Vascular Endothelial Growth Factor In Supernatant Research Articles

VEGF affects mitochondrial ROS generation in glioma cells and acts as a radioresistance factor.

Vascular endothelial growth factor (VEGF) is closely related to angiogenesis. Anticancer therapy by inhibiting VEGF signaling is well established. However, the role of VEGF in cell-cell communication during the response to ionizing radiation is not well understood. Here, we examined the role of VEGF on radiosensitivity of cells. The addition of recombinant VEGF (rVEGF) on cultured rat C6 glioma cells showed a radioprotective effects on X-ray irradiation and reduced oxidative stress. These effects were also observed by endogenous VEGF in supernatant of C6 glioma cells. Reduction of oxidative stress by VEGF is suggested to underlie the radioprotective effects. The mechanism of VEGF-induced reduction of oxidative stress was indicated by a decreased oxygen consumption rate (OCR) in mitochondria. However, the number of DNA double-strand breaks (DSB) immediately after irradiation was not reduced by the treatment with VEGF. These results suggest that VEGF plays a role in cell survival after irradiation by controlling the oxidative condition through mitochondrial function that is independent of the efficiency of DSB induction.

VEGF/Flk1 Mechanism is Involved in Roxarsone Promotion of Rat Endothelial Cell Growth and B16F10 Xenograft Tumor Angiogenesis

The potential angiogenic effect of roxarsone, a feed additive widely used to promote animal growth worldwide, was demonstrated recently. We explored the mechanism of vascular endothelial growth factor (VEGF) and its receptor (VEGFR) in roxarsone promotion of rat vascular endothelial cells (ECs) and B16F10 mouse xenografts. ECs were treated with 0.1–50 μM roxarsone or with roxarsone plus 10 ng/mL VEGF, VEGFR1 (Flt1), or VEGFR2 (Flk1) antibodies for 12–48 h to examine their role in cell growth promotion. Small interfering RNA (siRNA) targeting Vegf, Flt1, and Flk1 were transfected in the ECs, and we measured the expression level, cell proliferation, migration, and tube formation ability. The siRNA targeting Vegf or Flk1 were injected intratumorally in the B16F10 xenografts of mice that received 25 mg/kg roxarsone orally. Cell viability and VEGF expression following roxarsone treatment were significantly higher than that of the control (P < 0.05), peaking following treatment with 1.0 μM roxarsone. Compared to roxarsone alone, the VEGF antibody decreased cell promotion by roxarsone (P < 0.05), and the Flk1 antibody greatly reduced cell viability compared to the Flt1 antibody (P < 0.01). Roxarsone and Flk1 antibody co-treatment increased supernatant VEGF significantly, while cellular VEGF was obviously decreased (P < 0.01), whereas there was no significant difference following Flt1 antibody blockade. The siRNA against Vegf or Flk1 significantly attenuated the roxarsone promotion effects on EC proliferation, migration, and tube-like formation (P < 0.01), whereas the siRNA against Flt1 effected no obvious differences. Furthermore, the RNA interference significantly weakened the roxarsone-induced increase in xenograft weight and volume, and VEGF and Flk1 expression. Roxarsone promotion of rat EC growth, migration, and tube-like formation in vitro and of B16F10 mouse xenograft model tumor growth and angiogenesis involves a VEGF/Flk1 mechanism.

Overexpression of miR‑21 is involved in acute monocytic leukemia‑associated angiogenesis by targeting IL‑12.

Angiogenesis is important in pathophysiological processes, including the pathogenesis of acute monocytic leukemia (AML). MicroRNA‑21 (miR‑21) is overexpressed and exhibits oncogenic activity in cancer. However, the biological mechanism underlying the effect of miR‑21 in AML remains to be fully elucidated. In the present study, the expression levels of miR‑21 and vascular endothelial growth factor (VEGF) were determined in 26patients with AML and 28healthy individuals. The secretion of VEGF was also measured following the transfection of THP‑1 cells with miR‑21 mimic or inhibitor. The supernatants of the THP‑1 cells, which were transfected with miR‑21 mimic, inhibitor or small interfering RNA (si)VEGF, respectively, were used to incubate human umbilical vein endothelial cells (HUVECs), following which tube formation of the HUVECs was measured. miR‑21 targets were predicted using a biological target prediction website and confirmed using a luciferase assay. The effects of interleukin (IL)‑12 were investigated by examining the tube formation of HUVECs and the secretion of VEGF following recombinant human (rh) IL‑12 pretreatment. The results revealed that miR‑21 and VEGF expression was significantly increased in the peripheral blood monocytes of the patients, compared with the healthy controls. There was negative correlation between the expression of IL‑12 and miR‑21 in the serum of patients with AML. Furthermore, supernatant VEGF levels from the miR‑21 mimic‑transfected THP‑1 cells were increased, whereas a decreasing trend was observed in the miR‑21 inhibitor group. The angiogenic ability of the HUVECs pretreated with supernatant from the THP‑1 cells transfected with miR‑21 mimic was higher, and was lower in THP‑1 cells co‑transfected with miR‑21 mimic and siVEGF, compared with the miR‑21 mimic only group. A luciferase assay demonstrated that IL‑12 was the direct target of miR‑21, and the level of IL‑12 in the supernatant of THP‑1 cells transfected with miR‑21 mimic was increased. IL‑12 pretreatment increased VEGF expression and angiogenic ability in HUVECs. The inactivation of miR‑21 or activation of its target gene may be a potential therapeutic strategy in human AML.

