Vascular Endothelial Growth Factor Gene Expression Research Articles

Analysis of vascular endothelial growth factor gene expression in the tissues of patients with chronic venous insufficiency

Immunohistochemical techniques have revealed the presence of vascular endothelial growth factor (VEGF) in the epidermis of patients with chronic venous disease (CVD). Our objective was to perform a quantitative analysis of the VEGF gene transcription in tissues that are potential sources of this factor (skin, varicose veins [VV] and great saphenous vein [GSV]) in patients with CVD. In all, 212 skin and venous tissue samples were collected from patients diagnosed with CVD and controls. The VEGF gene expression was analysed using quantitative realtime polymerase chain reaction (PCR). The skin VEGF expression was lower in the CVD group than in the control group (P = 0.04). There were no significant differences between the insufficient GSV of the CVD group and the control healthy vein (P = 0.22). There was a greater expression of VEGF in the VV of the CVD group than in the control healthy vein (P = 0.03). Comparison of the VEGF expression between the different tissue types in the CVD group revealed significant differences between the skin and GSV (P = 0.02) and between the skin and the VV (P = 0.004), and between the VV and the GSV (P = 0.02). The results of the present study show an over-expression of VEGF gene in the VV tissue of patients with CVD. Based on the data in patients with C2 disease, the VVs appear to be the source of increased VEGF expression.

Cytokine production by infrapatellar fat pad can be stimulated by interleukin 1β and inhibited by peroxisome proliferator activated receptor α agonist

BackgroundInfrapatellar fat pad (IPFP) might be involved in osteoarthritis (OA) by production of cytokines. It was hypothesised that production of cytokines is sensitive to environmental conditions.ObjectivesTo evaluate cytokine production by...

Agonist and antagonist coast

The use of GnRH-a in ovarian stimulation permitted stronger stimulation resulting in an increased incidence of OHSS. The first Cochrane review comparing GnRH agonist and GnRH antagonist protocols for ovarian stimulation showed no significant difference in OHSS rate between the two protocols, however, a recent Cochrane review showed a highly significant decrease in the incidence in OHSS rate in the antagonist protocol. Coasting is a commonly used procedure for preventions of OHSS. The optimum time to start coasting is when the lead follicle reaches 16 mm in diameter and hCG should be given when E2 level drops below 3000 pg/ml. Coasting may act by diminishing the functioning granulosa cell cohort. Administration of daily GnRH antagonist in high risk patients for OHSS who were down-regulated by GnRH-a resulted in rapid drop of E2 and decrease in incidence of OHSS. A series of patients who developed early OHSS were treated by daily GnRH antagonist injections, all embryos were cryopreserved. No progression to severe OHSS was observed.

Enhanced Vascular Endothelial Growth Factor Gene Expression in Ischaemic Skin of Critical Limb Ischaemia Patients

Objectives. To perform a quantitative analysis of the vascular endothelial growth factor (VEGF) gene transcription in the skin of ischemic legs and provide information for VEGF in the pathogenesis in critical limb ischemia (CLI). Methods. Skin biopsies were obtained from 40 patients with CLI. Control samples came from 44 patients with chronic venous disease. VEGF gene expression was analysed using quantitative polymerase chain reaction. Results. Patients with CLI had higher skin VEGF expression than control group (RQ: 1.3 ± 0.1 versus 1, P = 0.04). Conclusions. We found an association between ischemic skin and an elevated VEGF expression in legs from patients with CLI. These data support that the mechanism for VEGF upregulation in hypoxia conditions is intact and acts appropriately in the ischaemic limbs from patients with CLI.

VEGF and metalloproteinase 2 (MMP 2) expression in gastric cancer tissue.

