Vascular Endothelial Growth Factor Receptor-2 mRNA Research Articles

SiRNA-mediated silencing of VEGFR-3 reduces proliferation, adhesion and migration of colorectal cancer cells

AIM: To investigate the effect of transfection with an adenovirus vector expressing short interfering RNA (siRNA) targeting vascular endothelial growth factor receptor-3 (VEGFR-3) on cell proliferation, adhesion and migration in colorectal cancer cell line LoVo. METHODS: An adenovirus vector expressing siRNA targeting VEGFR-3 was constructed and transfected into LoVo cells. The expression of VEGFR-3 was detected by RT-PCR and Western blot. Cell proliferation and migration were detected by MTT assay and Transwell assay, respectively. RESULTS: The expression of VEGFR-3 mRNA and protein was significantly decreased after transfection with the recombinant adenovirus (P < 0.05), compared with the blank control group and negative control group. The proliferation, adhesion and migration of LoVo cells were significantly decreased after transfection with the recombinant adenovirus (P < 0.05). CONCLUSION: SiRNA-mediated silencing of VEGFR-3 inhibits the proliferation, adhesion and migration of LoVo cells. © 2014 Baishideng Publishing Group Inc. All rights reserved.

Abstract 526: Angiogenesis and Kallikrein-kinin System Activation after Exercise Training in Rats with Sustained Adrenergic Hyperactivity

Several mechanisms are involved to exercise-related cardioprotection. Purpose Kallikrein-kinin system may contribute to this effect inducing endothelial vascular growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) expression. Methods In a model of β-adrenergic hyperactivity, male Wistar rats were assigned into four groups: sedentary non-treated (Con); sedentary isoproterenol-treated (Iso); exercised only (Ex); and exercised isoproterenol-treated (Iso+Ex). Left ventricles were processed for Real time PCR and Western blot analysis in order to quantify the expression of kinins and angiogenic molecules. Results In Iso+Ex, a significant increase of tissue kallikrein gene and protein expression was observed (p&lt;0.01). In the same group, an augmented eNOS (p&lt;0.05), VEGF and VEGF receptor mRNA expression (p&lt;0.01) was observed. The expression of angiogenic IL-1 and Il-8 were increased after exercise (p&lt;0.01). Expression of integrin linked kinase (ILK), another angiogenesis promoter, also augmented after training (p&lt;0.01). Conclusion Our results represent the first demonstration that exercise increases the tissue kallikrein gene and protein expression in rat myocardium after β-adrenergic hyperactivity. Together with the observed augmented eNOS and VEGF and angiogenesis-related molecules mRNA expression, a feasibly pathway might be drawn to understand the kallikrein-kinin system protective role to avoid the abnormalities developed due excessive catecholaminergic activation as observed in cardiac failure. Our data suggest a strong evidence of beneficial exercise action, particularly related to angiogenesis, in response of myocardium towards β-adrenergic hyperactivity.

Tumor Angiogenesis after Heated Lipiodol Infusion via the Hepatic Artery in a Rabbit Model of VX2 Liver Cancer

ObjectivesThis study aimed to observe the changes in tumor angiogenesis after heated lipiodol (60°C) infusion via the hepatic artery in a rabbit model of VX2 liver cancer.Materials and MethodsTwenty rabbits with VX2 hepatic tumors were randomly divided into 2 groups (10 rabbits in each group). Under anesthesia, a trans-catheter hepatic arterial infusion was performed, and lipiodol (37°C; control group) or heated lipiodol (60°C; treated group) was injected into the hepatic arteries of the animals. Then, changes in tumor angiogenesis were assessed using the following markers and methods. 1. Vascular endothelial growth factor receptor (VEGFR) and vascular endothelial growth factor (VEGF) expression levels in the tumor were assessed using western blotting and real-time quantitative polymerase chain reaction (PCR). 2. Proliferating cell nuclear antigen (PCNA) expression in the tumor was assessed through immunohistochemical staining. 3. The morphological changes in tumor vascular endothelial cells were observed using transmission electron microscopy (TEM).ResultsVEGFR and VEGF mRNA and protein expression levels were reduced in the treated group compared to the control group. PCNA protein showed reduced expression levels in the treated group compared to the control group. TEM indicated that the endothelial cell endoplasmic reticulum expanded, the chondriosome was swollen, and the endothelial cell microvilli were decreased after heated lipiodol infusion.ConclusionsThe tumor angiogenesis of rabbits with VX2 cancer was inhibited after arterial heated lipiodol infusion compared to lipiodol infusion.

Abstract 5254: Cedarinib inhibits VEGF-stimulated proliferation in NSCLC cell lines.

