Vascular Endothelial Cell Growth Factor mRNA Research Articles

In vitro antioxidant and anticancer activities of leaf extracts of Rhododendron arboreum and Rhododendron campanulatum from Uttarakhand region of India

Background: Rhododendron arboreum and Rhododendron campanulatum are two important trees of Uttarakhand, known for their ethnopharmacological importance. In the present study, the aqueous and methanolic extracts of leaves of both the plants were analyzed for phytochemical, antioxidant, and antiproliferative activities against cancer cell line and repression of vascular endothelial cell growth factor (VEGF) and hypoxia-inducible factor-1 (HIF-1α) transcription. Materials and Methods: Aqueous and methanolic leaf extracts of both the plants were prepared through pressurized liquid extraction method. Various assays were performed to analyze phytochemical and antioxidant potential. The antiproliferative activity was determined through 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxy anilide salt (XTT) assay. Further, the effect of plant extracts on the modulation of HIF-1α and VEGF mRNA expression was analyzed through quantitative real-time polymerase chain reaction. Results: A significantly high total phenol content, total flavonoid content, free radical scavenging, and reducing power activities were observed in both the plant extracts. High-performance thin-layer chromatography analysis indicated the presence of ascorbic acid, quercetin, gallic acid, and hesperidin in different extracts. XTT reduction assay confirmed the antiproliferative activities of aqueous extracts against cancer cell line, HeLa. Aqueous extract of R. arboreum (RAA), at a concentration of 31.25 μg/ml, inhibited 60.12% and 25.41% proliferation of HeLa and Vero cell lines, respectively. An equal concentration of aqueous extract of R. campanulatum (RCA) inhibited the growth by 48.04% and 15.17% for HeLa and Vero cell lines, respectively. Further, the RAA and RCA have downregulated the expression of two key angiogenic factors responsible for tumor neovascularization, HIF-1α and VEGF to a great extent. Conclusion: The present study affirms that the leaf extracts of R. arboreum and R. campanulatum have enormous potential to be developed as an effective natural antioxidant and anticancer drug. Abbreviations used: PLE: Pressurized liquid extraction; XTT: 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxy anilide salt; TPC: Total phenol content; TFC: Total flavonoid content.

Expression levels of vascular endothelial cell growth factor and microRNA-210 are increased in medulloblastoma and metastatic medulloblastoma.

The present study aimed to investigate the roles of the vascular endothelial cell growth factor (VEGF) and micro (mi)RNA-210 in the metastasis of primary medulloblastoma (MB) tumors. A total of 86 adult patients diagnosed with cerebellar MB were enrolled in the present study, of which 11 patients had metastatic MB in the subarachnoid space. The following samples were collected: MB primary tumor tissue, MB secondary tumor tissue, tumor adjacent tissues and cerebrospinal fluid (CSF). Immunohistochemical analyses of the tissue samples were conducted in order to detect patterns of VEGF expression. In addition, the expression levels of VEGF mRNA and miRNA-210 were analyzed using reverse transcription-quantititative polymerase chain reaction, and western blot analyses were used to investigate VEGF protein expression levels. The positive expression rate of VEGF was significantly higher in MB tumor tissue, as compared with adjacent tissues (P<0.01). In addition, VEGF mRNA and protein expression levels in MB primary and secondary tumor tissues, and in the CSF of patients with metastatic MB, were significantly upregulated, as compared with tumor adjacent tissues and the CSF of patients with non-metastatic MB, respectively (P<0.01). miRNA-210 expression levels were significantly upregulated in MB tumor tissues, the CSF of patients with metastatic MB and in tumor tissues of metastatic MB (P<0.01). In the present study, the expression levels of VEGF and miRNA-210 were upregulated in patients with MB and metastatic MB; thus suggesting that miRNA-210 may promote the metastasis of MB primary tumors by regulating the expression of VEGF.

