Vascular Endothelial Growth Factor Receptor 2 Protein Research Articles

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).

Moderate postnatal hyperoxia accelerates lung growth and attenuates pulmonary hypertension in infant rats after exposure to intra-amniotic endotoxin

To determine the separate and interactive effects of fetal inflammation and neonatal hyperoxia on the developing lung, we hypothesized that: 1) antenatal endotoxin (ETX) causes sustained abnormalities of infant lung structure; and 2) postnatal hyperoxia augments the adverse effects of antenatal ETX on infant lung growth. Escherichia coli ETX or saline (SA) was injected into amniotic sacs in pregnant Sprague-Dawley rats at 20 days of gestation. Pups were delivered 2 days later and raised in room air (RA) or moderate hyperoxia (O₂, 80% O₂ at Denver's altitude, ∼65% O₂ at sea level) from birth through 14 days of age. Heart and lung tissues were harvested for measurements. Intra-amniotic ETX caused right ventricular hypertrophy (RVH) and decreased lung vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2) protein contents at birth. In ETX-exposed rats (ETX-RA), alveolarization and vessel density were decreased, pulmonary vascular wall thickness percentage was increased, and RVH was persistent throughout the study period compared with controls (SA-RA). After antenatal ETX, moderate hyperoxia increased lung VEGF and VEGFR-2 protein contents in ETX-O₂ rats and improved their alveolar and vascular structure and RVH compared with ETX-RA rats. In contrast, severe hyperoxia (≥95% O₂ at Denver's altitude) further reduced lung vessel density after intra-amniotic ETX exposure. We conclude that intra-amniotic ETX induces fetal pulmonary hypertension and causes persistent abnormalities of lung structure with sustained pulmonary hypertension in infant rats. Moreover, moderate postnatal hyperoxia after antenatal ETX restores lung growth and prevents pulmonary hypertension during infancy.

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.

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

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

Abstract 374: VEGFR2/KDR polymorphisms and gene copy gain with outcome in non-small cell lung cancer

Abstract Background. The VEGF receptor 2 (VEGFR2) is the predominant mediator of VEGF-stimulated endothelial cell function. Recently, VEGFR2/KDR copy number gain and mutation, and VEGF copy number gain, have been described in lung adenocarcinoma tumor specimens. To better characterize the molecular changes of these two genes in non-small cell lung carcinoma (NSCLC), we investigated their abnormalities in NSCLC tumor tissue specimens and correlated with patients clinico-pathological characteristics. Methods. We extracted DNA from microdissected tissue obtained from 200 surgically resected NSCLCs. KDR single nucleotide polymorphism (SNP) 889G/A (rs2305948), SNP 1416A/T (rs1870377) and SNP −37A/G (rs2219471) were genotyped by PCR-based sequencing. KDR and VEGF copy number were examined by quantitative (q)-PCR. Protein expressions of VEGF and VEGFR2, and CD34 for microvascular density (MVD) analysis were studied by immunohistochemistry. SNP genotypes, genes copy number and protein expression were correlated with NSCLC clinico-pathological features, including overall survival (OS) and recurrence-free survival (RFS). Results. KDR 1416 AT/TT genotypes had significantly improved OS (HR = 0.56, 95% CI 0.33 to 0.96, P = 0.035) compared with KDR 1416 AA in all NSCLC patients in the multivariate analysis with adjustment of histology and tumor stage. In lung adenocarcinomas, KDR 1416 AT/TT genotypes and KDR −37AG/GG were associated with a favorable OS (HR = 0.43, 95% CI 0.20 to 0.92, P = 0.029; HR = 0.47, 95% CI 0.23 to 0.96, P = 0.039, respectively). Strikingly, KDR −37 AG/GG genotypes predicted a superior OS compared with KDR −37 AA genotype in the NSCLC patients treated with adjuvant therapy (HR = 2.45, 95% CI 1.06 to 5.66, P = 0.036). No genotype in KDR SNPs was associated with RFS in NSCLC patients. Gene copy gain of KDR and VEGF were detected respectively in 34/91 (37.4%) and 2/91 (2.2%) NSCLC tumors. Gene copy gain of KDR was associated with a poor overall survival in NSCLC patients (HR= 2.96, 95% CI 1.41 to 6.24, P = 0.004). Furthermore, tumors with gene copy gain of KDR showed significantly higher cytoplasmic (P = 0.013) and membrane (P = 0.009) VEGFR2 protein expression, lower cytoplasmic VEGF expression (P = 0.044), and higher MVD (P = 0.018) and larger vessel areas (P = 0.033) compared with tumors lacking KDR gene copy gain. Conclusion. Our findings suggest an association between KDR SNP genotypes and survival in NSCLC patients receiving adjuvant therapy. KDR copy number gain was frequently identified in NSCLC and was associated with worse survival in NSCLC patients. (Supported by grant US DoD W81XWH-07-1-0306). 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 374.