Valproic Acid Treatment Inhibits Vasopermeability and Improves Survival in Rats With Lethal Scald Injury.

The aim of this study was to examine whether administration of valproic acid (VPA), a histone deacetylase inhibitor, inhibits proinflammatory mediators and ameliorate visceral vasopermeability both in a rat model of major burn, and also in rat cultured endothelial cells stimulated with permeability evoking mediators. SD rats were subjected to a 50% TBSA full-thickness scald injury, and treated with either saline or VPA (300 mg/kg) intraperitoneally. Pulmonary vascular endothelial growth factor (VEGF), myeloperoxidase (MPO), pulmonary microvascular permeability, water content, and acetylation of histone H3K9 of lungs were evaluated. In addition, pulmonary microvascular endothelial cells (PMECs) from male SD rats were cultured. With then, MPO, VEGF, histone acetylation, and the permeability of PMECs were investigated. Lethal scald injury resulted in a significant increase in microvascular permeability and water content of lung, accompanied by a significant elevation of the content of VEGF and activity of MPO, and a decrease of histone acetylation. VPA treatment significantly alleviated the microvascular permeability and water content of lung, lowered the levels of VEGF and MPO, and promoted acetylation of histone H3K9 following scald injury. Moreover, VPA reduced permeability of monolayer PMECs subjected to scald serum challenge, reduced the level of MPO and VEGF in supernatants, and promoted acetylation of histone H3K9 in PMECs. These results indicated that VPA can protect pulmonary microvascular endothelial barrier, alleviate proinflammatory mediators-evoked vascular hyperpermeability and tissue edema and improve the survival rate of rats subjected to lethal scald injury.

MP71-16 INTERMITENT HYPOXIA INCREASES TUMOR ANGIOGENESIS IN A MOUSE MODEL OF KIDNEY CANCER

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I1 Apr 2016MP71-16 INTERMITENT HYPOXIA INCREASES TUMOR ANGIOGENESIS IN A MOUSE MODEL OF KIDNEY CANCER Antoni Vilaseca, Mireia Musquera, Marta Torres, Noelia Campillo, Josep M Montserrat, Karim Touijer, Ramon Farre, Isaac Almendros, and Antonio Alcaraz Antoni VilasecaAntoni Vilaseca More articles by this author , Mireia MusqueraMireia Musquera More articles by this author , Marta TorresMarta Torres More articles by this author , Noelia CampilloNoelia Campillo More articles by this author , Josep M MontserratJosep M Montserrat More articles by this author , Karim TouijerKarim Touijer More articles by this author , Ramon FarreRamon Farre More articles by this author , Isaac AlmendrosIsaac Almendros More articles by this author , and Antonio AlcarazAntonio Alcaraz More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1462AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Intermittent hypoxia (IH), a characteristic feature of obstructive sleep apnea (OSA), has been recently associated with an increased cancer aggressiveness and mortality. Renal cell carcinoma is commonly linked to an increased expression of hypoxia-inducible genes promoting neovascularization, and can result in poorest prognosis. We aim to assess the role of IH on tumor angiogenesis as a marker of malignancy in a mouse model of renal cell carcinoma and to study in vitro the role of isolated tumor cells on promoting angiogenesis under IH. METHODS A subcutaneous kidney cancer was induced in 24 Balb/c mice by injecting 105 RENCA cells in the left flank. Twelve mice were subjected to IH (cycles of 20s at 5% O2 followed by 40s at 21% O2, 6h/day) from 15 days before injection until 21 days after injection, when they were sacrificed. A control group (n=12) was kept under normoxia for the same period of time. After sacrifice, tumors were excised and processed to evaluate the presence of endothelial progenitor cells (CD309+Gr1+) and endothelial cells (CD31+) by flow cytometry. Circulating vascular endothelial growth factor (VEGF) was quantified by ELISA. We also tested in vitro the hypothesis that isolated RENCA cells subjected to IH increase VEGF production. We seeded 5x104 RENCA cells/well in two 6-well plates. After 12 hours we kept for 24 hours each 6-well plate under normoxia and IH, respectively. Supernatant VEGF quantification was done by ELISA. RESULTS Percentage of vascular progenitor cells in tumoral tissue was increased under IH compared to normoxia (6,1 ± 0,76 vs 4,5±1,1; p=0,001). Similarly, the percentage of endothelial cells was also promoted by IH (4±0,8 vs 2,5±1; p=0,013). Plasma VEGF was significantly higher among the IH group (306±93 vs 204±45 pg/mL; p=0,001). Tumor growth was not affected by IH (0,7±0,6 vs 0,8±0,42 gr; p=0,08). In the in vitro experiments, VEGF in the supernatant after 24h of culture did not differ between IH and normoxia (719±63 vs 729±192 pg/mL; p=0,912). CONCLUSIONS In the animal model, IH increases the production of circulating VEGF and the mobilization of vascular progenitor cells, resulting in an increased vascularization of the tumor. In vitro, isolated tumor cells do not increase VEGF production under IH compared to normoxia. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e921-e922 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Antoni Vilaseca More articles by this author Mireia Musquera More articles by this author Marta Torres More articles by this author Noelia Campillo More articles by this author Josep M Montserrat More articles by this author Karim Touijer More articles by this author Ramon Farre More articles by this author Isaac Almendros More articles by this author Antonio Alcaraz More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effects of Scaffold Material Used in Cardiovascular Surgery on Mesenchymal Stem Cells and Cardiac Progenitor Cells