HGF regulates VEGF expression via the c-Met receptor downstream pathways, PI3K/Akt, MAPK and STAT3, in CT26 murine cells

In the present study, we assessed the involvement of hepatocyte growth factor (HGF)/c-Met signalling with vascular endothelial cell growth factor (VEGF) and hypoxia inducible factor (HIF)-1α expression in the downstream pathways phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) in CT26 cells, to determine the mechanisms of the potent anti-angiogenic effect of NK4. We established genetically modified CT26 cells to produce NK4 (CT26-NK4). VEGF expression in subcutaneous CT26 tumours in vivo and in culture supernatants in vitro was determined by ELISA. HIF-1α expression in nuclear extracts was evaluated by western blot analysis. VEGF and HIF-1α mRNA levels were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR). The DNA binding activity of HIF-1α was evaluated using an HIF-1α transcription factor assay kit. Our results demonstrated that VEGF expression was reduced in homografts of CT26-NK4 cells, compared to those of the control cells. In vitro, VEGF expression, which was induced by HGF, was inhibited by anti-HGF antibody, NK4 and by kinase inhibitors (PI3K, LY294002; MAPK, PD98059; and STAT3, Stattic). HGF‑induced HIF‑1α transcriptional activity was also inhibited by the kinase inhibitors. Real-time RT-PCR demonstrated that HGF‑induced HIF‑1α mRNA expression was not inhibited by LY294002 and PD98059, but was inhibited by Stattic. These data suggest that the PI3K/Akt, MAPK and STAT3 pathways, downstream of HGF/c‑Met signalling, are involved in the regulation of VEGF expression in CT26 cells. HGF/c‑Met signalling may be a promising target for anti-angiogenic strategies.

Sphingosine 1-phosphate induces vesicular endothelial growth factor expression in endothelial cells

Angiogenesis is essential for tumor growth and vascular endothelial cell growth factor (VEGF) plays a key role in this process. Conversely, sphingosine 1-phosphate (S1P) is a biologically active sphingolipid known to play a key role in cancer progression by regulating endothelial cell proliferation and migration. In this study, the authors found that S1P increases the level of VEGF mRNA in human umbilical vein endothelial cells (HUVECs) and immortalized HUVECs (iHUVECs). Additionally, S1P was found to increase VEGF promoter activity in MS-1 mouse pancreatic islet endothelial cells. Furthermore, a pharmacological inhibitory study revealed that G(alpha i/o)-mediated phospholipase C, Akt, Erk, and p38 MAPK signaling are involved in this S1P-induced expression of VEGF. A component of AP1 transcription factor is important for S1P-induced VEGF expression. Taken together, these findings suggest that S1P enhances endothelial cell proliferation and migration by upregulating the expression of VEGF mRNA.

Insulin-like growth factor binding protein-7 (IGFBP7) blocks vascular endothelial cell growth factor (VEGF)-induced angiogenesis in human vascular endothelial cells

Insulin-like growth factor binding protein-7 (IGFBP7) and vascular endothelial growth factor (VEGF) are expressed in vascular endothelial cells in several tumor types. In this study, we examined the effect of IGFBP7 on VEGF-induced tube formation in cultured human umbilical vein endothelial cells (HUVECs) and its potential action in the modulation of VEGF signaling in vascular cells. IGFBP7 treatment suppressed VEGF-induced tube formation, proliferation, and the phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) 1/2 in HUVECs. IGFBP7 attenuated VEGF-enhanced cyclooxygenase (COX)-2 and VEGF mRNA expression, and prostaglandin E 2 secretion. Knocking down endogenous IGFBP7 enhanced COX-2 and VEGF mRNA expression. A significant increase in IGFBP7-induced caspases was not observed in the presence of VEGF. These findings indicate that IGFBP7 can modulate the stimulatory effect of VEGF on angiogenesis by interfering with VEGF expression as well as VEGF signaling and not by inducing apoptosis.

