Effects of autophagy inhibitor 3-methyladenine on a diabetic mice model.

Cellular senescence prevents the proliferation of cells at risk for neoplastic transformation. Nonetheless, the senescence response is thought to be antagonistically pleiotropic and thus contribute to aging phenotypes, including, ironically, late life cancers. The cancer-promoting activity of senescent cells is likely due to secreted molecules, the identity of which remains largely unknown. Here, we have shown that senescent fibroblasts, much more than presenescent fibroblasts, stimulate tumor vascularization in mice. Weakly malignant epithelial cells co-injected with senescent fibroblasts had larger and greater numbers of blood vessels compared with controls. Accordingly, increased vascular endothelial growth factor (VEGF) expression was a frequent characteristic of senescent human and mouse fibroblasts in culture. Importantly, conditioned medium from senescent fibroblasts, more than medium from presenescent cells, stimulates cultured human umbilical vein endothelial cells to invade a basement membrane, a hallmark of angiogenesis. Increased VEGF expression was specific to the senescent phenotype and increased whether senescence was induced by replicative exhaustion, overexpression of p16(Ink4a), or overexpression of oncogenic RAS. The senescence-dependent increase in VEGF production was accompanied by very little increase in hypoxic-inducible (transcription) factor 1 alpha protein levels, and hypoxia further induced VEGF in senescent cells. This result suggests the rise in VEGF expression at senescence is not a hypoxic response. Our findings may in part explain why senescent cells stimulate tumorigenesis in vivo and support the idea that senescent cells may facilitate age-associated cancer development by secreting factors that promote malignant progression.

Etiology of OHSS and use of dopamine agonists

Vascular endothelial growth factor (VEGF) is a potent mitogen and permeability factor for endothelial cells that plays a central role in angiogenesis, vascular maintenance, inflammation, and cancer. VEGF also mediates the homeostatic adaptation to hypoxic conditions by promoting an increase in vascular density to compensate for decreased oxygenation. This process is triggered by an oxygen-sensitive transcription factor, hypoxia-inducible factor-1 (HIF1alpha), which becomes active in hypoxic tissues, leading to the synthesis and secretion of VEGF. The role of HIF1alpha in other processes that involve angiogenesis such as in inflammation is less clear. Of interest, endothelial cells not only respond to but also store and secrete VEGF, which is required for the maintenance of the integrity of the vascular system. How this intracellular pool of VEGF is regulated is still not understood. Here, we found that CXCL8/IL8, a potent proangiogenic and inflammatory chemokine, up-regulates VEGF mRNA and protein levels in endothelial cells by acting on its cognate receptor, CXCR2, and that this results in the autocrine activation of VEGFR2. Surprisingly, this process does not involve HIF1alpha but instead requires the activation of the transcription factor NFkappaB. Furthermore, we identified the components of the CBM complex, Carma3, Bcl10, and Malt1, as key mediators of the CXCL8/IL8-induced NFkappaB activation and VEGF up-regulation. Together, these findings support the existence of an NFkappaB-mediated pathway by which the proinflammatory chemokine CXCL8/IL8 controls the expression of VEGF in endothelial cells, thereby promoting the activation of VEGF receptors in an autocrine fashion.