Polytetrafluoroethylene (PTFE) and porcine small intestinal submucosa (pSIS) are patch materials used in congenital heart surgery. Porcine SIS is an extracellular-matrix scaffold that may interact with stem or progenitor cells. To evaluate this, we determined the in vitro effects of pSIS and PTFE on human bone marrow mesenchymal stromal cells (MSCs) and cardiac progenitor cells (CPCs) in 3 areas; cell proliferation, angiogenic growth-factor production, and differentiation. Human MSCs and CPCs were seeded onto pSIS and PTFE patches. Cell-seeded patches were cultured and then assessed for cell viability and proliferation and supernatant vascular endothelial growth factor A (VEGFA) levels. Cell proliferation was quantified by MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). Quantitative real-time polymerase chain reaction was performed on cell-seeded scaffolds to determine relative changes in gene expression related to angiogenesis and cardiogenesis. The MSCs and CPCs were able to attach and proliferate on pSIS and PTFE. The proliferation rate of each cell type was similar on pSIS. Total RNA isolation was only possible from the cell-seeded pSIS patches. The MSC VEGFA production was increased by pSIS. Porcine SIS promoted an angiogenic gene profile in MSCs and an early cardiogenic profile in CPCs. Both PTFE and pSIS allow for varying degrees of cell proliferation. Porcine SIS elicits different phenotypical responses in MSCs as compared with CPCs, which indicates that pSIS may be a bioactive scaffold that modulates stem cell activation and proliferation. These findings highlight the differences in scaffold material strategies and suggest potential advantages of bioactive approaches.

In vitro and ex vivo angiogenic effects of roxarsone on rat endothelial cells

Roxarsone, a feed additive, is being used worldwide to promote animal growth. However, the potential effect of roxarsone on angiogenesis has not been extensively characterized. We examined the ability of roxarsone to promote angiogenesis of rat endothelial cells in vitro and from rat aorta rings ex vivo. Endothelial cells from rats were exposed to 0.01–10.00μM roxarsone, 5ng/mL vascular endothelial growth factor (VEGF) as a positive control or phosphate buffer saline (PBS) as a negative control. Cell proliferation was measured by MTT assay, and the content of VEGF in supernatants was measured by enzyme-linked immunosorbent assay and Western blotting. A Matrigel-induced tube formation assay was used to evaluate the effects of roxarsone on endothelial cells. Additionally, the total number and length of microvessels sprouted from rat aortic rings were measured for ex vivo investigation of angiogenesis. Results showed that the cell viability and total number and length of capillary-like tube formations after roxarsone treatment was significantly higher than that of negative (P<0.05), with a maximum effect at 1.00μM exposure. Furthermore, the number of microvessels sprouted from aortic rings treated for 4h with 0.1–10.0μM roxarsone was significantly higher than that of PBS treatment, with a peak value of 1.0μM. These results further demonstrate the potential of roxarsone to promote angiogenesis in vitro and ex vivo.