The Dipeptide Arg-Gln Inhibits Retinal Neovascularization in the Mouse Model of Oxygen-Induced Retinopathy

Premature infants undergoing intensive care are highly vulnerable to amino acid deprivation. Supplementation of glutamine or arginine has resulted in beneficial effects in human neonates. This study was conducted to examine the effect of the dipeptide arginyl-glutamine (Arg-Gln) on vascular endothelial cell growth factor (VEGF) levels in primary human retinal pigment epithelial (hRPE) cell cultures and on inhibition of neovascularization in the oxygen-induced retinopathy (OIR) model. The effects of Arg-Gln on VEGF levels were measured in supernates from hRPE cells by using ELISAs. For in vivo studies, mouse pups received twice-daily intraperitoneal injections of Arg-Gln, a control dipeptide (Ala-Gly) or were not injected. Retinal flatmounts from one cohort were prepared and retinal vessel morphology examined. The contralateral eyes were embedded, sectioned, and stained to count preretinal neovascular nuclei. RNA was isolated from retinas of selected animals and was used to quantify VEGF mRNA by real-time RT-PCR. Treatment of hRPE cells with Arg-Gln decreased VEGF levels in a dose-dependent manner. In the OIR model, Arg-Gln at 5 g/kg per day reduced preretinal neovascularization by 82%+/-7% (P<0.005), when compared with the control dipeptide Ala-Gly, and reduced VEGF mRNA by 64%+/-9% (P<0.001). Arg-Gln dramatically inhibited retinal neovascularization in the OIR model. This effect was associated with a reduction in retinal VEGF mRNA levels. Similarly the dipeptide reduced VEGF expression in hRPE cells, a cell type likely to respond to retinal hypoxia by expressing VEGF. Arg-Gln appears to be safe and, with future studies in human infants, may prove beneficial in the prevention of ROP.

PACAP-27 tyrosine phosphorylates mitogen activated protein kinase and increases VEGF mRNAs in human lung cancer cells.

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) on human lung cancer cell line NCI-1299 mitogen activated protein kinase (MAPK) tyrosine phosphorylation and vascular endothelial cell growth factor (VEGF) expression were investigated. PACAP-27 (100 nM) increased MAPK tyrosine phosphorylation 3-fold, 5 min after addition to NCI-H1299 cells. PACAP caused tyrosine phosphorylation in a concentration-dependent manner being half-maximal at 10 nM PACAP-27. PACAP-27 or PACAP-38 (100 nM) but not PACAP28-38 or VIP caused increased MAPK tyrosine phosphorylation using NCI-H1299 cells. Also, the increase in MAPK tyrosine phosphorylation caused by PACAP-27 was totally inhibited by 10 microM PACAP(6-38), a PAC(1) receptor antagonist or 10 microM PD98059, a MAPKK inhibitor. These results suggest that PAC(1) receptors regulate tyrosine phosphorylation of MAPK in a MAPKK-dependent manner. PACAP-27 (100 nM) caused increased VEGF mRNA in NCI-H1299 cells after 8 h. The increase in VEGF mRNA caused by PACAP-27 was partially inhibited by PACAP(6-38), PD98059 and H-89. Addition of VIP to NCI-H1299 cells caused increased VEGF mRNA, which was totally inhibited by H89, a PKA inhibitor. These results suggest that PAC(1) and VPAC(1) receptors regulate VEGF expression in lung cancer cells.

Capsaicin regulates vascular endothelial cell growth factor expression by modulation of hypoxia inducing factor-1alpha in human malignant melanoma cells.

Capsaicin (8-Methyl-N-Vanillyl-6-nonenamide), a known natural dietary chemopreventive agent, inhibits malignant melanoma cell proliferation. In the present study, we examined the effect of capsaicin on constitutive and induced vascular endothelial cell growth factor (VEGF) expression in malignant melanoma cells. Capsaicin treatment resulted in enhanced VEGF protein secretion in malignant melanoma cells independent of IL-1beta and TNF-alpha. The observed up-regulation of VEGF production by capsaicin was concentration- and duration-dependent and was inversely associated with inhibition of melanoma cell proliferation. We observed an increase in hypoxia-inducible factor (HIF)-1alpha and VEGF mRNA expression in capsaicin-treated melanoma cells. Further, an electrophoretic mobility shift assay (EMSA) from nuclear extracts from melanoma cells showed a decrease in constitutive activation of NF-kappaB and enhanced HIF-1alpha binding activity to hypoxia response element (HRE) in melanoma cells treated with different concentrations of capsaicin. These data suggest that inhibition of cellular proliferation by capsaicin follows enhanced VEGF production by remaining melanoma cells by enhancing HIF-1alpha expression and binding to HRE.