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 ...

Accumulation of VEGFR2 in zoledronic acid-treated endothelial cells

Nitrogen-containing bisphosphonates (BIS) are potent inhibitors of bone resorption and are used in the treatment of a number of medical conditions, including multiple myeloma, breast cancer and osteoporosis. Recent experimental evidence demonstrates that BIS also affect endothelial cell functions and angiogenesis; however, the molecular mechanism(s) are unclear. Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic signal for endothelial cells. BIS inhibit VEGF responses in endothelial cells. The VEGF receptor-2 (VEGFR2) is the main signaling receptor for VEGF in endothelial cells. We hypothesized that altered VEGFR2 expression in BIS-treated endothelial cells may account for these attenuated responses to VEGF. The affect of the BIS zoledronic acid (ZOL) was investigated in human umbilical vein endothelial cells using confocal microscopy, Western blotting, real-time PCR and flow cytometry. VEGFR2 accumulated within the ZOL-treated endothelial cells (p=0.0002), though not on the cell surface (p>0.05). ZOL did not induce VEGFR2-specific mRNA (p>0.05). ZOL inhibited endothelial cell chemotaxis towards VEGF (p=0.001). VEGF stimulation significantly reduced the amount of VEGFR2 in the endothelial cells (p=0.01). This response to VEGF was reduced by ZOL (p>0.05). The effects of ZOL on endothelial cell migration, VEGFR2 protein expression and response to VEGF were attenuated by geranylgeranyl pyrophosphate. Two- and one-way ANOVAs with Tukey or Dunnett's multiple comparison adjustments were used. The data suggest that ZOL induces aberrant VEGFR2 accumulation. This is not likely due to the induction of mRNA transcription, but rather to the disruption of the mevalonate pathway.

Inhibiting tumor growth of colorectal cancer by blocking the expression of vascular endothelial growth factor receptor 3 using interference vector-based RNA interference

Many reports show that vascular endothelial growth factor receptor 3 (VEGFR3) plays an essential role in tumor metastasis and is a promising target for cancer therapy. The present study was designed to determine the role of VEGFR3 in tumor growth using RNA interference (RNAi) technology. Three small interfering RNA (siRNA) sequences for the VEGFR3 gene were cloned into expression plasmids (pSUPER) and transfected into human colorectal carcinoma (CRC) LoVo cells. Stable transfection of these plasmids decreased VEGFR3 protein expression, leading to the potent suppression of tumor cell proliferation and lymphangiogenesis in vitro. Furthermore, we selected the most effective silenced expressor vector and injected it and pSUPER vector into a tumor xenograft model in nude mice. The tumor growth of LoVo cells expressing VEGFR3 siRNA were significantly inhibited compared with cells transfected with control vector alone. Immunohistochemical analyses of tumor sections revealed a decreased vessel density and decreased VEGFR3 expression in animals when siRNA against VEGFR3 was expressed. These results showed that RNAi of VEGFR3 is an effective tool to reduce lymphangiogenesis in CRC.

Endothelial VEGFR-3 expression in colorectal carcinomas is associated with hematogenous metastasis