SummaryBackgroundNeoplasms are the second leading cause of death in Poland after vessel diseases, despite the huge progress in medical sciences in the last 20 years. Recently, gastric cancer morbidity has decreased, but mortality is still at a high level.Material/MethodsTissues from 24 patients with a histopathologically diagnosed mucosal and adenomucosal gastric cancer were tested. Patients were divided into 2 equal groups: patients without metastases (G1) and patients with metastases in the liver (G2). In all tested tissues of G1 and G2, the expression of VEGF (vascular endothelial growth factor) and metalloproteinase 2, respectively, were estimated.ResultsResults revealed a statistically significant increase in the VEGF expression for G1 and G2 in relation to the margin (p1<0.001; p2<0.001). The increase of gene expression for VEGF did not significantly differ statistically in G1 and G2. The obtained results revealed a statistically significant difference in the increase of gene expression for MMP-2 in G1 in relation to the margin (p<0.05) and a very high one in G2 in relation to the average margin value (p<0.001). A highly statistically significant correlation was obtained for VEGF and MMP-2 in the tissue of patients with metastases (p<0.001; r=0.714). The highly elevated expression of MMP-2 in the tissue of gastric cancer in patients with metastases confirms its participation in the invasiveness of the neoplasmatic process.ConclusionsThe highly significant correlation between VEGF and MMP-2 suggests a connection between both mechanisms in the progression of gastric cancer.

Aloe emodin inhibits colon cancer cell migration/angiogenesis by downregulating MMP-2/9, RhoB and VEGF via reduced DNA binding activity of NF-κB

Aloe emodin (AE), a natural anthraquinone, is reported to have antiproliferative activity in various cancer cell lines. In this study we analyzed molecular mechanisms involved in the antimigratory and antiangiogenic activity of this hydroxy anthraquinone in colon cancer cell, WiDr. Our results show that a relatively non toxic concentration of AE suppressed the phorbol-12-myristyl-13-acetate (PMA) induced migration and invasion of tumor cells. On analysis for the molecules involved in the migration/invasion, we found AE downregulated mRNA expression and promoter/gelatinolytic activity of Matrix Metalloproteinase (MMP)-2/9, as well as the RhoB expression at gene and protein level. It was also a strong inhibitor of Vascular Endothelial Growth Factor (VEGF) expression, promoter activity and endothelial cell migration/invasion and in vitro angiogenesis. AE suppressed the nuclear translocation and DNA binding of NF-κB, which is an important transcription factor for controlling MMP-2/9 and VEGF gene expression. Taken together these data indicate that AE target multiple molecules responsible for cellular invasion, migration and angiogenesis. Inhibitory effect on angiogenic and metastatic regulatory processes make AE a sensible candidate as a specific blocker of tumor associated events.

The effect of connexin43 on the level of vascular endothelial growth factor in human retinal pigment epithelial cells

Connexins (Cx) are the basic units of gap junctions and contribute to cellular integrity by promoting intercellular communication. Disruption of the retinal pigment epithelial monolayer may be an early event in the pathogenesis of age-related macular degeneration, a condition in which vascular endothelial growth factor (VEGF) is known to be of importance. This study was designed to assess the effect of connexin43 (Cx43) expression and gap junctional intercellular communication (GJIC) on the expression and secretion of VEGF from the retinal pigment epithelium under normal cell culture and oxidative stress conditions. Stable cell lines of ARPE-19 were produced in which wild-type Cx43 was either over-expressed, down-regulated by targeted shRNA, or functionally inhibited by co-expression of a disease-linked dominant-negative mutant (G21R). Pharmacologic blockade of GJIC was accomplished with flufenamic acid. Oxidant challenge was performed with tert-butyl hydroperoxide (tBH). VEGF gene expression and secretion were assessed by real-time PCR and ELISA respectively. Over-expression of Cx43 in ARPE-19 cells reduced both gene expression and secretion of VEGF. Down-regulation of Cx43 increased gene expression and secretion of VEGF. Increased secretion of VEGF was also observed in ARPE-19 cells expressing a dominant-negative mutant of Cx43, and when GJIC was blocked. Over-expression of Cx43 reduced tBH-induced secretion of VEGF from ARPE-19 cells. These studies show that Cx43 protects against oxidative stress-induced VEGF secretion in ARPE-19 cells, and thus has important implications in understanding the pathogenesis of age-related macular degeneration.