Effects of Radix Ginseng and Radix Notoginseng formula on secretion of vascular endothelial growth factor and expression of vascular endothelial growth factor receptor-2 in human umbilical vein endothelial cells

To investigate the effects of Radix Ginseng and Radix Notoginseng formula on secretion of vascular endothelial growth factor (VEGF) and expression of vascular endothelial growth factor receptor-2 (VEGFR-2) in human umbilical vein endothelial cells (HUVECs) in vitro. HUVECs were cultured in vitro. Bovine basic fibroblast growth factor (bFGF) at concentration of 320 U/mL and Radix Ginseng and Radix Notoginseng formula at concentrations of 0.1, 0.2 and 0.4 mg/mL were used to culture with HUVECs. And HUVECs in blank control group were cultured with culture solution only. After 24-hour culture, the content of VEGF in supernatant was detected by enzyme-linked immunosorbent assay and the expression of VEGFR-2 was detected by immunocytochemical staining and Western-blotting. Radix Ginseng and Radix Notoginseng formula at 0.4 mg/mL, the same as bFGF, increased VEGF content in the HUVEC supernatant and the number of VEGFR-2-positive HUVECs. Expression of VEGFR-2 protein in high-dose Radix Ginseng and Radix Notoginseng formula group was up-regulated as compared with the blank control group. Radix Ginseng and Radix Notoginseng formula can promote HUVEC proliferation and secretion of VEGF, as well as the expression of VEGFR-2 protein, which may be one of the mechanisms of Radix Ginseng and Radix Notoginseng formula in promoting angiogenesis.

High concentrations of vascular endothelial growth factor reduce stretch‐induced apoptosis of alveolar type II cells

Vascular endothelial growth factor (VEGF) has protective as well as injurious effects in ARDS/acute lung injury. The influence of VEGF was investigated in a model of stretch-induced apoptosis. High-amplitude mechanical stretch induced the secretion of VEGF. High VEGF concentrations may prevent stretch-induced apoptosis by restoring stretch-impaired phospatidylinositol-3 kinase signalling. Vascular endothelial growth factor (VEGF) is strongly expressed in the alveolar epithelium. VEGF has been shown to exhibit protective as well as injurious effects in ARDS/acute lung injury. We therefore investigated the influence of VEGF in a model of stretch-induced apoptosis. Isolated rat alveolar type II (ATII) cells were subjected to high-amplitude cyclic mechanical stretch (40 per minute, 30% change in surface area) for 24 h. VEGF gene expression was investigated by real-time reverse transcription-PCR. Concentrations of VEGF in culture supernatants of stretched cells were determined by ELISA. Apoptosis of cells following stretching was assessed by flow cytometry. Vascular endothelial growth factor gene expression increased during the first 4 h of stretching and then declined to a similar level to that of static control cells. VEGF concentrations in cell supernatants increased in response to mechanical stretch, as compared with those in supernatants of static control cells. Incubation of ATII cells with higher concentrations of VEGF (50 ng/mL) during stretching inhibited apoptosis, presumably by restoring stretch-impaired phosphatidylinositol-3 kinase signalling. However, blocking free VEGF in the supernatant with an anti-VEGF antibody did not influence stretch-induced apoptosis. These findings suggest that high-amplitude mechanical stretch induced secretion of VEGF, which in high concentrations, may prevent stretch-induced apoptosis. In this model, however, the protective influence of VEGF was not essential for survival of ATII cells subjected to high-amplitude mechanical stretch.

Effection of ultraviolet on pterygial cell.

Objective To detect the relationship between ultraviolet and pathogenesis of pterygium through study at effect of irradiated on pterygial cell models. Methods Pterygial samples were extracted and collected and the pterygial endothelial cells （PE） and pterygial fibroblasts （PF） were cultured alone, condi- tional and co-cultured to form different culture systems. The methods included that to select the suitable intensity of uhraviolet（UV） by MTT, to detect the changes of curves of growth about two kinds of cells by MTT and to explore the developments of protein and RNA of vascular endothelial growth factor （VEGF） and fibroblast growth factor-basic（bFGF） in three culture systems under ultraviolet whose intensity is 20 mJ/cm2 by ELISA and RT-PCR. Results 1. The suitable intensity of ultraviolet selected was 20 mJ/cm2. 2. We found that there were many changes of proliferation of PE and PF, included that the activities of cells are declined, speeds of growth are decreased and growth cycles are blocked. 3. We found that the protein of VEGF in supernatant and RNA of VEGF in PEC cultured alone system after irradiated were much lower than un-irradiated. The protein of VEGF in supernatant and RNA of VEGF in PF cultured alone and conditional system after irradiated were much lower than un-irradiated. 4. The protein of bFGF in supernatant and RNA of bFGF in PE and PF cultured alone, conditional and co-cultured systems after irradiated were much lower than un-irradiated. Conclusion The ultraviolet has inhibited the growth of pterygialPE and PF cultured in vitro. Key words: pterygium; endothelial cell; interaction; uhravi olet; VEGF; bFGF

Chronic Lymphocytic Leukemia (CLL) Cells Require Microenvironmental Stimuli to Resist Apoptosis through Activation of STAT3 Mediated by VEGF.