Vascular endothelial growth factor receptor 3 (VEGFR-3) is a major inducer of lymphangiogenic signalling and seems to be involved also in angiogenesis. Since both processes are closely linked with tumor metastasis this study investigated the expression of VEGFR-3 in tumor-associated vessels in colorectal carcinomas and evaluated its relevance for lymphogenous and hematogenous metastasis. In a comparative study between microvascular endothelial cells isolated from the tumor (HCTEC) and the corresponding non-neoplastic tissue (HCMEC) from five patients with colorectal cancer VEGFR-3 expression was measured using a specific ELISA. The expression pattern was individually different, with cases showing reduced, elevated and unchanged protein levels. Under hypoxic culture conditions (3% O2 for 24 h), which are more realistic for the tumor situation, the levels remained unchanged. In contrast, hypoxia exposure of macrovascular human umbilical vein endothelial cells (HUVEC) led to a consistent downregulation of VEGFR-3 protein. These data indicate a 'hypoxia-resistant' behaviour of VEGFR-3 in colonic microvasculature. Using immunohistochemistry the endothelial expression pattern of VEGFR-3 in 74 non-metastatic, lymphogenously-metastatic and hematogenously-metastatic colorectal carcinoma specimens was assessed. Positive VEGFR-3 expression was highly significantly associated with those cases showing distant metastasis (p=0.0003). In contrast, significant differences in the expression of VEGFR-3 between non-metastatic tumors and carcinomas with lymph node metastasis were not found. The majority of the detectable intratumoral VEGFR-3-positive vessels were of blood vascular origin (CD31 positive, D2-40 negative). Whereas intratumoral lymphatic vessels were collapsed, VEGFR-3 positive peritumoral lymphatic vessels had mostly open lumina. These morphological observations provide evidence for a predominant significance of VEGFR-3-positive, possibly angiogenesis-mediated, tumor-associated blood vessels in hematogenous metastasis of colorectal cancer. In addition, due to their patency VEGFR-3-positive peritumoral, but not intratumoral lymphatics could be the vascular substrate functionally mediating lymphogenous metastasis.

Inhibition of Rac1 GTPase downregulates vascular endothelial growth factor receptor‐2 expression by suppressing Sp1‐dependent DNA binding in human endothelial cells

RhoA, Rac1 and CDC42 are small GTP-binding proteins of the Rho family that play a crucial role in regulation of the actin-based cytoskeleton. In addition to cell growth regulation, they are implicated in transcriptional activation, oncogenic transformation and angiogenesis. The small Rho-GTPases have been linked to vascular endothelial growth factor (VEGF)-induced signalling pathways, but their role has not yet been elucidated. As signalling via the VEGF receptor-2 (VEGFR2) pathway is critical for angiogenic responses in cancer, wound repair and ischaemic and inflammatory diseases, we investigated whether the small Rho-GTPase Rac1 influences VEGFR2 expression in human endothelial cells. In this study, we show that a dominant negative Rac1 expression vector led to a pronounced decrease in VEGFR2 mRNA and protein expression. To identify minimal promoter requirements and potential applications of the small Rho-GTPases, we used VEGFR2 promoter-reporter gene constructs containing various deletions. The inhibitory effects of dominant negative Rac1 on the transcriptional activity of the VEGFR2 promoter localized to an element between -77 and -60 that contains an Sp1 transcription factor binding site. Electrophoretic mobility shift assays demonstrated that constitutive Sp1-dependent DNA binding decreased with Rac1 inhibition. Hence, repression of the small Rho GTPase Rac1 seems to be an additional critical molecular mechanism in the regulation of VEGFR2 expression.

Oral N‐acetylcysteine attenuates pulmonary emphysema and alveolar septal cell apoptosis in smoking‐induced COPD in rats

The role of apoptosis in lung destruction in emphysema/COPD is increasingly being recognized. The relationship between anti-oxidants and alveolar septal cell apoptosis in COPD lungs remains to be elucidated. The aim of this study was to investigate the effects of the anti-oxidant, N-acetylcysteine (NAC), on the development of emphysema and alveolar septal cell apoptosis in smoking-induced COPD in rats. Sprague-Dawley rats (n = 48) were randomly assigned to normal, COPD, sham and NAC groups. The effects of treatment were assessed by measuring the levels of vascular endothelial growth factor (VEGF) in BAL fluid by ELISA, VEGF and VEGF receptor-2 (VEGFR2) protein expression by western blotting, and the apoptotic index (AI) of alveolar septal cells by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay. Histopathological evaluations (mean linear intercept (MLI), destructive index (DI)) and lung function measurements were performed. FEV(0.3)/FVC and PEF were lower in the COPD group than in the normal group. MLI and DI were lower in the NAC-treated group than in the COPD or sham-treated groups. As confirmed by western blotting, the levels of VEGF in BAL fluid were higher in the NAC-treated group than in the COPD group. VEGFR2 protein expression was higher in the NAC-treated group than in the COPD group. The AI was significantly lower in the NAC-treated group than in the COPD group. There was an inverse correlation between levels of VEGF in BAL fluid and the AI of alveolar septal cells. NAC attenuates lung damage, pulmonary emphysema and alveolar septal cell apoptosis by partly reversing the decrease in VEGF secretion and VEGFR2 protein expression in smoking-induced COPD in rats.