Abstract Background: Vascular endothelial growth factor (VEGF) is a key stimulator of physiological and pathological angiogenesis. VEGF signals primarily through VEGF receptor 2 (VEGFR2), a receptor tyrosine kinase whose expression is generally considered to be restricted to endothelial cells. Recently, tumor cell VEGFR2 expression has been reported in several tumor settings, including non-small cell lung cancer (NSCLC). The purpose of this study was to determine the role of VEGFR2 in NSCLC cells, and to investigate the impact of inhibiting VEGFR2 signaling. Methods: NSCLC tissue sections (n=52) were stained for VEGFR2 and CD31 expression by immunohistochemistry (IHC). Total and phosphorylated VEGFR2, AKT and MAPK protein levels in human NSCLC cell lines (n=8) were determined by Western blotting. VEGFR2 mRNA levels were measured by RT-PCR. For cell proliferation assays, cells were seeded (4x10ˆ3 per well) and 24 h later treated with VEGF (0-100 ng/mL) +/- cediranib (a potent and selective inhibitor of VEGF signaling). Alternatively, cells were treated with VEGFR2 siRNA 24 h post plating (4x10ˆ3 per well) and then treated with VEGF (0-100 ng/mL). Cell growth was quantified 5 d after VEGF treatment using crystal violet staining. For cytotoxicity assays, cells (2x10ˆ4 per well) were plated and 24 h later treated with VEGF, chemotherapy agents or cedarinib and incubated for 48 h. For radiation, cells were exposed to 137Cs (10 Gy, 1.958 Gy/min) 4 h after VEGF treatment. 48 h after VEGF treatment Hoechst 33258 (25 μM, 30 min) was added and apoptotic cells quantified by fluorescence (INCELL Analyzer 1000). Results: IHC analysis of tissue sections demonstrated VEGFR2 expression in both adenocarcinoma and squamous cell carcinoma, and in endothelial cells. Western blot analysis showed expression of VEGFR2 protein in 3/8 NSCLC cell lines that correlated with VEGFR2 mRNA expression levels. VEGF-dependent VEGFR2 activation (increased pVEGFR2 and pMAPK but not pAKT) was apparent in NSCLC cells, and was associated with increased proliferation (1.4 - 1.9 fold, p&amp;lt;0.05) in 2/3 of the VEGFR2-expressing NSCLC cell lines. Cedarinib inhibited both VEGF-dependent increases in cell proliferation and VEGFR2 downstream signaling. Knock down of VEGFR2 protein expression using siRNA also reduced VEGF-stimulated NSCLC cell proliferation. Inhibition of VEGFR2 tyrosine kinase activity using cediranib was more effective than inhibition of AKT (MK2206) or MEK (AZD6244) for overcoming VEGFR2-driven cell proliferation. VEGF treatment did not affect cell survival following treatment with radiation, cisplatin, docetaxel or gemcitabine. Conclusion: Our data suggest that a subset of NSCLC patients may express functional tumor cell VEGFR2 which can act to promote VEGF-dependent tumor cell growth. In this patient subset, therapies targeting VEGFR2 signaling, such as cedarinib, may have the potential to inhibit both tumor cell proliferation and angiogenesis. Citation Format: Aoife M. Devery, Rekha Wadeka, Sivan M. Bokobza, Anika M. Weber, Yanyan Jiang, Anderson J. Ryan. Cedarinib inhibits VEGF-stimulated proliferation in NSCLC cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5254. doi:10.1158/1538-7445.AM2013-5254

Vascular Endothelial Growth Factor Receptor Family in Ascidians, Halocynthia roretzi (Sea Squirt). Its High Expression in Circulatory System-Containing Tissues

The vascular endothelial growth factor (VEGF)-VEGF Receptor (VEGFR) system is an important pathway for regulation of angiogenesis. However, its evolutionary development, particularly the step from invertebrates to vertebrates, is still largely unknown. Here, we molecularly cloned the VEGFR-like gene from Halocynthia roretzi, a species belonging to the Tunicata, the chordate subphylum recently considered the sister group of vertebrates. The cDNA encoded a homolog of human VEGFR, including the transmembrane domain, and the tyrosine kinase domain with a kinase-insert region, which was designated S. sq VEGFR (GenBank AB374180). Similar to Tunicates including ascidians in the phylogenetic tree, the Amphioxus, another chordate, is located close to vertebrates. However, S. sq VEGFR has a higher homology than the Amphioxus VEGFR-like molecule (GenBank AB025557) to human VEGFR in the kinase domain-2 region. The S. sq VEGFR mRNA was expressed at highest levels in circulatory system-containing tissues, suggesting that S. sq VEGFR plays an important role in the formation or maintenance of circulatory system in Tunicates, Halocynthia roretzi.