Aberrant p53 Expression Correlates With Expression of Vascular Endothelial Growth Factor mRNA and Interleukin-8 mRNA and Neoangiogenesis in Non–Small-Cell Lung Cancer

To evaluate interactions between expressions of tumor suppressor gene p53 and angiogenic factors vascular endothelial cell growth factor (VEGF) and interleukin-8 (IL-8) and their effect on tumor angiogenesis and patient prognosis in non--small-cell lung cancer (NSCLC). p53, VEGF, IL-8, and the microvessel endothelium were immunostained, and VEGF and IL-8 mRNA expression were quantified using the real-time quantitative reverse-transcription polymerase chain reaction in 65 NSCLC surgical specimens. Aberrant p53 expression was correlated with VEGF and IL-8 mRNA expression, microvessel count (MVC), other clinical-pathologic variables, and patients' survival. Tumors with high aberrant p53 expression showed significantly higher VEGF and IL-8 mRNA expression and MVC than those with low aberrant p53 expression (P <.001). When tested as a continuous variable, aberrant p53 expression correlated strongly and positively with VEGF and IL-8 mRNA expression and MVC (P <.0001). Tumors with high aberrant p53 expression were associated with mediastinal or distant lymph node metastasis (P =.006). Survival and postoperative relapse time were significantly shorter in patients with high aberrant p53 expression tumors than in those with low aberrant expression tumors (P <.0001). A significant difference in survival was also seen between patients with high and low tumoral VEGF mRNA expression and between those with high and low tumoral IL-8 mRNA expression (P <.0001). We report here for the first time that aberrant p53 expression is strongly positively correlated with VEGF mRNA and IL-8 mRNA expression in NSCLC. This result indicates that aberrant p53 expression may play a significant role in regulation of VEGF and IL-8 expression and be involved in controlling angiogenesis and explains the adverse prognosis of cancers with high aberrant p53 expression.

Aberrant p53 Expression Correlates With Expression of Vascular Endothelial Growth Factor mRNA and Interleukin-8 mRNA and Neoangiogenesis in Non-Small-Cell Lung Cancer

PURPOSE: To evaluate interactions between expressions of tumor suppressor gene p53 and angiogenic factors vascular endothelial cell growth factor (VEGF) and interleukin-8 (IL-8) and their effect on tumor angiogenesis and patient prognosis in non–small-cell lung cancer (NSCLC). PATIENTS AND METHODS: p53, VEGF, IL-8, and the microvessel endothelium were immunostained, and VEGF and IL-8 mRNA expression were quantified using the real-time quantitative reverse-transcription polymerase chain reaction in 65 NSCLC surgical specimens. Aberrant p53 expression was correlated with VEGF and IL-8 mRNA expression, microvessel count (MVC), other clinical-pathologic variables, and patients’ survival. RESULTS: Tumors with high aberrant p53 expression showed significantly higher VEGF and IL-8 mRNA expression and MVC than those with low aberrant p53 expression (P < .001). When tested as a continuous variable, aberrant p53 expression correlated strongly and positively with VEGF and IL-8 mRNA expression and MVC (P < .0001). Tu...

Control of VEGF expression by progestins

A recently published report suggests that, in human breast cancer cells, the expression of vascular endothelial cell growth factor (VEGF) (one of the most potent angiogenesis factors currently known) is stimulated by the presence of progestins. Salman M. Hyder et al. (University of Texas Health Science Center, Houston, TX, USA), writing in the International Journal of Cancer (15th May), suggest that anti-progestins might play a role in preventing tumor progression in patients with breast cancer.

Vascular endothelial growth factor secreted by replicating hepatocytes induces sinusoidal endothelial cell proliferation during regeneration after partial hepatectomy in rats