Requirement of Fut8 for the expression of vascular endothelial growth factor receptor-2: a new mechanism for the emphysema-like changes observed in Fut8-deficient mice

alpha1,6-Fucosylation plays key roles in many biological functions, as evidenced by the study of alpha1,6-fucosyltransferase (Fut8) knockout (Fut8(-/-)) mice. Phenotypically, Fut8(-/-) mice exhibit emphysema-like changes in the lung, and severe growth retardation. Fut8(-/-) cells also show marked dysregulation of the TGF-beta1 receptor, EGF receptor, integrin activation and intracellular signalling, all of which can be rescued by reintroduction of Fut8. The results of the present study demonstrated that vascular endothelial growth factor receptor-2 (VEGFR-2) expression was significantly suppressed in Fut8(-/-) mice, suggesting that Fut8 was required for VEGFR-2 expression. The expression of VEGFR-2 mRNA and protein was consistently down-regulated by knockdown of the Fut8 gene with small interference RNA in A549 cells, as well as in TGP49 cells, suggesting that suppression occurs at the level of transcription. In contrast, the expression level of ceramide, an inducer of cell apoptosis, was increased in the lungs of Fut8(-/-) mice. The terminal transferase dUTP nick end-labelling (TUNEL) assay was used to identify apoptotic cells. The number of TUNEL-positive septal epithelia and endothelia cells was significantly increased in the alveolar septa of lungs from Fut8(-/-) mice when in comparison with lungs from wild-type mice. It is well known that, in emphysema, ceramide expression can be greatly enhanced by blockade of the VEGFR-2. Thus, suppression of VEGFR-2 expression may provide a novel explanation for the emphysema-like changes in Fut8(-/-) mice.

The role of VEGFR-2 genetic variation in breast cancer progression.

Abstract Abstract #904 Background: Substantial laboratory and clinical data have demonstrated the critical role of angiogenesis in breast tumor progression. A significant correlation between vascular endothelial growth factor receptor-2 (VEGFR-2) expression and cell proliferation has been described in invasive breast carcinomas, suggesting that VEGF stimulates mammary cell growth through VEGFR-2. We sought to examine whether variability in VEGFR-2 expression and activation in tumors might be due to individual genetic variations, which may also play a role in response to anti-angiogenic therapy. To our knowledge, no study has correlated genetic variation in VEGFR-2 to expression and activity in primary breast tumors.&amp;#x2028; Methods: DNA from 42 primary breast tumors was extracted from fresh frozen tissue. The core promoter, 5'-untranslated region (UTR), 3'-UTR, exons and intron-exon boundary regions of VEGFR-2 were sequenced for all tumors. Tissue microarrays were constructed, and tumor and paired normal breast tissue were stained with anti-VEGFR-2 antibody (Calbiochem). Microvessel density (MVD) was determined by immunohistochemical staining using a primary antibody against platelet endothelial cell adhesion molecule (anti-CD34, Novocastra). Semiquantitative analysis was performed independently by two pathologists. VEGFR-2 expression was correlated with genotype and MVD using the Mann-Whitney test. VEGFR-2 expression in normal and tumor tissue was compared using the Wilcoxon signed-rank test.&amp;#x2028; Results: Two-thirds of tumors were from self-reported African Americans (AA), and the majority were ER positive. Twenty-three different single nucleotide polymorphisms (SNPs) were identified; ten were previously reported in dbSNP. Three of these SNPs were common (minor allele frequency &amp;gt;10%): one was located in the core promoter region and the other two were located in exons 7 and 11 (both non-synonymous SNPs). Using PolyPhen prediction software (http://genetics.bwh.harvard.edu/pph/), the two non-synonymous SNPs were predicted to affect protein function. Of the 23 different SNPs identified, 11 were only seen in tumors from AA and 3 were only observed in tumors from Caucasians. Thirty-six of the 42 tumors (86%) had at least one SNP. VEGFR-2 expression in tumor was significantly higher than in paired normal tissue (p=0.0002). VEGFR-2 expression was significantly lower in tumors with the AA genotype of the 4032 A/G core promoter SNP as compared to those with the AG and GG genotypes combined (p=0.02). VEGFR-2 expression was significantly associated with MVD in tumor tissues (p=0.04).&amp;#x2028; Discussion: Our preliminary study suggests an association between genetic variations in the VEGFR-2 gene and protein expression in tumor tissue. Future work will examine the spectrum of these genetic variations in diverse populations and their potential role in predicting response to anti-angiogenic therapy.&amp;#x2028; This study was funded by the University of Chicago Breast SPORE NCI P50 CA125183. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 904.