Effect of minocycline for expression of vascular endothelial growth factor receptor-1 and vascular endothelial growth factor receptor-2 in hypoxia chorioretinal endothelial cells of monkeys

Objective To observe the expression of vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 in hypoxic chorioretinal endothelial cells of monkeys (RF/6A),and to evaluate the effect of minocycline.Methods RF/6A was cultured and divided into four groups:control group,hypoxia group,hypoxia and low dose of minocycline group (0.5 μmol/L),hypoxia and medium dose of minocycline group (5 μmol/L),and hypoxia and high dose of minocycline group (50 μmol/L).Real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistopathological staining were used to measure the mRNA and protein expression of VEGFR-1 and VEGFR-2,respectively.Results RT-PCR showed that the expression of VEGFR-1 mRNA did not vary significantly between groups (F24 h =0.17,F48 h =1.53,F72 h =2.04; P＞0.05).Compared with hypoxia group,the expression of VEGFR-2 mRNA in all minocycline treated groups were significantly down-regulated (low minocycline,medium minocycline,high minocycline:t=4.69,20.16,17.12; P＜0.001).The immunohistopathological study showed the cells with positive staining of VEGFR-1 can be observed in all groups,and the staining was relatively weak and mainly located in cell membrane and cytoplasm.The optical density value analysis showed that the protein expression of VEGFR-1 did not vary significantly between groups at all time points(F24 h =0.251,F48 h=0.340,F72 h =0.589; P＞0.05).The VEGFR-2 positive staining cells were also observed in all groups,and the staining was relatively high.Brown staining particles of VEGFR-2 were observed in the cell membrane with minor staining particles in cytoplasm.The staining density of VEGFR-2 was significantly higher in hypoxia group than control group.Compared with the hypoxia group,the protein expression of VEGFR 2 in minocycline treated groups was significantly lower (F24 h =19.147,F48 h =14.893,F72 h =11.984; P＜0.05).Conclusion The expression of VEGFR-2 is up-regulated in RF/6A,and minocycline somewhat shows an inhibition effect. Key words: Minocycline; Endothelial cells/pathophysiology; Vascular endothelial growth factor receptor-l; Vascular endothelial growth factor receptor-2; Cell hypoxia

Mechanisms of apple polyphenols-induced proliferation inhibiting and apoptosis in a metastatic oral adenoid cystic carcinoma cell line

Adenoid cystic carcinoma (ACC) is characterized by intensive local invasion and high incidence of distant metastases. Conventional chemotherapy for ACC produces a poor result. We aimed to evaluate the effect of apple polyphenols (APs), a novel nutraceutical agent, on the proliferation and apoptosis levels in a metastatic oral ACC cell line. A metastatic ACC (ACC-M) cell line and control cells (MRC-5 cells derived from normal lung tissue) were treated with APs at different concentrations. MTT assay was used to determine the in vitro cytotoxicity. The cell cycle distribution and apoptosis levels were measured by flow cytometry. To evaluate the mechanism of APs, vascular endothelial growth factor receptor-2 (VEGFR-2) and caspase-3 messenger ribonucleic acid (mRNA) and protein levels were evaluated by reverse transcription-polymerase chain reaction and Western blots, respectively. After cells were cultured for 24 hours or 48 hours, the critical concentration of cytotoxicity of APs in MRC-5 cells was found to be 250 μg/mL. In contrast, in the concentration range of 100–250 μg/mL, the cytotoxicity of APs in ACC-M cells was time- and dose-dependent: ACC-M cell proliferation declined at 100 μg/mL when cultured for 48 hours, whereas growth was not inhibited at the concentrations of APs below 200 μg/mL when cultured for 24 hours. In selected time and dose patterns (ACC-M cells cultured at the concentrations of 150 and 250 μg/mL for 48 hours), the flow cytometry performance showed that apoptosis and necrosis occurred in APs-treated ACC-M cells. Also, in these patterns, VEGFR-2 mRNA and protein levels decreased whereas the levels of caspase-3 increased. In summary, APs could inhibit proliferation and induce apoptosis in ACC-M cells in vitro. These effects may be related to the downregulation of VEGFR-2 expression and the activation of caspase-3 expression.

Upregulation of Vascular Endothelial Growth Factor Receptor-3 in the Spinal Cord of Lewis Rats with Experimental Autoimmune Encephalomyelitis

We investigated the spatiotemporal expression of vascular endothelial growth factor receptor-3 (VEGFR-3) in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. VEGFR-3 mRNA and protein were constitutively expressed in gray matter neurons and in a few white matter astrocytes. Induction of VEGFR-3 occurred predominantly in perivascular infiltrated macrophages in the spinal cord white matter during the inductive phase of EAE. VEGFR-3 expression was also induced in activated microglial cells in the gray and white matter, mainly in the peak phase. In addition, reactive astrocytes in the white matter, but not in the gray matter, expressed VEGFR-3 as disease severity increased. These data suggest that VEGFR-3 is involved in the recruitment of monocytic macrophages and in glial reactions during EAE.