Background : The aim of this study was to investigate regulatory mechanisms of sinusoidal endothelial cell (SEC) proliferation after hepatectomy in rats. Methods : We investigated expressions of vascular endothelial cell growth factor (VEGF) and its receptors, flt-1 and KDR/flk-1, in regenerating liver after 70% hepatectomy. Proliferation of both hepatocytes and SECs was also monitored by evaluating the proliferating cell nuclear antigen (PCNA) labeling index. Furthermore, VEGF production by cultured hepatocytes isolated at different times after hepatectomy was measured in vitro. Results : The expression of VEGF mRNA was increased markedly between 48 and 72 h after hepatectomy, and thereafter decreasing gradually. The immunohistochemical staining revealed that expression of VEGF started to increase 24 h after hepatectomy, with a peak at 72 h, and the majority of the VEGF-positive cells were hepatocytes located in periportal areas. Meanwhile, expression of flt-1 and KDR/flk-1 was observed along the sinusoids even before hepatectomy, but was increased between 72 and 120 h. Furthermore, VEGF production by cultured hepatocytes isolated 72 h after hepatectomy was significantly increased. The PCNA labeling index of the SECs exhibited a delayed and slower regenerative response in comparison to the hepatocytes, reaching a peak at 72 h. Conclusions : These data strongly suggest that VEGF secreted by proliferating hepatocytes may represent an important stimulator of SEC proliferation.

Prostaglandin E2 and vasoactive intestinal peptide increase vascular endothelial cell growth factor mRNAs in lung cancer cells

The effects of prostaglandin E2 (PGE2) and vasoactive intestinal peptide (VIP) on vascular endothelial cell growth factor (VEGF) mRNAs were investigated using lung cancer cells. By RT-PCR, VEGF121, VEGF165, and VEGF189, but not VEGF206 isoforms were detected in all lung cancer cell lines and biopsy specimens examined. By Northern blot, VEGF mRNA was detected in all small cell lung cancer (SCLC) and non-SCLC (NSCLC) cell lines examined. PGE2, VIP and forskolin caused increased VEGF expression in a time- and concentration-dependent manner using NSCLC cell line NCI-H157. Approximately 1 μM PGE2, 0.1 μM VIP and 50 μM forskolin caused cAMP elevation, 64-, 33- and 128-fold, respectively, using NCI-H157 cells after 5 min. The increase in cAMP caused by PGE2 and VIP was reversed by somatostatin (SST). Also 1 μM PGE2, 0.1 μM VIP and 50 μM forskolin increased the VEGF mRNA 2.0-, 1.5- and 2.3-fold, respectively, after 4 h. The increase in VEGF mRNA caused by PGE2, VIP and forskolin was inhibited by H-89, a protein kinase A inhibitor. A VIP receptor antagonist, VIPhybrid, inhibited the increase in cAMP and VEGF mRNA caused by VIP. By ELISA, VEGF was detected in the conditioned media exposed to the lung cancer cell lines. These results suggest that VEGF synthesis in and secretion from lung cancer cells can be regulated by agents, which cause adenylyl cyclase activation.

Induction of VEGF Gene Transcription by IL-1 β is Mediated Through Stress-activated MAP Kinases and Sp1 Sites in Cardiac Myocytes

Interleukin-1 β (IL-1 β) is a multipotent cytokine participating in a variety of cardiovascular diseases. In this study, we examined the effects of IL-1 β on the expression of vascular endothelial cell growth factor (VEGF) and pursued the molecular mechanisms underlying this effect. Treatment of cultured neonatal rat cardiac myocytes with IL-1 β increased the levels of VEGF mRNA in a time- and a concentration-dependent manner. These effects were completely abolished by SB203580 and SB202190 (p38 MAPK inhibitors) but not by PD98059 (MEK1 inhibitor), calphostin C (protein kinase C inhibitor), or genistein (tyrosine kinase inhibitor). While IL-1 β phosphorylated c-Jun N-terminus protein kinase (JNK) rapidly and transiently, the effect of IL-1 β on p38 mitogen-activated protein kinase (MAPK) was gradual and persistent. Transient transfection assays showed that IL-1 β increases the transcription from the VEGF promoter. A series of 5-deletion and site-specific mutation analyses indicated that IL-1 β as well as overexpression of p38 MAPK and JNK activate VEGF promoter activity through two G+C-rich sequences located at -73 and -62. Electrophoretic mobility shift and supershift assays showed Sp1 and Sp3 proteins specifically bind to the G+C-rich sequences. The half-life of VEGF mRNA was significantly increased in cells treated with IL-1 β. Together, these results indicate that IL-1β induces VEGF gene expression at both transcriptional and post-transcriptional levels, and IL-1 β evokes p38 MAPK and JNK signalings, which in turn stimulate the transcription of the VEGF gene through Sp1-binding sites. These findings suggest the role of IL-1 β as a cytokine inducing VEGF in cardiac myocytes, and imply that activation of stress-activated MAP kinases regulate Sp1 sites-dependent transcription.