The hemopexin domain of MMP-9 inhibits angiogenesis and retards the growth of intracranial glioblastoma xenograft in nude mice.

Matrix Metalloproteinase-9 (MMP-9) consists of a prodomain, catalytic domain with 3 fibronectin-like type II modules and C-terminal hemopexin-like (PEX) domain. These domains play distinct roles in terms of proteolytic activity, substrate binding and interaction with inhibitors and receptors. To assess the potential of the MMP-9-PEX domain to interfere with tumor progression, we stably transfected human glioblastoma cells with an expression vector containing a cDNA sequence of the MMP-9-PEX. The selected clones exhibited decreased MMP-9 activity and reduced invasive capacity. We assessed how secretion of MMP-9-PEX by glioblastoma cells affects angiogenic capabilities of human microvascular endothelial cells (HMECs) in vitro. MMP-9-PEX conditioned medium treatment caused a reduction in migration of HMECs and inhibited capillary-like structure formation in association with suppression of vascular endothelial growth factor (VEGF) secretion and VEGF receptor-2 protein level. The suppression of HMECs survival by conditioned medium from MMP-9-PEX stable transfectants was associated with apoptosis induction characterized by an increase in cells with a sub-G0/G1 content, fragmentation of DNA, caspase-3, -8 and -9 activation and poly (ADP-ribose) polymerase (PARP) cleavage. A significant tumor growth inhibition was observed in intracranial implants of MMP-9-PEX stable transfectants in nude mice with attenuation of CD31 and MMP-9 protein expression. These results demonstrate that MMP-9-PEX inhibits angiogenic features of endothelial cells and retards intracranial glioblastoma growth.

Correlation Between Tumor Growth Delay and Expression of Cancer and Host VEGF, VEGFR2, and Osteopontin in Response to Radiotherapy

To determine the late effects of radiotherapy (RT) on vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR2), and osteopontin (OPN) expression in cancer and stromal cells. LS174T xenografted athymic mice were used as a tumor model. Radiation was delivered in two equivalent fractionation schemes: 5 x 7 Gy and 1 x 20 Gy, the latter at two dose rates. Tumor growth arrest was similar in all treatment groups, with the exception of a better response of small-size tumors in the 5 x 7-Gy group. The host VEGF and OPN levels were directly proportional to the tumor doubling time and were independent of the fractionation scheme. The host and cancer cell VEGFR2 levels in tumor were also directly related to the tumor response to RT. Upregulated VEGFR2 in cancer cells suggest paracrine signaling in the VEGFR2 pathway of cancer cells as the factor contributing to RT failure. The transient activation of the host VEGF/VEGFR2 pathway in tumor supports the model of angiogenic regeneration and suggests that radiation-induced upregulation of VEGF, VEGFR2, and downstream proteins might contribute to RT failure by escalating the rate of vascular repair. Coexpression of host OPN and VEGF, two factors closely associated with angiogenesis, indicate that OPN can serve as a surrogate marker of tumor recovery after RT. Taken together, these results strongly support the notion that to achieve optimal therapeutic outcomes, the scheduling of RT and antiangiogenic therapies will require patient-specific post-treatment monitoring of the VEGF/VEGFR2 pathway and that tumor-associated OPN can serve as an indicator of tumor regrowth.