Expression of VEGF-receptors in TMJ synovium of rabbits with experimentally induced internal derangement

Our aim was to evaluate the expression of vascular endothelial growth factor receptors (VEGFRs) in the synovium of the temporomandibular joints (TMJ) of rabbits with experimentally induced internal derangement. Internal derangement was experimentally induced in 52 rabbit TMJ, and established on the right side of TMJ while the left side was used as the control. Each joint and its control was evaluated by magnetic resonance imaging (MRI) and endoscopy. The synovial tissues on both sides were harvested after one, two, three, and four weeks. The expression of VEGFRs mRNA was investigated in the experimental joint and its control using real-time polymerase chain reaction (PCR). Internal derangement was successfully confirmed in 45 of the 52 of the experimental joints (87%) on the right side by MRI and endoscopy. In the first and fourth week, the VEGFR-2 mRNA expression was higher in the experimental joints than in the controls (P=0.008 and P=0.02). Meanwhile, the VEGFR-1 mRNA expression was up-regulated in the experimental group compared with the controls during the fourth week (P=0.02). However, we found no significant differences in VEGFR-3 mRNA expression in the two groups during the first and fourth weeks. During the second and third weeks, the mRNA expression of the three receptors did not differ significantly among the groups. Our data have shown increased expression of VEGFR-1 and VEGFR-2 mRNA in the synovium of rabbit TMJ with internal derangement, which indicates that VEGFR-1 and VEGFR-2 may have important roles in the processes of internal derangement and formation of adhesions.

Expression of Hypoxia-Inducible Factors, Regulators, and Target Genes in Congenital Diaphragmatic Hernia Patients

Congenital diaphragmatic hernia (CDH) is associated with lung hypoplasia and pulmonary hypertension and has high morbidity and mortality rates. The cause and pathophysiology of CDH are not fully understood. However, impaired angiogenesis appears to play an important role in the pathophysiology of CDH. Therefore, we examined different components of an important pathway in angiogenesis: hypoxia-inducible factors (HIFs); HIF regulators von Hippel-Lindau (VHL) and prolyl 3-hydroxylase (PHD3); and HIF target genes vascular endothelial growth factor A ( VEGF-A ) and vascular endothelial growth factor receptor 2 ( VEGFR-2 ). Quantitative polymerase chain reaction of lung tissue showed a significantly decreased expression of VEGF-A mRNA in the alveolar stage of lung development in CDH patients compared with matched control patients. In the canalicular stage, no differences for VEGF-A were seen between the lungs of CDH patients and those of control patients. Other components of angiogenesis (VHL, HIF-1α, HIF-2α, HIF-3α, VEGFR-2 mRNA, PHD3 protein) that were analyzed showed no differences in expression between CDH and control patients, independent of the developmental stage. A lower expression of VEGF mRNA in CDH patients in the alveolar stage, possibly as a result of downregulation of HIF-2α might indicate a role for these factors in the pathophysiology of CDH.

Elevated microRNA-126 is associated with high vascular endothelial growth factor receptor 2 expression levels and high microvessel density in colorectal cancer

MicroRNAs (miRNAs) are involved in a number of biological processes, including tumour biology. Pre-clinical studies have shown that miRNA-126 regulates signalling downstream of vascular endothelial growth factor receptor 2 (VEGFR-2) and, consequently, angiogenesis. The aim of this study was to analyse the possible relationship between miRNA-126, VEGFR-2 and angiogenesis in tumour tissue from patients with colorectal cancer (CRC). Tumour tissue was obtained from 81 patients. The miRNA-126 and VEGFR-2 gene expression levels were analysed by PCR and the protein concentrations of VEGFR-2 were analysed by ELISA. Angiogenesis, visualised by the endothelial cell marker CD105 combined with caldesmon, was assessed by immunohistochemistry and the microvessel density (MVD) technique. In situ hybridisation was performed for miRNA-126. Tumours were classified as low or high miRNA-126-expressing using the median as the cut-off. The median gene expression levels of VEGFR-2 were significantly lower in the tumours expressing low levels of miRNA-126, 0.30 (95% CI, 0.24-0.36), compared to those expressing high levels of miRNA-126, 0.48 (95% CI, 0.28-0.60), p=0.02. A positive association was observed with VEGFR-2 protein concentrations, p=0.06. The median MVD was significantly lower in the tumours expressing low levels of miRNA-126, 5.8 (95% CI, 5.33-6.67), compared to those expressing high levels, 8.0 (95% CI, 6.33-9.00), p<0.01. miRNA-126 was detected in endothelial cells by in situ hybridisation analysis. These results suggest that high levels of miRNA-126 in CRC are associated with high VEGFR-2 mRNA and protein levels and a higher density of newly formed microvessels. However, further studies should be conducted to analyse the clinical value of miRNA-126 in CRC.