B-cell chronic lymphocytic leukemia (CLL) is characterized by an accumulation of mature, but incompetent B-cells due to a decrease of apoptosis rather than an increase in proliferation. Vascular endothelial growth factor (VEGF) has been suggested to play an important role in this so called apoptotic block. However, so far little is understood whether VEGF is acting mainly as a microenvironmental stimulus and/or whether CLL cells themselves contribute to the enhanced apoptotic resistance by maintaining an autocrine VEGF loop. Moreover, it is unknown by which mechanisms VEGF prevents apoptosis and whether this can be circumvented by inhibition of VEGF signaling. By quantitative real time PCR we found no significant difference in mRNA VEGF levels in B-cells from CLL patients and healthy donors after isolation from blood. In contrast, ELISA revealed clearly increased levels of secreted VEGF in plasma of CLL patients and in the supernatant under culture conditions compared to healthy individuals. In addition, we found the VEGF receptor 2 (VEGFR2), which is existent in CLL and healthy B-cells, in a phosphorylated, hence activated state, to a significantly higher extent in CLL cells as assessed by intracellular phospho flow cytometry. In conclusion, despite its expression in healthy B-cells VEGF does not seem to be secreted and therefore, no VEGF receptor phosphorylation takes place. Whereas CLL cells exhibit a long life span in vivo, they die rapidly in vitro, suggesting major survival factors being existent in the CLL cells microenvironment. We found levels of secreted VEGF in supernatant decreasing with time in culture, going along with decreasing levels of phosphorylated VEGFR2 and increasing cell death as assessed by Annexin V-FITC/PI staining. This further supports the role of VEGF in CLL cell survival. Coculturing primary CLL cells with the bone marrow stromal derived cell line HS5 dramatically increased VEGF transcription and secretion and improved cell survival. Hence, VEGF expression in CLL cells is not only mediated by autocrine, but also paracrine stimuli involving bone marrow stromal. Knocking down VEGF in HS5 cells and subsequent coculture with CLL cells might prove the major role of VEGF in this survival supporting coculture setting. Besides coculturing also supplement of culture medium with recombinant human VEGF (rhVEGF) increased survival, but to a lesser extent than coculture, indicating a direct cell-cell interaction as advantageous. Furthermore, we found a downregulation of anti apoptotic proteins, such as X-linked inhibitor of apoptosis protein (XIAP), myeloid cell leukemia 1 (MCL1) and BclXL upon VEGF stimulation. Also cyclinD1 was upregulated as seen by immunoblotting. We further tried to discover the underlying mechanism of how VEGF mediates its pro survival effect and found STAT3 to become phosphorylated on tyrosine 705 upon VEGF stimulation. In CLL STAT3 is known to be constitutively phosphorylated on serine 727. This phosphorylation is not sufficient to induce target gene expression though. We could show that Y705 phosphorylation of STAT3 is responsible for upregulation of anti apoptotic BCLXL and cyclinD1. A PCR array detecting mRNA levels of 84 transcription factors in untreated and VEGF stimulated CLL cells shall provide more information about mechanistical details how VEGF mediates it pro survival effect. Since VEGF seems to be a major player in CLL cell survival it might be a suitable target to overcome the apoptotic block. In first experiments we found an induction of apoptosis after neutralization of VEGF or inhibition of the VEGF receptor. This additionally highlights the severe importance of VEGF in the apoptotic block in CLL cells. Therefore, VEGF might serve as an excellent therapeutic target in CLL.

Vascular remodeling is a feature of asthma and nonasthmatic eosinophilic bronchitis

Increased vascularity and expression of vascular endothelial growth factor (VEGF) are recognized features of the asthmatic airway. The association of vascular remodeling with airway hyperresponsiveness (AHR) is unclear. To assess vascular remodeling and sputum VEGF concentration in subjects with asthma, subjects with nonasthmatic eosinophilic bronchitis (EB), and healthy controls. In cohort 1, 19 patients with asthma (Global Initiative for Asthma [GINA] 1-2, n = 9; GINA 3-5, n = 10), 10 patients with EB, and 11 healthy matched controls were recruited. Expression of the endothelial marker EN4 was assessed in bronchial biopsy samples. Vessels were counted using the validated mean Chalkley count by a blind observer. For cohort 2, a second independent cohort of 31 patients with asthma (GINA 1-2, n = 11; GINA 3-5, n = 20), 14 patients with EB, and 15 matched controls was recruited. Induced sputum supernatant VEGF was measured by ELISA. The mean chalkley count was significantly greater in GINA 3-5 asthma (5.2 [0.4]) and EB (4.8 [0.3]) compared with controls (3.5 [0.5]) and demonstrated a significant inverse correlation with the postbronchodilator FEV(1)% predicted in patients with asthma (R(2) = 0.28; P = .02). Sputum VEGF concentration was also increased in GINA 3-5 asthma (2365 [1361-4110] pg/g) and EB (4699 [2818-7834] pg/g) compared with controls (1094 [676-1774] pg/g) and was inversely related to postbronchodilator FEV(1)% predicted in asthma (R(2) = 0.2; P = .01). Vascular remodeling is a feature of asthma, and EB and is inversely associated with the postbronchodilator FEV(1) in asthma, suggesting that vascular remodeling is associated with airflow obstruction but not AHR. Vascular remodeling is dissociated from AHR in asthma and associated with airflow limitation.