Construction and identification of recombinant adenovirus vectors expressing GFP and human vascular endothelial growth factor short hairpin RNA

Objective To construct and identify recombinant adenovirus vectors expressing GFP and human vascular endothelial growth factor (VEGF) iRNA and to inhibit the gene expression of VEGF in human nasopharyngeal carcinoma cell line CNE with the RNA interference (RNAi) technique,and explore the expression of VEGF in CNE and its proliferation before and after transfection.Methods By the method of restriction endonuclease digestion,the GFP and human VEGF genes were subcloned from pGenesil-1 plasmid into the shuttle vector pGSadeno-GFP-VEGF.After correct identification of the recombinant plasmid pGSadeno-GFP-VEGF,it was then digested by Pacl and transfected into HEK293 cells to be packaged and amplification for rAd-VEGF.Meanwhile,the recombinant adenovirus was identified by PCR method.The expression of GFP was detected to evaluate the titre and transfective ratio.Results Digestion and PCR identification showed the construction of recombinant adenovirus rAd-VEGF was successful via LR recombinant.The titre was 2×109 PFU/ml while the transfective ratio in CNE cell line was more than 90 percent which met the need of experiment.Cell growth was inhibited compared with non-siRNA transfected CNE cell line.Conclusion The recombinant virus expressing GFP and VEGF are successfully constructed; it layed a foundation for further relevant study. Key words: Nasopharyngeal neoplasms; Vascular endothelial growth factors; RNA interference; Adenoviridae

Inhibition of mTOR with Everolimus (RAD001) and Silencing by Vascular Endothelial Cell Growth Factor Specific siRNA Induces Additive Antitumor Activity in Multiple Myeloma Cells,

Abstract 3984 Background:Angiogenesis plays an important role in the pathogenesis and progression in multiple myeloma (MM). MM cells secrete vascular endothelial growth factor (VEGF), which further promotes proliferation of the tumor cells. Several RNA interference (RNAi) methodologies are rapidly being established and hold promise to specifically inhibit gene expression in mammals. RNAi is the sequence-specific, posttranscription gene silencing methods initiated by double-stranded RNAs, which are homologous to the gene being suppressed. Mammalian target of rapamycin (mTOR) is an essential part of tumour growth being capable of integrating proliferative, antiapoptotic and angiogenic signalling by connecting VEGF, hypoxia-inducible factor 1 (HIF-1) and HER family receptors. Several reports have demonstrated the anti-tumor activity of everolimus (RAD001), an mTOR inhibitor, in a variety of cancers, including various leukemias and lymphomas. Aims:Therefore, we evaluated the anti-myeloma effect of VEGF siRNA silencing in MM cells and whether it can be augmented by the additional application of everolimus.Methods and Results: After transfection with VEGF siRNA we observed a reduction of VEGF expression in all studied cell lines: OPM-2, RPMI-8226, INA-6, Jurkat, Raji and Karpas-299, as well as in cells of MM- and lymphoma patients. Next, using the MM cell line OPM-2 we studied the time courses of VEGF siRNA transfection in order to investigate the knock-down efficiency of VEGF expression. The efficiency of VEGF siRNA transfection in treated OPM-2 cells showed a reduction of 75.5% VEGF protein levels after 24 h compared to the untreated OPM-2 cells (p<0.001). Further, VEGF siRNA both significantly induced apoptosis and inhibited proliferation in OPM-2 cells (p<0.001), RPMI-8226 (p<0.001), and in INA-6 (p<0.01) versus controls. To assess whether everolimus treatment of OPM-2 and RPMI-8226 affects the viability of these cells, cells were treated with various doses (1–20 nM) of everolimus for 24 hours, harvested, and analyzed for cell viability by MTT assay. Everolimus significantly decreased the viability of OPM-2 cells (IC50 = 5.9 nM) and of RPMI-8226 cells (IC50 = 0.6 nM), respectively. Everolimus and siRNA both together might to reduce the VEGF gene expression up to 33% (p<0.001) compare to siRNA VEGF (61%) alone or everolimus alone (39%), that demonstrated additive effects of everolimus and siRNA. Conclusions:These findings suggest that mTOR inhibition and silencing by VEGF specific siRNA may be associated with an additive antitumor activity and might be a suitable target for new therapeutic strategies using RNA interference in MM. Disclosures:No relevant conflicts of interest to declare.