Effects of tumour necrosis factor-alpha on activity and nitric oxide synthase of endothelial progenitor cells from peripheral blood

The aim of this investigation was to determine whether tumour necrosis factor-alpha (TNF-α) has any effect on endothelial progenitor cells (EPCs). Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with tumour necrosis factor-α (final concentrations: 0, 10, 20, 50 and 100 mg/l) for 0, 6, 12, 24 and 48 h. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding, by direct fluorescence staining. EPC proliferation and migration were assayed using the MTT assay and modified Boyden chamber assay, respectively. EPC adhesion assay was performed by re-plating those cells on fibronectin-coated dishes, and adherent cells were counted. Tube formation activity was assayed using a tube formation kit. Levels of apoptosis were revealed using an annexin V apoptosis detection kit. Vascular endothelial growth factor Receptor-1 (VEGF-R1) and stromal derived factor-1 (SDF-1) mRNA, assessed by real-time RT-PCR inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were assayed by western blot analysis. Incubation of EPCs with tumour necrosis factor-α reduced EPC proliferation, migration, adhesion, tube formation capacity, iNOS and eNOS in concentration- and time-dependent manners. Tumour necrosis factor-α reduced proliferation, migration, adhesion and tube formation capacity of EPCs. TNF-α increased EPC apoptosis level, reduced VEGF-R1 and SDF-1 mRNA expression; tumour necrosis factor-α also reduced iNOS and eNOS in the EPCs.

Effects of polydeoxyribonucleotide on the histological damage and the altered spermatogenesis induced by testicular ischaemia and reperfusion in rats

The effects of polydeoxyribonucleotide (PDRN), an agonist of the A2A adenosine receptors which when activated positively influences sperm activity, were tested in an experimental testicular ischaemia/reperfusion injury model. Anaesthetized male Sprague-Dawley rats were subjected to testicular torsion-induced ischaemia, followed by reperfusion (TI/R). Immediately after detorsion, randomized animals, including SHAM, received intraperitoneal injections of: (i) vehicle (1 mL/kg 0.9% NaCl solution); (ii) PDRN (8 mg/kg); (iii) DMPX (3,7-dimethyl-1-propargilxanthine, 0.1 mg/kg); or (iv) PDRN (8 mg/kg) + DMPX (0.1 mg/kg). Animals were euthanized at 1, 7 and 30 days following reperfusion. Vascular endothelial growth factor (VEGF) expression is normally associated with adenosine A2A receptor stimulation. After treatment, VEGF mRNA/protein expression quantified by qPCR and Western blot, vascular endothelial growth factor receptor-1 (VEGFR1) and endothelial nitric oxide synthase (eNOS) mRNA measured by qPCR, VEGF and VEGFR1 assessed using immunohistochemical methods, histological staining and spermatogenic activity were all analysed. Testis ischaemia-reperfusion (TI/R) injury caused increases in VEGF mRNA and protein, VEGFR1 and eNOS mRNA, histological damage and reduced spermatogenic activity. Immunostaining showed a lower expression of VEGF in germinal epithelial cells and a strong expression of VEGFR1 in Leydig cells after TI/R. PDRN administration increased significantly VEGF message/protein, VEGFR1 and eNOS message, decreased histological damage and ameliorated spermatogenic activity. PDRN might be useful in the management of testicular torsion.

VEGF receptor mRNA expression by ACL fibroblasts is associated with functional healing of the ACL

Recent advances in the treatment of ACL ruptures employ platelet-rich plasma combined with collagen to modulate growth factor release from platelets to stimulate healing. Among the most notable of these growth factors is VEGF, which is a potent mitogen and stimulator of vascular growth and healing. However, the effect of such a growth factor on healing depends on the cellular ability to bind with its receptor. The purpose of this study was to test (1) whether the strength of a tissue-engineered ACL repair is associated with VEGF receptors' mRNA expression of ACL cells and (2) whether age influences this association. Nineteen female Yucatan pigs underwent enhanced ACL repair. Biomechanical testing was performed after 15weeks of healing. Messenger RNA of VEGF receptors 1 and 2 in ACL fibroblasts was assessed by RT-PCR. The ACL structural properties were regressed on receptor expression levels in a multivariate model including serum levels of VEGF, age, and weight as potential confounders. While maximum load and linear stiffness were independent of VEGF receptor expression, VEGF receptor 1 was associated with displacement (positively) and yield load (negatively). In a multivariate model of VEGF receptor expression and biomechanics, age was associated with maximum load and yield load. These findings suggest that high VEGF receptor expression, even more so at higher age, results in a more compliant scar, which in turn may lead to greater knee laxity and a compromised clinical result.