Activation or overexpression of HER-2/neu is associated with up-regulation of vascular endothelial growth factor (VEGF) in human breast cancer cells in vitro. Preclinical experiments indicate that increased expression of VEGF may in part mediate the biologically aggressive phenotype of HER-2/neu-overexpressing human breast cancer. It was the purpose of this study to: (a). evaluate the association between HER-2/neu and VEGF expression in a large clinical cohort of primary breast cancer patients; (b). compare the prognostic significance of VEGF isoforms; and (c). analyze the combined effects of HER-2/neu and VEGF on clinical outcome. HER-2/neu and VEGF were measured by ELISA in primary breast tumor tissue lysates from 611 unselected patients with a median clinical follow-up of 50 months. At least six VEGF isoforms consisting of 121, 145, 165, 183, 189, or 206 amino acids are generated as a result of alternative splicing. The VEGF(121-206) ELISA uses antibodies that bind to VEGF(121) and, therefore, detects all of the VEGF isoforms with 121 and more amino acids. The VEGF(165-206) ELISA uses antibodies that bind to VEGF(165) and, therefore, detects all of the VEGF isoforms with 165 and more amino acids. VEGF(121-206) and VEGF(165-206) were analyzed both as continuous and categorical variables, using detectable expression as a cutoff for positivity. Cell lines with defined HER-2/neu expression levels were used to establish a cutoff point for HER-2/neu overexpression in breast tumor samples. Our findings indicate a significant positive association between HER-2/neu and VEGF expression. VEGF(121-206) and VEGF(165-206) expression was detectable in 88 (77.2%) and 100 (87.7%), respectively, of the 114 patients with HER-2/neu-overexpressing tumors, in contrast to 271 (54.5%) and 353 (71.0%), respectively, of the 497 patients with nonoverexpressing tumors (chi(2) test: P < 0.001 for both VEGF(121-206) and VEGF(165-206)). VEGF(121-206) and VEGF(165-206) demonstrate a comparable prognostic significance for survival in unselected primary breast cancer patients (univariate analysis: VEGF(121-206), P = 0.0068; VEGF(165-206), P = 0.0046; multivariate analysis: VEGF(121-206), P = 0.1475; VEGF(165-206), P = 0.1483). When the analyses were performed separately for node-negative and node-positive patients, VEGF(121-206) and VEGF(165-206) were of prognostic significance for survival only in node-positive patients (univariate analysis: VEGF(121-206), P = 0.0003; VEGF(165-206), P = 0.0038; multivariate analysis: VEGF(121-206), P = 0.0103; VEGF(165-206), P = 0.0150). A biological concentration-effect relationship between VEGF expression and survival (VEGF(121-206), P = 0.0280; VEGF(165-206,) P = 0.0097) suggests that VEGF levels, as determined by ELISA, could be of importance as a predictive marker for therapeutic strategies that target VEGF. Combining HER-2/neu and VEGF(121-206)/VEGF(165-206) results in additional prognostic information for survival (VEGF(121-206), P = 0.0133; VEGF(165-206), P = 0.0092). The positive association between HER-2/neu and VEGF expression implicates VEGF in the aggressive phenotype exhibited by HER-2/neu overexpression, and supports the use of combination therapies directed against both HER-2/neu and VEGF for treatment of breast cancers that overexpress HER-2/neu.

Vascular Endothelial Growth Factor Localization in the Adult

Vascular endothelial growth factor expression up-regulated by endometrial ischemia in secretory phase plays an important role in endometriosis

Objective: To investigate the expression of von Hippel-Lindau (VHL), vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1α (HIF-1α) in endolymphatic sac tumor (ELST) and its clinical significance, and to analyze its association with VHL gene mutation. Methods: Twenty-four cases of ELST, which were surgically resected and diagnosed by pathological examination in Beijing Tongren Hospital Affiliated to Capital Medical University, Beijing, China during 2012-2020, were recruited as the ELST group, and 24 cases of otitis media diagnosed in the same hospital were selected as the control group. The expression of VHL, VEGF, and HIF-1α was assessed using EnVision immunohistochemical staining and compared between the ELST and control groups. Sanger sequencing was performed to detect the VHL mutation status in 24 ELSTs. The correlations among VHL, VEGF and HIF-1α expression were analyzed. The associations of VHL, VEGF and HIF-1α expression with age of onset, gender, tumor size, bone invasion and clinical stage in ELST were also analyzed. Results: The expression rate of VHL in the ELST group was significantly lower than that in the control group (P<0.05), but the expression rates of VEGF and HIF-1α in the ELST group were significantly higher than those in the control group (P<0.05). VHL expression was inversely correlated with VEGF and HIF-1α expression. The expression of VEGF and HIF-1α was associated with bone invasion and clinical stage (P<0.05), but the expression of VHL, VEGF and HIF-1α had no significant associations with the age of onset, gender, or tumor size of ELST (P>0.05). Conclusions: The expression of VHL is decreased while that of VEGF and HIF-1α increased in ELST. Expression of VHL is inversely correlated with that of VEGF and HIF-1α. The expression of VEGF and HIF-1α is correlated with bone invasion and clinical stage. Thus, VEGF and HIF-1α may be therapeutic targets of ELST.