Angiogenic Factors eNOS and VEGF Have Increased Expression in Granulocytes of JAK2V617F Homozygous Forms of Polycythemia Vera,

Abstract 3847The microvessel density of bone marrow is increased in myeloproliferative neoplasms (MPN) parallel with vascular endothelial growth factor (VEGF). VEGF-mediated angiogenesis requires nitric oxide (NO) production from activated endothelial NO synthase (eNOS). NO as well as hypoxia stimulate the VEGF gene expression and angiogenesis by enhancing hypoxia inducible factor (HIF)-1 activity. We studied 126 newly diagnosed patients with BCR-ABL− MPN: 64 polycythemia vera (PV), 36 essential thrombocythemia (ET), 26 primary myelofibrosis (PMF) and 12 healthy individuals. We performed a combined analysis of hematopoietic CD34+ progenitor cells and granulocytes in peripheral blood of these individuals. The eNOS protein level is more than three-fold elevated in granulocytes of JAK2V617F homozygous PV patients. The essential inducer of angiogenesis VEGF-A has also about three-fold elevation at the protein level in granulocytes of PV patients, with major increases in JAK2V617F homozygous forms. Immunohistochemical analysis reveal that the percentage of VEGF-A-positive cells is increased in bone marrow of PV (5.58±0.7%) compared to normal controls (2.78±0.7%) and VEGF-A mRNA levels are increased in hematopoietic progenitor cells of PV origin. Transcription factor HIF-1α gene expression is decreased in hematopoietic progenitor cells and increased in granulocytes of PV patients. Negative regulator of HIF-1α activity, a transcription factor HIF-3α, has decreased expression in hematopoietic progenitor cells and not changed in granulocytes. In contrast to PV patients, PMF and ET disorders with a minor JAK2 mutation burden demonstrate reduced eNOS and VEGF protein levels and decreased HIF-1a gene expression in peripheral blood granulocytes, although the increase in percentage of VEGF-A-positive cells in bone marrow observed in PV patients is also evident. The present results expand the significance of JAK2V617F mutation in induction of angiogenic factors eNOS and VEGF in granulocytes of PV patients with enhanced HIF-1α presence. Moreover, the stromal and hematopoietic cells also show increased VEGF protein expression in bone marrow of PV patients. Therefore, we find that variations in angiogenic factors expression among MPN patients appear to be related to JAK2V617F mutation allele burden. Disclosures:No relevant conflicts of interest to declare.

Down-regulation of the human VEGF gene expression by perylene monoimide derivatives

The proximal promoter region of the human vascular endothelial growth factor (VEGF) gene contains a guanine-rich strand that can act as a transcriptional silencer by forming an intramolecular G-quadruplex. In this study, we compared two perylene monoimide derivatives, PM1 and PM2, with the well-studied perylene diimide derivative, PIPER, and the well-studied porphyrin derivative, TmPyP(4), with regard to G-quadruplex formation, G-quadruplex binding selectivity, and human VEGF gene silencing in A549 lung cancer cells. The results show that these perylene derivatives can preferentially induce intramolecular G-quadruplex formation from a duplex containing the VEGF G-quadruplex motif in vitro. Incubating A549 lung cancer cells with these perylene derivatives, especially PM2, led to the reduction of both VEGF mRNA and VEGF protein. This study might provide the foundation for the rational design and development of new perylene derivatives as effective anti-angiogenesis agents for cancer therapy.