Neuropilin-1 Is Essential for Enhanced VEGF165-Mediated Vasodilatation in Collateral-Dependent Coronary Arterioles of Exercise-Trained Pigs

Background/Aims: Exercise training enhances vasodilatation to vascular endothelial growth factor (VEGF₁₆₅) in collateral-dependent coronary arterioles. Interaction of VEGF receptor 2 (VEGFR-2) and the non-tyrosine-kinase receptor, neuropilin-1 has been reported to potentiate VEGF₁₆₅-mediated signaling. In the current study, we tested the hypotheses that neuropilin-1 mediates the exercise-enhanced VEGF₁₆₅-mediated vasodilatation in collateral-dependent arterioles and that neuropilin-1 and/or VEGFR-2 protein levels are increased in these arterioles. Methods: Ameroid occluders were surgically placed around the proximal left circumflex coronary artery of miniature swine. Eight weeks after surgery, the animals were randomized into sedentary or exercise training (treadmill run; 5 days/week; 14 weeks) protocols. Coronary arterioles (∼100 μm diameter) were isolated from both collateral-dependent and control (left anterior descending) myocardial regions and studied by in vitro videomicroscopy or frozen for immunoblot analysis. Results: Exercise-enhanced VEGF₁₆₅-mediated vasodilatation in collateral-dependent arterioles was reversed by inhibition of the VEGF₁₆₅-neuropilin-1 interaction. VEGF₁₂₁, which does not interact with neuropilin-1, induced similar vasodilatation in arterioles from all treatment groups. Immunoblot revealed significantly elevated VEGFR-1, VEGFR-2 and neuropilin-1 protein levels in collateral-dependent arterioles of exercise-trained pigs. Conclusions: Neuropilin-1 plays a vital role in the exercise-enhanced VEGF₁₆₅-mediated vasodilatation of collateral-dependent coronary arterioles and is associated with increased neuropilin-1 receptor protein levels.

Human CD4+ T Lymphocytes Recognize a Vascular Endothelial Growth Factor Receptor-2–Derived Epitope in Association with HLA-DR

Given the multiple escape mechanisms of tumor cells, immunotherapy targeting tumor-dependent stroma may be an effective cancer treatment strategy. Animal models indicate that inducing immunity to tumor endothelia engenders potent antitumor effects without significant pathology. Recently, the first human tumor endothelial antigen vascular endothelial growth factor receptor-2 (VEGFR-2) recognized by HLA class I-restricted CD8(+) T cells has been characterized. In this study, we sought to investigate specific recognition of this molecule by human CD4(+) T cells. To identify HLA-DR-restricted antigenic peptides on VEGFR-2 recognized by CD4(+) T cells of healthy donors and cancer patients. Nine candidate VEGFR-2 peptides with high binding probability to six common HLA-DRB1 alleles were synthesized using the SYFPEITHI algorithm. One 15-mer peptide (EKRFVPDGNRISWDS), mapping to the 167-181 region of VEGFR-2, stimulated CD4(+) T cells in association with several HLA-DR alleles, including DR4 and DR7. Importantly, the epitope could be naturally processed and presented both by HLA-DR-matched antigen-expressing proliferating endothelial cells and by dendritic cells loaded with the native antigen. Furthermore, circulating VEGFR-2-specific CD4(+) T cells were detected in 4 of 10 healthy donors and 12 of 40 cancer patients even after single-round peptide stimulation in short-term culture. Patient's T cells could recognize antigen-expressing proliferating endothelial cells in a HLA-DR-restricted fashion. These findings indicate an important role for the 167-181 region of VEGFR-2 in the stimulation of CD4(+) T cell responses to VEGFR-2 protein, and may be instrumental both for the development and monitoring of upcoming antitumor vessel vaccines against different cancers based on VEGFR-2 immunogens.