The Janus tyrosine kinase and signal transducers and activators of transcription (JAK/STAT) pathway is involved in vascular endothelial growth factor (VEGF) expression, but the role of this pathway in diabetic retinopathy (DR) remains unclear. We investigated the role of the JAK/STAT pathway on DR and VEGF expression using a streptozotocin (STZ)-induced DR mouse model. Cultured ARPE-19 cells were exposed to high-glucose conditions and treated with JAK/STAT inhibitors (JAK inhibitor I [JAKiI], tofacitinib, STAT3 inhibitor [STAT3i]) for 48h. Reverse-transcription polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay were used to investigate p-JAK/STAT and VEGF expression. Diabetes was induced by intraperitoneal injection of STZ (50mg/kg) in C57BL/6 mice for 5days. DR development was evaluated every 4weeks. JAK/STAT inhibitors were administered for 8weeks. Immunofluorescence was used to measure the activation status of the JAK/STAT pathway and VEGF production in the retinal tissue. In ARPE-19 cells exposed to high-glucose conditions, the mRNA and secretory protein levels of VEGF, p-JAK1, p-JAK2, p-STAT3, and p-STAT5 levels were significantly increased. Treatment with JAKiI, tofacitinib, and STAT3i significantly suppressed VEGF to basal levels at both the mRNA and secretory levels in vitro. In STZ-induced mice, retinal vascular leakage, p-JAK1, p-JAK2, p-JAK3, p-STAT3, and VEGF were significantly increased after diabetes induction. Diabetes-induced retinal vascular leakage was significantly reduced by treatment with JAKiI and tofacitinib. Increased p-JAK1 and VEGF in STZ-induced mice were significantly reduced by JAKiI (p < 0.05, p < 0.001) and tofacitinib (p < 0.001, respectively). JAK1 may be more involved in VEGF production and DR progression in mice than JAK2, JAK3, and STAT3.

Oral squamous cell carcinoma (OSCC) is one kind of invasive malignancy arising from oral epithelial cells. According to the statistical data of the Department of Health in Taiwan, OSCC is most common cancer in head and neck and also the fifth leading cause of cancer mortality in 2010. In order to promote their own growth and dispersion, malignant tumors will secrete variant signaling molecules to recruit host blood vessels to grow into the vicinity of the tumor (so-called tumor angiogenesis). Among these signaling molecules, vascular endothelial growth factor (VEGF) is the most important and usually overexpresses in head and neck squamous cell carcinoma. In OSCC, VEGF highly correlates with microvessel density (MVD) of oral cancer micro-environment, and negatively correlates with the survival rates of OSCC patients. Besides, endothelin-1 (ET-1) composed of 21 amino acids plays an important role in tumor growth, metastases, and angiogenesis. Furthermore, it is proved that ET-1 released by tumors would activate endothelin A receptor (ETAR) of the tumor cells and induce VEGF expression in ovarian. Moreover, studies indicated that the expression of ET-1 is increased in OSCC cell lines. However, the relationship between ET-1 and VEGF in OSCC is still not clear. Therefore, in this study we want to testify that whether ET-1 will modulate the expression of VEGF in OSCC via ETAR, and also confirm this phenomenon in clinical samples at the same time. Firstly, we find the significant correlation between ET-1 and VEGF expressions with immunohistochemical stain in clinical OSCC samples. In survival analysis, the patients with high VEGF expressions, instead of ET-1, have lower survival rates, and there is a statistical significance. In the studies of OSCC cell lines, after ET-1 stimulated, the expression of VEGF was increased in OSCC cells, and was inhibited by ETAR antagonist (BQ-123). This phenomenon indicated that ET-1 would induce the expression of VEGF via ETAR in OSCC, and the basal expressions of ET-1 and VEGF would regulate this result. On the other hand, VEGF expression did not inhibit completely by ETAR antagonist when compared with the control group or the group with BQ-123 only. We speculated that ET-1 might be a strong agonist, or the other receptor of ET-1, endothelin B receptor (ETBR), had the similar function as ETAR did. However, the expression of VEGF is regulated by many factors, so therefore we need to study the underlying mechanism of carcinogenesis. Finally, the basal expressions of mRNA of ET-1 and VEGF were varied between selected nine OSCC cell lines. There was a positive correlation between ET-1 and VEGF when we omitted OEC-M1 and OC-2 which expressed great levels of VEGF mRNA. This result matched the finding of clinical samples. According to the experimental results, ETAR antagonist will inhibit the expression of VEGF in OSCC; however, it still further studies whether ETAR antagonist can become a candidate of the anti-cancer drug.