Dual action of a selective cyclooxygenase-2 inhibitor on vascular endothelial growth factor expression in human hepatocellular carcinoma cells: novel involvement of discoidin domain receptor 2

Vascular endothelial growth factor (VEGF) greatly contributes to the progression of hepatocellular carcinoma (HCC). It is reported that a selective cyclooxygenase-2 (COX-2) inhibitor inhibits cellular proliferation and may attenuate VEGF expression in HCC. We propose that different cascades in the VEGF pathway respond to COX-2 inhibition, depending on the cell types. The six human HCC cell lines--Hep3B, SNU387, SNU182, SNU423, SNU449, and PLC/PRF5--were cultured under normoxic and hypoxic conditions. Cells were treated with a selective COX-2 inhibitor (NS-398) and discoidin domain receptor 2 (DDR2) siRNA, and microarray analysis was performed. NS-398 inhibited HCC proliferation and decreased the expression level of VEGF in HCC cells only under normoxia conditions. In hypoxia conditions, VEGF expression level in Hep3B cell was suppressed, while that in SNU387 cell was increased by NS-398 (P < 0.001). The NS-398-induced increase in VEGF expression in SNU387 cell was associated with the up-regulation of the DDR2 gene. NS-398-treated SNU series cells and PLC/PRF5 cells displayed a robust increase in DDR2 mRNA expression. Also, transfection with DDR2 siRNA decreased the VEGF expression level of SNU387, 423, 449 cells under hypoxia conditions (P < 0.05). In vivo chromatin immunoprecipitation assay demonstrated that NS-398 induces the enhancement of HIF-1α binding on VEGF promoter, leading to the increase in VEGF gene expression in hypoxic conditions. There is strong evidence that it is related to the DDR2 gene expression in SNU387 cells. These findings disclose a novel cell-dependent regulatory mechanism of VEGF involving DDR2 gene in HCC cells.

Vascular endothelial growth factor A protein level and gene expression in intracranial meningiomas with brain edema

Meningiomas are the second most common primary intracranial tumors in adults. Although meningiomas are mostly benign, more than 50% of patients with meningioma develop peritumoral brain edema (PTBE), which may be fatal because of increased intracranial pressure. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and angiogen. VEGF-A protein, which is identical to vascular permeability factor, is a regulator of angiogenesis. In this study, 101 patients with meningiomas, and possible co-factors to PTBE, such as meningioma subtypes and tumor location, were examined. Forty-three patients had primary, solitary, supratentorial meningiomas with PTBE. In these, correlations in PTBE, edema index, VEGF-A protein, VEGF gene expression, capillary length, and tumor water content were investigated. DNA-branched hybridization was used for measuring VEGF gene expression in tissue homogenates prepared from frozen tissue samples. The method for VEGF-A analysis resembled an ELISA assay, but was based on chemiluminescence. The edema index was positively correlated to VEGF-A protein (p = 0.014) and VEGF gene expression (p < 0.05). The capillary length in the meningiomas was positively correlated to the PTBE (p = 0.038). If VEGF is responsible for the formation of PTBE, the edema may be treated with the anti-VEGF drug Bevacizumab (Avastin), which has been shown to reduce PTBE in patients with glioblastoma multiforme.

CCK-2/gastrin receptor signaling pathway is significant for gemcitabine-induced gene expression of VEGF in pancreatic carcinoma cells

AimsAs activation and overexpression of the cholecystokinin-2 (CCK-2)/gastrin receptor can lead to carcinogenesis, it has been explored as a therapeutic target in pancreatic cancer. We demonstrated that Z-360, a CCK-2/gastrin receptor antagonist, combined with gemcitabine prolonged survival and reduced gemcitabine-induced vascular endothelial growth factor (VEGF) expression in a pancreatic carcinoma orthotopic xenograft mouse. In this study, we investigated the role of the CCK-2/gastrin signaling pathway on gemcitabine-induced VEGF expression in PANC-1 human pancreatic carcinoma cells. Main methodsIn PANC-1 cells treated with Z-360, anti-gastrin IgG or kinase inhibitors, the gene expression levels were analyzed by quantitative real-time RT-PCR, and the protein levels of Akt and phosphorylated Akt (p-Akt) in cellular extracts were measured by ELISA. Key findingsGemcitabine-induced expression of VEGF and hypoxia-inducible factor-1 alpha (HIF-1 alpha) were suppressed by the treatment with an anti-gastrin antibody. In addition, VEGF and HIF-1 alpha gene expression was inhibited by treatment with an inhibitor of phosphatidylinositol 3-kinase (PI3K), which is involved in the downstream signaling pathway of the CCK-2/gastrin receptor, and was also suppressed by treatment with Z-360. Moreover, although Akt phosphorylation was increased by treatment with gemcitabine, this elevation was partially, but significantly, inhibited by an exposure of Z-360. SignificanceGemcitabine might induce gene expression of VEGF via the PI3K/Akt signaling pathway in the downstream of the CCK-2/gastrin receptor. The suppression of the CCK-2/gastrin signaling pathway by treatment with Z-360 could be a useful approach for potentiating prolonged survival of pancreatic cancer patients receiving gemcitabine therapy.