PPARδ agonists suppress angiogenesis in a VEGFR2-dependent manner

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that have a pleiotropic impact on the regulation of differentiation, cell growth, and the metabolism of lipids and glucose. PPARδ agonists display a variety of effects on pro- and anti-tumor processes, and seem to have pro-angiogenic activity at very low concentrations. We analyzed the influence of higher concentrations of PPARδ agonists on angiogenesis and its underlying mechanisms. We found that treatment with PPARδ agonists inhibited the formation of capillary-like structures and endothelial cell migration. Since signaling via the vascular endothelial growth factor receptor-2 (VEGFR2) pathway is critical for angiogenic responses during chronic inflammation and tumor development, we explored whether PPARδ agonist inhibition acted by diminishing VEGFR2 expression. PPARδ agonists inhibited endothelial VEGFR2 protein expression in a time- and concentration-dependent manner. In contrast, neither tie-2, neuropilin-1 nor VEGFR1 expression was significantly affected by PPARδ agonist treatment. We also demonstrated that PPARδ agonists significantly suppressed accumulation of VEGFR2 mRNA. Consistent with these results, promoter luciferase assays showed that the inhibitory effects of PPAR agonists occur through suppression of VEGFR2 promoter activity. Hence, VEGFR-2 expression may be a critical molecular target of PPAR δ agonists, which may be responsible for their anti-angiogenic effects. These results may help to define the optimal therapeutic doses of PPARδ agonists in prospective therapeutic applications.

Effect of mitomycin C on concentrations of vascular endothelial growth factor and its receptors in bladder cancer cells and in bladders of rats intravesically instilled with mitomycin C

• To examine, using in vitro and in vivo models, the largely unexamined effect of mitomycin C (MMC), an effective intravesical treatment for superficial bladder cancer and carcinoma in situ, on expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2), which mediates many of the angiogenic properties of VEGF. • To measure, as a positive control, concentrations of the inhibitor of apoptosis, survivin, as an assessment of MMC effectiveness. • To measure MMC-induced changes in proliferation in the presence and absence of VEGF-A small interfering RNA (siRNA). • After treatment with increasing MMC concentrations (5-200 µg/mL), we measured proliferation, as well as VEGF, survivin, VEGF receptor-1 (VEGFR-1) and VEGFR-2 concentrations in RT-4 and T-24 bladder cancer cells. • The effect of pre-treatment of VEGF siRNA and survivin siRNA on MMC-induced decreases in proliferation was measured. • Urinary VEGF concentrations and bladder and kidney concentrations of VEGF-A, VEGFR-1, VEGFR-2 and interleukin-6 (IL-6) mRNA were measured in rats intravesically instilled with saline or MMC (200 µg/mL). • Although MMC treatment inhibited cell proliferation and decreased survivin mRNA expression in T-24 and RT4 cells, MMC (12-50 µg/mL) increased VEGF-A mRNA and VEGFR-2 mRNA and protein expression. • Pre-treatment with VEGF-A siRNA or survivin siRNA before MMC treatment reduced proliferation more than MMC alone. • MMC-induced reductions in proliferation were reduced additively by pre-treatment with survivin siRNA, but were potentiated by pre-treatment with VEGF-A siRNA. • VEGFR-2 mRNA and protein concentrations and urinary VEGF concentrations were increased in bladders of rats instilled with MMC. • Intravesically instilled MMC increases urinary VEGF and bladder VEGFR-2 protein and mRNA in rats. • MMC increases VEGF mRNA and VEGFR-2 protein and mRNA concentrations in bladder cancer cells. Therefore, we speculate that MMC could increase the angiogenic potential of both cancer and normal cells. • In cancer cells this effect is largest at lower MMC concentrations. • Combining MMC with agents that reduce EGF concentrations could be of value in treatment of transitional cell carcinoma of the bladder (TCC).

Effects of Radix Ginseng and Radix Notoginseng formula on expressions of vascular endothelial growth factor receptor-2 and hypoxia-inducible factor-1α in ischemic myocardium of rats with acute myocardial infarction

To investigate the effects of Radix Ginseng and Radix Notoginseng formula on expressions of vascular endothelial growth factor receptor-2 (VEGFR-2) and hypoxia-inducible factor-1alpha (HIF-1alpha) in ischemic myocardium of rats with acute myocardial infarction. A total of 100 Wistar rats were randomly divided into normal control group, sham-operated group, untreated group, metoprolol (Betaloc) group, and high- and low-dose Radix Ginseng and Radix Notoginseng formula groups. Acute myocardial infarction was induced in the untreated group, Betaloc group, and high- and low-dose Radix Ginseng and Radix Notoginseng formula groups by left anterior descending coronary artery ligation. After 12-day treatment, microvessel density (MVD) in ischemic myocardium was detected by immunohistochemical method, while expressions of VEGFR-2 and HIF-1alpha proteins were detected by Western blotting, and expressions of VEGFR-2 and HIF-1alpha mRNAs were detected by real-time fluorescent quantitative polymerase chain reaction. MVD in the untreated group was increased significantly, higher than those in the normal control group and the sham-operated group (P<0.05) and lower than those in the high- and low-dose Radix Ginseng and Radix Notoginseng formula groups and Betaloc group (P<0.01). VEGFR-2 and HIF-1alpha protein and mRNA expressions in the untreated group were higher than those in the normal control group and the sham-operated group (P<0.05). VEGFR-2 and HIF-1alpha protein and mRNA expressions in the high- and low-dose Radix Ginseng and Radix Notoginseng formula groups and Betaloc group were higher than those in the untreated group (P<0.05). There was a significant difference between the high- and low-dose Radix Ginseng and Radix Notoginseng formula groups (P<0.05). Radix Ginseng and Radix Notoginseng extract can up-regulate the protein and mRNA expressions of VEGFR-2 and HIF-1alpha and increase MVD in ischemic myocardium to improve myocardial ischemia so as to promote the development of collateral circulation.