Expression and secretion of VEGF in solid tumor cells is mediated by the mammalian target of rapamycin (mTOR)

14123 Background: Tumor progression and metastasis formation are often associated with enhanced angiogenesis and with the formation of malignant effusions. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and a mediator of vascular permeability. We here describe that VEGF is produced and secreted by neoplastic cells in various solid tumors and its production mediated through mTOR. Methods and Results: As assessed by ELISA, the VEGF protein was detected in supernatants of cell lines derived from breast cancer (MDA-MB231), pancreatic carcinoma (BxPC-3), lung cancer (A-427), colon carcinoma (HCT8), and cholangiocellular carcinoma (EGI-1). In addition, VEGF was detected in supernatants of primary tumor cells obtained from malignant effusions in various malignancies (breast cancer, n=4; pancreatic cancer, n=1; ovarial cancer, n=1; parotic carcinoma, n=1; oesophageal carcinoma, n=1). In each case, VEGF protein was detectable in neoplastic cells by immunocytochemistry, and was found to accumulate in supernatants of cultured tumor cells over time, suggesting constant production and secretion. Correspondingly, as assessed by RT-PCR, primary tumor cells as well as the cell lines tested were found to express VEGF mRNA in a constitutive manner. Since mTOR is a well known regulator of VEGF synthesis, we applied rapamycin on primary neoplastic cells and on tumor cell lines. Rapamycin (20–200 nM) was found to counteract the production and secretion of VEGF in all tumor cells tested (VEGF in supernatants in cultures supplemented with rapamycin at 100 nM compared to control=100% on day 6: MDA-MB231: 11.8±0.2%; BxPC-3: 23.6±18.8%; A-427: 30.1±3.4%; HCT8 17.2±0.5%; EGI-1 28.4±1.1%; p&lt;0.05). By contrast, neither rapamycin nor VEGF were found to modulate growth of primary tumor cells or the growth of the tumor cell lines tested. Conclusions: Various human tumor cells express and secrete VEGF. VEGF production is mediated through mTOR. These observations may have implications for the design of new treatment approaches attempting to counteract VEGF production/secretion and thus VEGF-dependent angiogenesis and effusion- formation in solid tumors. No significant financial relationships to disclose.

Implication of Vascular Endothelial Growth Factor (VEGF) in Human Head and Neck Cancer

Metastatic activity is one parameter indicating the malignancy of tumor cells. Angiogenesis has now been extensively studied to clarify the mechanisms of tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is an angiogenic cytokine expressed by many human and animal tumors. We studied the role of VEGF in tumor growth by transfecting the VEGF gene into tumor cells and analyzing the survival period of nude mice implanted with these transfected tumor cells. Cell line: The tumor cell line, OKK-LN, was established from human maxillary squamous cell carcinoma and used in this study. The tumor cells did not produce VEGF in the culture supernatant. Transfection: OKK-LN cells were stably transfected with sense VEGF165 cDNA or with the vector alone. The full-length VEGF165 cDNA was cloned into an expression vector (pCIneo). The DNA transfection was performed by the lipofection method, and the limiting dilution method was used for cloning. ELISA was used to measure VEGF in the culture supernatant. As a control, OKK-LN cells were transfected with the vector alone without VEGF (OKK-LN/pCIneo). The tumor cells were subcutaneously injected into nude mice (Balb/c nu/nu, 6W), and the survival period and tumor volume were analyzed. Effects of angio-suppressive agent, TNP-470, and anti-VEGF antibody on tumor growth and angiogenesis: TNP-470 (supplied by Takeda Pharmaceutical Co., Ltd.) and monoclonal anti-human VEGF antibodies were intraperitoneally administered to mice implanted with tumor cells once a week and twice a week for 5 weeks, respectively. The effects of TNP-470 and anti-VEGF antibodies were analyzed by examining tumor size and survival rate and immunohistologically using CD31 monoclonal antibody. Tumor cells transfected with sense VEGF 165 cDNA (referred to as OKK-LN/pCIneo VEGF ) produced VEGF in the supernatant permanently, confirming the establishment of a VEGF-producing human cancer cell line. We observed marked tumor growth and a shortened survival period by nine days in the OKK-LN/pCIneo-VEGF group, compared to the control group. The administration of TNP-470 and anti-VEGF antibody significantly suppressed tumor growth. The immunohistological study showed the significant suppression of a number of tumor vessels in anti-VEGF antibody-administered mice. Our data strongly suggests that VEGF plays an important role in tumor growth and that treatment by anti-VEGF antibody may be a promising strategy against head and neck cancers.