Expression and mechanism of granulocyte-macrophage colony stimulating factor in acute skin defect of the mice

Objective To investigate the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its associated mechanism during the wound healing. Methods The animal model with the full-thickness skin injury was used in the study. Fifty male mice were involved in the study and divided randomly into control group (n = 25) and GM-CSF group (n = 25). Each group had five time points (5 mice per time point). All the mice received full-thickness skin defect (1 cm × 1 cm) through the panniculus camosus on the midline of the back near the neck after anesthesia. Recombinant human granulocyte-macrophage colony stimulating factor (RhGM-CSF) gel (10 μg/cm2) were applied in the GM-CSF group and gel matrix without RhGM-CSF applied in the control group. The wound healing time and rate were observed at days 3, 5, 7, 10 and 14 after injury. The wound specimens were collected to detect the histopathological change. The microvessel density of the wound was counted based on the results of CD31 immunohistochemistry. RT-PCR was employed to detect the expression changes of GMCSF, platelet-derived growth factor (PDGF) , vascular endothelial growth factor ( VEGF) and stromal cell derived factor-1 (SDF-1). Results RT-PCR results showed that the gene expression of GM-CSF reached the peak at day 3 after injury (P＜0. 01) and kept the high level at days 3-10 after injury (P＜ 0. 05) , followed by a sharp decrease to a normal level at day 14 after wound. The wound healing time was average (2.4 ±0. 3) days earlier than the control mice after application of rhGM-CSF, with significant increase of the wound healing rate during 7-14 days after injury ( P ＜ 0. 05 ). In the GM-CSF group, the early histology of trauma wound showed a small number of neutrophils, obvious epithelial cell proliferation in the wound margin, marked hyperplasia of the granulation tissue, high cell density with quantity of spindle-shaped and oval-shaped cells and increased number of new blood vessels. The microvessel density was also increased significantly (P ＜ 0. 05) at days 7-14 after injury. The gene expressions of VEGF and SDF-1 were significantly increased at day 7 and day 10 respectively after injury (P＜0.05) and the gene expression of pro-healing factor PDGF was significantly increased in every time point (at days 5, 7 and 10,P＜0.05;at day 14,P＜0.01). Conclusion GM-CSF expresses highly in the early stage after injury and can promote the wound healing, when the mechanism may relate to the up-regulated expressions of pro-angiogenic factors and pro-healing growth factors. Key words: Wound healing; Granulocyte-macrophage colony stimulating factor; Microvessel density; Growth factor; Gene expression

Plantaricin A synthesized by Lactobacillus plantarum induces in vitro proliferation and migration of human keratinocytes and increases the expression of TGF-β1, FGF7, VEGF-A and IL-8 genes