Abstract 3620: Efficacy of Vandetanib on EGFR mutant lung cancer cells with PTEN loss

Abstract Introduction EGFR gene is frequently mutated in non-small cell lung cancer (NSCLC) of never smokers, especially in Asian ethnicity. The mutations often predict dramatic response to EGFR-tyrosine kinase inhibitors (TKIs) like gefitinib. PTEN is a tumor-suppressor gene deactivating PI3K in downstream of EGFR signaling. Approximately 2% to 9% in NSCLC tumors were considered to be PTEN loss. PTEN loss and EGFR mutation co-occurred in 1 out of 24 EGFR mutant patients with lung adenocarcinoma (Sos, et al. Cancer Res 69, 2009). PTEN loss was considered to represent primary or acquired resistance to EGFR-TKIs. Therefore, a new strategy is needed to break through for the resistance due to PTEN loss. Objective We clarify the effectiveness of vandetanib, EGFR and vascular endothelial growth factor receptor (VEGFR) inhibitor, against lung cancer cell lines harboring EGFR mutations with PTEN loss. Materials and methods: Two EGFR mutant (deletion in exon19) NSCLC cell lines, PC-9 (PTEN wild type) and NCI-H1650 (PTEN loss) were used. Other two cell lines with PTEN loss, prostate cancer cell line (PC-3) and glioblastoma cell line (U-87MG) were also examined. We transfected intact PTEN gene into H1650 cells and knocked down PTEN expression in the PC-9 cells using shRNA. Antiproliferative activity was determined by MTT assay in terms of 50% inhibitory concentration (IC50) values. We also examined the effectiveness of the drugs (15 mg/kg/day) in xenograft model. Protein expression profile was examined by Western blotting. Expression of VEGFR mRNA levels was also assessed by RT-PCR. Results PC-9 cells were more resistant to vandetanib (IC50: 0.08 μM) than to gefitinib (IC50: 0.01 μM). However, mean IC50 values of vandetenib were significantly lower than those of gefitinib against H1650 cells (2 μM vs 25 μM), PC-3 cells (8 μM vs 25 μM), and U87MG cells (6 μM vs 28 μM) with PTEN loss. EGFR downstream signals including pAKT, pMAPK, and pSTAT3, and VEGFR were not different in the H1650 cells treated with vandetanib or gefitinib. In the xenograft model using H1650 cells, vandetanib was also more effective than gefitinib. Although PTEN transfected H1650 cells did not restore sensitiveness to gefitinib in vitro, the xenograft tumors responded to gefitinib as well as vandetanib. Knockdown of PTEN lead six times more resistance to gefitinib in PC-9 cells. Conclusion PTEN loss may play a role to determine the sensitivity to gefitinib in NSCLC with EGFR mutations. Vandetanib is more effective in NSCLC with EGFR mutation and PTEN loss. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3620.

Abstract 5206: VEGF-A/VEGFR-2 signaling might be a promising target for the tumor invasion of pancreatic cancer

Abstract Introduction: Pancreatic cancer is one of the most lethal solid tumors. Vascular endothelial growth factor receptors (VEGFRs) are expressed not only by endothelial cells but also by some types of cancer cells, including pancreatic cancer cells. VEGFRs might play an important role for the development of cancer cells. The purpose of this study was to evaluate the efficacy of VEGF/VEGFR-2 targeted therapy in pancreatic carcinoma. We here demonstrated that VEGFR-2 signaling on pancreatic cancer cells might be a promising therapeutic target for the invasion of pancreatic cancer. Materials &amp; Methods: Three pancreatic carcinoma cell lines, MiaPaCa-2, OCUP-AT and Panc-1, were used. The effect of anti-VEGF antibody, bevacitumab, a tyrosine kinase inhibitor against VEGFRs, sunitinib, and VEGF-R2 siRNA on cancer invasion was examined by invasion assay. The expression level of VEGFR mRNA of cancer cells was examined by RT-PCR. The effect of VEGF, bevacitumab and sunitinib on MAPK and PI3K signaling pathway was examined. Result: VEGF-A significantly increased the number of migration cells of MiaPaCa-2 and OCUP-AT, but not that of Panc-1. Bevacitumab and sunitinib significantly decreased the migration ability of MiaPaCa-2 and OCUP-AT cells. The expression levels of VEGF-R2 were high in MiaPaCa-2 and OCUP-AT cells, in compared with Panc-1 cells. Invasion and migration ability of MiaPaCa-2 cells significantly (p&amp;lt;0.05) decreased by VEGFR-2 siRNA treatment. The level of VEGFR-2 autophosphorylation, p-ERK and p-Akt expression was increased by VEGF-A, and decreased by bevacitumab and sunitinib. Conclusion: The invasion of pancreatic cancer cells was closely associated with VEGF-A/VEGFR-2 signaling. VEGF-A/VEGFR-2 inhibition by bevacitumab or sunitinib appears therapeutically promising in pancreatic cancer with VEGFR-2 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5206.