Phase I and Pharmacokinetic Study of Flavopiridol followed by 1-β-d-Arabinofuranosylcytosine and Mitoxantrone in Relapsed and Refractory Adult Acute Leukemias

The serine/threonine kinase inhibitor flavopiridol targets multiple cyclin-dependent kinases, induces checkpoint arrest, and interrupts transcriptional elongation. We designed a phase I clinical trial using a timed sequential therapy approach where flavopiridol was given for the dual purpose of initial cytoreduction and enhancing cell cycle progression of the remaining leukemia cell cohort followed by cycle-dependent drugs 1-beta-D-arabinofuranosylcytosine (ara-C) and mitoxantrone. Flavopiridol was given by 1-hour infusion daily for 3 days beginning day 1 followed by 2 g/m2/72 h ara-C beginning day 6 and 40 mg/m2 mitoxantrone beginning day 9. In vivo correlates included pharmacokinetics, modulation of blast cycle regulators, and serum and marrow supernatant vascular endothelial growth factor levels. Of 34 adults receiving induction therapy, 16 (47%) evinced direct leukemia cytotoxicity with > or =50% drop in peripheral blast counts and tumor lysis in 9 (26%). Four (12%) died during therapy (two fungal infections and two sudden death). Dose-limiting toxicity occurred at 60 mg/m2/d with profound neutropenia >40 days duration, and maximal tolerated dose was 50 mg/m2/d. Overall response rate was 31% in 26 acute myelogenous leukemia and 12.5% in acute lymphoblastic leukemia. Pharmacokinetics showed that a linear two-compartment model with first-order elimination provided the best fit of the observed concentration versus time data. Flavopiridol down-regulated one or more target proteins in marrow blasts in vivo. Vascular endothelial growth factor was detected in sera and marrow supernatant pretreatment, and sera obtained on day 3 inhibited bovine aortic endothelial cell proliferation by a mean of 32% (range, 10-80%). Our data suggest that flavopiridol is cytotoxic to leukemic cells and, when followed by ara-C and mitoxantrone, exerts biological and clinical effects in patients with relapsed and refractory acute leukemias. These findings warrant continuing development of flavopiridol at 50 mg/m2/d x 3 days in combination with cytotoxic and biological agents for acute leukemias.

Vascular Endothelial Growth Factor May Contribute to Increased Vascular Permeability in Acute Respiratory Distress Syndrome

The development of noncardiogenic pulmonary edema is a characteristic feature of acute respiratory distress syndrome (ARDS). We hypothesized that vascular endothelial growth factor (VEGF) would play an important role in this process. Plasma VEGF was measured in 40 patients with ARDS, 28 at-risk patients, 14 normal control subjects, and 9 ventilated control subjects. Cultured peripheral blood mononuclear cells (PBM) supernatant VEGF was measured in 21 patients with ARDS and 12 at-risk patients, respectively. The functional importance of VEGF as a mediator of endothelial permeability was assessed by measuring albumin flux across human pulmonary endothelial cell monolayers. Plasma VEGF was significantly elevated in patients with ARDS compared with at-risk patients, normal control subjects, and ventilated control subjects (p = 0.01, p = 0.0001, and p = 0.002, respectively). PBM from patients with ARDS produced significantly more VEGF in vitro than at-risk patients (p = 0.05). Albumin flux across human pulmonary endothelial cell monolayers was significantly increased following the addition of plasma from patients with ARDS compared with plasma from normal control subjects (p = 0.008). When VEGF activity in plasma was neutralized by the addition of a soluble VEGF inhibitor, the albumin flux induced by ARDS plasma was reduced by 48%. We conclude that VEGF makes a significant contribution to the endothelial cell permeability-inducing activity in plasma from patients with ARDS, and may play an important role in the development of noncardiogenic pulmonary edema in ARDS.

Blockade of GpIIb/IIIa inhibits the release of vascular endothelial growth factor (VEGF) from tumor cell-activated platelets and experimental metastasis

