lung, heart, brain, kidney, and liver. VEGF itself was never found to be up-regulated in endothelial cells under hypoxic conditions, consistent with its paracrine action during normoxia. Our results show that the response to hypoxia in vivo is differentially regulated at the level of specific cell types or layers in certain organs. In these tissues, up- or down-regulation of VEGF and VEGFR-1 during hypoxia may influence their oxygenation after angiogenesis or modulate vascular permeability.     

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.     

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.     

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。