1153 EFFECT OF MITOMYCIN C ON LEVELS OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND ITS RECEPTORS IN BLADDER CANCER CELLS AND IN BLADDERS OF RATS INTRAVESICALLY INSTILLED WITH MITOMYCIN C

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 20101153 EFFECT OF MITOMYCIN C ON LEVELS OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND ITS RECEPTORS IN BLADDER CANCER CELLS AND IN BLADDERS OF RATS INTRAVESICALLY INSTILLED WITH MITOMYCIN C Avanti Verma, Marcia A. Wheeler, Justin DeGrado, Hristos Z. Kaimakliotis, Adam B. Hittelman, and Robert M. Weiss Avanti VermaAvanti Verma More articles by this author , Marcia A. WheelerMarcia A. Wheeler More articles by this author , Justin DeGradoJustin DeGrado More articles by this author , Hristos Z. KaimakliotisHristos Z. Kaimakliotis More articles by this author , Adam B. HittelmanAdam B. Hittelman More articles by this author , and Robert M. WeissRobert M. Weiss More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.652AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The chemotherapeutics, mitomycin C (MMC) and Bacillus Calmette-Guérin (BCG) are effective intravesical treatments for superficial bladder cancer and carcinoma in situ. Intravesical instillation of BCG does not change vascular endothelial growth factor (VEGF) immunoreactivity in bladders from TCC patients and upregulates VEGF receptors in rats. The impact of MMC on expression of the soluble form of VEGF (VEGF-A) and on VEGF receptor-2 (VEGFR-2) which mediates many angiogenic properties of VEGF, has been largely unexamined. We plan to examine the effect of MMC on proliferation and on levels of VEGF-A, VEGF receptor-1 (VEGFR-1) and VEGFR-2 in in vitro and in vivo models. METHODS One day after MMC treatment (6-100 μg/ml), we measured proliferation, and VEGF-A, VEGFR-1 and VEGFR-2 levels in T-24 and RT4 bladder cancer cells. The effect of pretreatment of VEGF siRNA on MMC induced decreases in proliferation was measured. Rats were intravesically instilled with saline or MMC (200 μg/rat). Urinary VEGF levels, and bladder levels of VEGFR-2 protein relative to actin and VEGF, VEGFR-1, and VEGFR-2 mRNA relative to GAPDH mRNA were measured. RESULTS Although MMC treatment inhibited cell proliferation, it did not decrease VEGF protein expression in T-24 and RT4 cells. In fact, in T-24 cells, MMC (6-50 μg/ml) increased VEGF mRNA expression normalized to GAPDH 3-13 fold and increased VEGFR-2 mRNA and protein expression, 2-12 fold and 2-5 fold, respectively. Levels of VEGFR-1 were unchanged. In T-24 cells, MMC (25 μg/ml) reduced proliferation 40 ± 5%, while increasing VEGF mRNA levels 3.4 fold. However, pretreatment of MMC treated cells with VEGF siRNA blocked VEGF mRNA production and potentiated MMC induced reductions in proliferation by an additional 27 ± 4%. Similar results were seen with RT-4 cells. In rats intravesically instilled with MMC, urinary VEGF/creatinine was increased 80% and VEGFR-2 mRNA and protein levels were increased 80% and 150%, respectively, compared to levels in saline instilled rats (n = 4-6). CONCLUSIONS MMC treatment increases levels of VEGF mRNA and VEGFR-2 mRNA and protein in bladder cancer cells. VEGF siRNA potentiates mitomycin inhibitory effects on proliferation in bladder cancer cells. Intravesical Instillation of MMC increases urinary VEGF and bladder VEGFR-2 levels. We speculate that MMC increases the angiogenic potential of both cancer and normal cells. Combining MMC with agents that reduce VEGF levels may be of value in treatment of transitional cell carcinoma of the bladder. New Haven, CT© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e446-e447 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Avanti Verma More articles by this author Marcia A. Wheeler More articles by this author Justin DeGrado More articles by this author Hristos Z. Kaimakliotis More articles by this author Adam B. Hittelman More articles by this author Robert M. Weiss More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...