Evidence that platelets play a role in tumor metastasis includes the observation of circulating tumor cell-platelet aggregates and the anti-metastatic effect of thrombocytopenia and anti-platelet drugs. Platelets have recently been shown to contain vascular endothelial growth factor (VEGF) which is released during clotting. We therefore studied the effects of (1) tumor cell-platelet adherence and tumor cell TF activity on platelet VEGF release; and (2) the effects of GpIIb/IIIa blockade on tumor cell-induced platelet VEGF release, tumor cellinduced thrombocytopenia and experimental metastasis. Adherent A375 human melanoma cells (TF+) and KG1 myeloid leukemia (TF-) cells were cultured in RPMI containing 10% fetal bovine serum. Platelet-rich plasma was obtained from normal citrated whole blood and the presence of VEGF (34 and 44 kDa isoforms) confirmed by immunoblotting. Platelet-rich plasma with or without anti-GpIIb/IIIa (AbciximabTM) was added to A375 monolayers and supernatant VEGF measured by ELISA. Tumor cell-induced platelet activation and release were determined by CD62P expression and serotonin release respectively. In vitro, tumor cell-platelet adherence was evaluated by flow cytometry. In vivo, thrombocytopenia and lung seeding were assessed 30 min and 18 days, respectively, after i.v. injection of Lewis Lung carcinoma (LL2) cells into control or murine 7E3 F(ab') 2 (6 mg/ kg) athymic rats. Maximal in vitro platelet activation (72% serotonin release) occurred 30 min after adding platelets to tumor cells. At this time, 87% of the A375 cells had adhered to platelets. AbciximabTM significantly (P<0.05) reduced platelet adherence to tumor cells as evidenced by flow cytometry. Incubation of A375 cells with platelets induced VEGF release in a time-dependent manner. This release was significantly inhibited by AbciximabTM (81% at 30 min; P<0.05). In the presence of fibrinogen and FII, VEGF release induced by A375 (TF+) cells was significantly higher than that induced by KG1 (TF-) cells (105.5+/-24 vs. 42+/-7 pg/ml; P<0.001). Omitting fibrinogen or FII from the reaction mixture markedly decreased VEGF release. In vivo, GpIIb/IIIa blockade with murine 7E3 F(ab') 2 reduced LL2 tumor cell-induced thrombocytopenia by 90% (P<0.001) and lung seeding by 82% (P<0.05). We conclude that TF-bearing tumor cells can activate platelets largely via thrombin generation, and that such activation is associated with release of VEGF. This may enhance metastasis, possibly by increasing extravasation at points of adhesion to vascular endothelium.

ACTIVATED HUMAN NEUTROPHILS EXPRESS VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF)

The neutrophil (PMN) influx in the acute inflammatory response is associated with a local increase in vascular permeability and oedema. Vascular endothelial growth factor (VEGF), a growth factor known to have potent vascular permeability-enhancing properties in addition to being an endothelial cell mitogen and a chemo-attractant for mononuclear cells, has previously been shown to be expressed by mononuclear cells and platelets, though PMN VEGF expression has not been reported.PMNs isolated from healthy adult volunteers (n=16) were incubated for 4 h at 37°C in the presence of tumour necrosis factor α (TNF-α) (5 ng/ml) and serum opsonized zymozan (SOZ) (500 μg/ml). Supernatant VEGF levels were measured using a sandwich antibody capture immuno-assay. Median (interquartile range) VEGF levels were significantly increased in PMN supernatants following stimulation with both TNF-α [347 pg/ml (264–385 pg/ml)] and SOZ [506 pg/ml (407–593 pg/ml)] compared with control values [78 pg/ml (78–87 pg/ml)]. Time course experiments with SOZ stimulated PMNs showed that the majority of VEGF production occurred within the first hour (1 h mean VEGF level 318 pg/ml, 4 h mean VEGF level 451 pg/ml). RT-PCR studies showed that PMNs express mRNA for the two common VEGF splice variants, VEGF121and VEGF165.PMN VEGF production may be central to the classic acute phase response to injury and the chemo-attraction of other leukocytes to the source of injury.

Measurement of bio- and chemiluminescence in liquid scintillation counters or dedicated luminometers (plenary lecture): F. Berthold, Laboratium Prof. Dr. Berthold D-7547 Wildbad, Germany

The neutrophil (PMN) influx in the acute inflammatory response is associated with a local increase in vascular permeability and oedema. Vascular endothelial growth factor (VEGF), a growth factor known to have potent vascular permeability-enhancing properties in addition to being an endothelial cell mitogen and a chemo-attractant for mononuclear cells, has previously been shown to be expressed by mononuclear cells and platelets, though PMN VEGF expression has not been reported.PMNs isolated from healthy adult volunteers (n=16) were incubated for 4 h at 37°C in the presence of tumour necrosis factor α (TNF-α) (5 ng/ml) and serum opsonized zymozan (SOZ) (500 μg/ml). Supernatant VEGF levels were measured using a sandwich antibody capture immuno-assay. Median (interquartile range) VEGF levels were significantly increased in PMN supernatants following stimulation with both TNF-α [347 pg/ml (264–385 pg/ml)] and SOZ [506 pg/ml (407–593 pg/ml)] compared with control values [78 pg/ml (78–87 pg/ml)]. Time course experiments with SOZ stimulated PMNs showed that the majority of VEGF production occurred within the first hour (1 h mean VEGF level 318 pg/ml, 4 h mean VEGF level 451 pg/ml). RT-PCR studies showed that PMNs express mRNA for the two common VEGF splice variants, VEGF121and VEGF165.PMN VEGF production may be central to the classic acute phase response to injury and the chemo-attraction of other leukocytes to the source of injury.