Targeting the vascular endothelial growth factor system to prevent ovarian hyperstimulation syndrome

Ovarian hyperstimulation syndrome (OHSS) typically occurs when ovaries are primed with FSH/LH and subsequently exposed to hCG. The ultimate pathophysiological step underlying this clinical picture is increased vascular permeability (VP). A search of the literature was carried out using PubMed and the authors' files. In rodents and humans, the expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR-2) mRNA increases during ovarian stimulation. With the administration of hCG, the expression of each rises to a maximum. Expression of VEGF/VEGFR-2 mRNAs correlates with enhanced VP, with both peaking 48 h following an injection of hCG. Immunohistochemistry shows the presence of VEGF and VEGFR-2 proteins in the granulosa-lutein and endothelial cells of the entire corpus luteum. Increased VP may be mediated through adhesion molecules such as VE-cadherin, which is involved in the loosening of endothelial intercellular junctions. These findings regarding the pathophysiology of OHSS suggest that the syndrome can be prevented by inducing ovulation with LH or GnRH analogues, which prevent VEGF overexpression. Also, co-administration of a dopamine agonist inhibits phosphorylation of the receptor VEGFR-2. In a trial of 69 oocyte donors, the incidence of moderate OHSS was 20% with the dopamine agonist cabergoline and 44% with a placebo (P = 0.04). The pathophysiological mechanisms involved in OHSS suggest potential preventive approaches, but larger trials are necessary for evaluating the efficacy and safety of the pharmaco-prevention of OHSS.

Differential Regulation of VEGF Signaling by PKC-α and PKC-ε in Endothelial Cells

Vascular endothelial growth factor (VEGF) stimulates proangiogenic signal transduction and cell function in part through activation of protein kinase C (PKC). Our aim was to examine how individual isoforms of PKC affect VEGF action. Transfection of bovine aortic endothelial cells with small interfering RNA (siRNA) targeting either PKC-alpha, delta, or epsilon caused a reduction in the cognate PKC protein by 76% to 89% without changing expression of nontargeted isoforms. Downregulation of PKC-epsilon abrogated VEGF-stimulated phosphorylation of Akt at Ser473 and eNOS at Ser1179 and decreased VEGF-stimulated NO synthase activity in intact cells. In contrast, PKC-alpha knockdown increased Akt and eNOS phosphorylation, whereas PKCdelta knockdown had no significant effect. PKC-epsilon knockdown also decreased VEGF-stimulated Erk1/2 phosphorylation and abolished VEGF-stimulated DNA synthesis. Consistent with an effect on several pathways of VEGF signaling, VEGF receptor-2 (VEGFR2) tyrosine phosphorylation and expression of VEGFR2 protein and mRNA was decreased by 81, 90, and 84%, respectively, during knockdown of PKC-epsilon, but increased during PKC-alpha knockdown. By regulating VEGFR2 expression and activation, PKC-epsilon expression is critical for activation of Akt and eNOS by VEGF and contributes to VEGF-stimulated Erk activation, whereas PKC-alpha has opposite effects.

Z-Guggulsterone, a constituent of Ayurvedic medicinal plant Commiphora mukul, inhibits angiogenesis in vitro and in vivo

Our previous studies have shown that z-guggulsterone, a constituent of Indian Ayurvedic medicinal plant Commiphora mukul, inhibits the growth of human prostate cancer cells by causing apoptosis. We now report a novel response to z-guggulsterone involving the inhibition of angiogenesis in vitro and in vivo. The z-guggulsterone treatment inhibited capillary-like tube formation (in vitro neovascularization) by human umbilical vein endothelial cells (HUVEC) and migration by HUVEC and DU145 human prostate cancer cells in a concentration- and time-dependent manner. The z- and E-isomers of guggulsterone seemed equipotent as inhibitors of HUVEC tube formation. The z-guggulsterone-mediated inhibition of angiogenesis in vitro correlated with the suppression of secretion of proangiogenic growth factors [e.g., vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor], down-regulation of VEGF receptor 2 (VEGF-R2) protein level, and inactivation of Akt. The z-guggulsterone-mediated suppression of DU145 cell migration was increased by knockdown of VEGF-R2 protein level. Ectopic expression of constitutively active Akt in DU145 cells conferred protection against z-guggulsterone-mediated inhibition of cell migration. Oral gavage of 1 mg z-guggulsterone/d (five times/wk) to male nude mice inhibited in vivo angiogenesis in DU145-Matrigel plug assay as evidenced by a statistically significant decrease in tumor burden, microvessel area (staining for angiogenic markers factor VIII and CD31), and VEGF-R2 protein expression. In conclusion, the present study reveals that z-guggulsterone inhibits angiogenesis by suppressing the VEGF-VEGF-R2-Akt signaling axis. Together, our results provide compelling rationale for further preclinical and clinical investigation of z-guggulsterone for its efficacy against prostate cancer.