This work showed the effect of pheromone plantaricin A (PlnA) on the proliferation and migration of the human keratinocytes NCTC 2544. PlnA was chemically synthesized and used as pure peptide or biologically synthesized during co-cultivation of Lactobacillus plantarum DC400 and Lactobacillus sanfranciscensis DPPMA174. The cell-free supernatant (CFS) was used as the crude preparation containing PlnA. The inductive effect of PlnA on the proliferation of NCTC 2544 cells was higher than that found for hyaluronic acid, a well known skin protective compound. As shown by scratch assay and image analyses, PlnA enhanced the migration of NCTC 2544 cells. Compared to the basal serum free medium (control), the highest inductive effect was found using 10μg/ml of chemically synthesized PlnA. Similar results (P>0.05) were found for CFS. In agreement, the percentage of the starting scratch area was decreased after treatment (24h) with PlnA. The expression of transforming growth factor-β1 (TGF-β1), keratinocyte growth factor 7 (FGF7), vascular endothelial growth factor (VEGF-A), and interleukin-8 (IL-8) genes was affected by PlnA. Compared to control, TGF-β1 gene was under expressed in the first 4h of treatments and up-regulated after 8–24h. On the contrary, FGF7 gene was strongly up-regulated in the first 4h of treatments. Compared to control, VEGF-A and IL-8 genes were always up-regulated during the 4–24h from scratching. Since capable of promoting the proliferation and migration of the human keratinocytes and of stimulating IL-8 cytokine, the use of PlnA for dermatological purposes should be considered.

The regulatory effect of norepinephrine on connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF) expression in cultured cardiac fibroblasts

BackgroundConnective tissue growth factor (CTGF) and vascular endothelial cell growth factor (VEGF) have been implicated as important effectors during cardiac remodeling. This study tested the hypothesis that norepinephrine (NE) induces CTGF and VEGF gene and protein expression in cardiac fibroblasts (CF) and the CTGF/VEGF complex will have an effect on angiogenesis. Methods and resultsRats CF were cultured in NE (0.01 to 100μM) for 24h. CTGF and VEGF gene expression were measured by quantitative-PCR. CTGF protein and CTGF/VEGF complex were detected by Western blot. The effect of CTGF/VEGF complex on angiogenesis was detected by endothelial cell tube formation assay. VEGF antigen level, reactive oxygen species (ROS) production were measured by ELISA and DCFH-DiOxyQ assay respectively. NE at 0.01μM up-regulated CTGF mRNA and secretory protein expression significantly whereas at 100μM both gene and protein were down-regulated significantly when compared with controls. At 0.01 to 0.1μM of NE, there was no change in VEGF gene and protein level. NE at 100μM increased VEGF gene and antigen level and ROS production significantly when compared with controls. CTGF/VEGF complex was found to inhibit the angiogenesis of endothelial cells. ConclusionsNE regulates CTGF and VEGF expression in a dose-dependent manner and via VEGF can induce angiogenesis. This work suggests NE may have an important role in ventricular remodeling.

VEGF in the Development of Ovarian Malignancy

BackgroundIt is well known that the vascular endothelial growth factor (VEGF) system is essential to the growth of tumors, but its possible changes during tumor development are still sparsely elucidated. The current study, therefore, investigated the possible differences of VEGF and VEGFR-1 and VEGFR-2 protein concentration in tissue from patient groups with normal ovaries and those with benign, borderline, and malignant ovarian tumors. Furthermore, we investigated VEGF gene expression of the same groups. Material and MethodsWe analyzed tissue samples from 157 patients undergoing surgery of the ovaries. Tissue VEGF and VEGFR-1 and VEGFR-2 were measured by enzyme-linked immunosorbent assay, and gene expression was analyzed by reverse transcriptase polymerase chain reaction regarding VEGF isotypes 121, 165, and 189. ResultsSignificantly higher VEGF protein concentration was found in malignant tissue compared with normal ovarian tissue (P < 10-6) and benign ovarian tumors (P < 10-6). Similar results were found with respect to VEGFR-1. The gene expression differed among normal ovaries, benign, and malignant tumors for all Three VEGF isotypes (P < 10-6), with significantly higher levels of VEGF mRNA in malignant tumors. Malignant tumors with VEGFR-2 protein levels above the median had decreased progression-free survival (PFS) (P = .0005) and overall survival (P = .002). Median PFS was 26.2 months in the group with low VEGFR-2 levels vs. 7.8 months in the other group. Multivariate analysis confirmed the results (hazard ratio, 1.94; P = .04). ConclusionThe current study showed major changes in the VEGF system during tumor development. A significant difference was found in protein concentration and gene expression among the different patient groups. In addition, VEGFR-2 was correlated with PFS and overall survival.