Sequential development of an angiogenic phenotype by human fibroblasts progressing to tumorigenicity.

As normal cells progress to malignancy they must acquire an angiogenic phenotype that will enable them to attract the blood vessels necessary to support their progressive growth. Here we define the mechanism by which human fibroblasts cultured from Li Fraumeni patients and progressing to tumorigenicity in vitro become angiogenic. Initially cells were anti-angiogenic due to the secretion of high levels of inhibitory thrombospondin that overrode the modest amounts of the major inducer, vascular endothelial cell growth factor (VEGF), that were also produced. Cells became fully angiogenic in two steps, the first dependent on the loss of both alleles of wild-type p53 which caused a drop of at least 20-fold in secreted thrombospondin and a fourfold increase in secreted VEGF. Angiogenic activity increased again upon transformation by activated ras due to a further twofold increase in VEGF. Changes in relative levels of VEGF mRNA were sufficient to account for changes in secreted protein levels and in overall angiogenic activity. These studies demonstrate that an angiogenic phenotype able to support tumorigenicity can arise in a step-wise fashion in response to both oncogene activation and tumor suppressor gene loss and involve both a decrease in the secretion of inhibitors and the sequential ratcheting up of the secretion of inducers of angiogenesis.

Expression of mRNA for vascular endothelial growth factor in human placenta

Implantation and growth of the placenta requires extensive angiogenesis to establish the vascular structures involved in exchange. Failure to establish adequate blood supply to the fetus may have serious clinical consequences such as intrauterine growth retardation. Vascular endothelial cell growth factor (VEGF) is a recently identified growth factor with significant angiogenic properties. We have demonstrated the presence of four species of mRNA encoding VEGF in both first trimester and term placenta. In situ hybridization was used to localize the sites of expression of VEGF mRNA in these tissues. VEGF expression was seen in villous trophoblast in the first trimester and in extravillous trophoblast at term, and in both fetal macrophages within the villi and maternal macrophages in the decidua. Glandular epithelium in maternal decidua also expressed VEGF mRNA. The strongest site of expression was in maternal macrophages adjacent to Nitabuch's stria, a zone of necrosis at the site of implantation. This complex pattern of expression suggests that VEGF is involved in angiogenesis on both maternal and fetal sides of the placenta and that macrophages are the primary source of VEGF. However, VEGF may also play a role in term placenta, when extensive angiogenesis has diminished, possibly regulating vascular permeability.

Alterations of growth factor transcripts in rat lungs during development of monocrotaline-induced pulmonary hypertension

Although pathologic and hemodynamic changes in monocrotaline (MCT)-induced pulmonary hypertension have been studied extensively, relatively little is known about the inter- and intracellular signaling mechanisms underlying such alterations. As a first step to delineating signaling mechanisms governing adverse structural alterations in the hypertensive lungs, we examined changes in the steady-state levels of mRNAs encoding several growth factors including transforming growth factors (TGF), platelet-derived growth factors (PDGF), vascular endothelial cell growth factor (VEGF) and endothelin (ET) as a function of time in MCT-induced pulmonary hypertension in rats. These studies demonstrated a very diverse pattern of growth factor gene expression in response to MCT administration. In general, alterations in the steady-state levels of mRNAs encoding the growth factors preceded the onset of MCT-induced pulmonary hypertension. TGF-β1, -β2 and -β3 transcripts were seen to be elevated, whereas that of TGF-α and PDGF-A remained unchanged. Transcripts for PDGF-B and ET were increased in the early stages but declined to less than controls in the latter stages of MCT-induced hypertension. In contrast, levels of VEGF mRNA decreased to less than controls as the disease progressed. Viewed collectively, the diverse pattern of expression suggests that alterations in the levels of the growth factor transcripts may have a significant role in the development of pulmonary hypertensive disease and may be relevant to the pathological and structural changes in MCT-induced pulmonary hypertension.