Neural Stem Cells Modified by a Hypoxia-Inducible VEGF Gene Expression System Improve Cell Viability under Hypoxic Conditions and Spinal Cord Injury

An in vitro neural hypoxia model and rat spinal cord injury (SCI) model were used to assess the regulation of therapeutic vascular endothelial growth factor (VEGF) gene expression in mouse neural stem cells (mNSCs) by the EPO (erythropoietin) enhancer or RTP801 promoter. To increase VEGF gene expression in mNSCs under hypoxic conditions in SCI lesions but avoid unwanted overexpression of VEGF in normal sites, we developed a hypoxia-inducible gene expression system consisting of the EPO enhancer and RTP801 promoter fused to VEGF or the luciferase gene, then transfected into mNSCs. On the basis of the ischemic response in the injured area, poor cell survival at the transplantation site is a consistent problem with NSC transplantation after SCI. Although VEGF directly protects neurons and enhances neurite outgrowth, uncontrolled overexpression of VEGF in uninjured tissue may cause serious adverse effects. To effectively improve NSC survival in ischemic sites after transplantation, we evaluated mNSCs modified by a hypoxia-inducible VEGF gene expression system in an SCI model. Hypoxia-inducible luciferase or VEGF plasmids were constructed using the EPO enhancer or RTP801 promoter. The effect of these systems on targeted gene expression and cell viability was evaluated in mNSCs in both hypoxic in vitro injury and a rat SCI model in vivo. The gene expression system containing the EPO enhancer or RTP801 promoter significantly increased the expression of the luciferase reporter gene and therapeutic VEGF gene under hypoxic conditions. The Epo-SV-VEGF plasmid transfection group had significantly fewer apoptotic cells in vitro. This system also augmented cell viability in the in vivo SCI model. These results strongly suggest the potential utility of mNSCs modified by a hypoxia-inducible VEGF gene expression system in the development of effective stem cell transplantation protocols in SCI.

Tobacco carcinogen mediated up‐regulation of AP‐1 dependent pro‐angiogenic cytokines in head and neck carcinogenesis

Tobacco is notably genotoxic and associated with head and neck carcinogenesis. Cigarette carcinogens have the capacity to alter early response gene expression in tobacco-related malignancies via genes such as nuclear factor kappa B (NFκB). A number of early response gene activation events are also facilitated by fos/jun activator protein 1 (AP-1) associated pathways. In the present study, we hypothesize that tobacco products may induce microenvironment alterations, promoting angiogenesis and providing a permissive environment for head and neck cancer progression. In an in vitro analysis, we employed immortalized oral keratinocyte (HOK-16B) and laryngeal squamous carcinoma (UM-SCC-11A) cells to investigate interleukin (IL)-8 and vascular endothelial growth factor (VEGF) induction by cigarette smoke condensate (CSC). IL-8 and VEGF expression is based on interactions between NFκB, AP-1, and NF-IL6. We identified at least 1.5-fold dose-dependent induction of AP-1, VEGF, and IL-8 promoter/reporter gene activity after 24 h exposure to CSC. Next, we stably transfected UM-SCC-11A cells with A-Fos, a dominant negative AP-1 protein. Treatment with CSC of the A-Fos cell lines compared to empty vector controls significantly down-regulated AP-1, VEGF, and IL-8 promoter/reporter gene expression. We also performed ELISAs and discovered significant up-regulation of IL-8 and VEGF secretion by UMSCC 11A after treatment with phorbol 12-myristate 13-acetate, tumor necrosis factor alpha, and CSC, which was down-regulated by the A-Fos dominant negative protein. We conclude tobacco carcinogens up-regulate AP-1 activity and AP-1 dependent IL-8 and VEGF gene expression in head and neck cancer. This up-regulation may promote an angiogenic phenotype favoring invasion in both premalignant and squamous cancer cells of the head and neck.