Objective To investigate the expression and clinical significance of vascular endothelial growth factor (VEGF) and its receptor Flk-1 in nephroblastoma. To assess whether tumor microvessel density (MVD) immunoreactivity, determined by the CD34 antigen, is related to the expression of VEGF and Flt-1. Methods Immunohistochemical staining (SABC) was used to examine MVD and the expression of VEGF, Flk-1 in 33 nephroblastoma tissues, 33 paracancer renal tissues and 6 normal kidney tissues. The relation between the expressions of VEGF, Flk-1 and MVD was analyzed in different clinical stage and pathology type, Patients were treated preoperatively with chemotherapy and mean follow up was 34 months. Results The positive rates of VEGF and Flk-1 in nephroblastoma tissues were 81.8% and 69.7%, the positive rates of VEGF and Flk-1 in paracancer renal tissues were 9.1% and 6.1%, the expressions of VEGF and Flk-1 were negative in all normal kidney. The differences between the three groups were statistically significant (P＜0.05). In nephroblastoma tissues,the expression of VEGF correlated to both Flt-1 and MVD (P＜0.05), and the expression of Flt-1 correlated to MVD (P＜0.05). In addition, the expressions of VEGF and Flk-1 were increased in clinical stage Ⅲ and poor prognosis. Conclusions These results indicate that VEGF and Flk-1 may play an important role in nephroblastoma angiogenesis. Increased expression of VEGF and Flk-1 in nephroblastoma correlated with tumor stage, clinical progression and tumor related death. VEGF and Flk-1 protein expression are closely related to MVD and seem to be a important predictor for poor prognosis in treated patients with nephroblastoma. Key words: Nephroblastoma; Vascular endothelial growth factor; Receptors,vascular endothelial growth factor

BackgroundAngiogenesis plays a role in the progression of osteosarcoma, as well as in other mesenchymal tumors and carcinomas, and it is most commonly assessed by vascular endothelial growth factor (VEGF) expression or tumor CD31-positive microvessel density (MVD). Tumor VEGF expression is predictive of poor prognosis, and chemotherapy can affect the selection of angiogenic pattern. The aim of the study was to investigate the clinical and prognostic significance of VEGF and CD31 in osteosarcoma, both at diagnosis and after neoadjuvant chemotherapy, in order to identify a potential role of chemotherapy in angiogenic phenotype.MethodsA retrospective analysis was performed on 16 patients with high grade osteosarcoma. In each case archival pre-treatment biopsy tissue and post-chemotherapy tumor specimens were immunohistochemically stained against CD31 and VEGF, as markers of angiogenic proliferation both in newly diagnosed primary osteosarcoma and after multidrug chemotherapy including high-dose methotrexate (HDMTX). The correlation between clinicopathological parameters and the degree of tumor VEGF and CD31 expression was statistically assessed using the χ2 test verified with Yates' test for comparison of two groups. Significance was set at p < 0,05.ResultsExpression of VEGF was positive in 11 cases/16 of cases at diagnosis. Moreover, 8 cases/16 untreated osteosarcomas were CD31-negative, but the other 8 showed an high expression of CD31. VEGF expression in viable tumor cells after neoadjuvant chemotherapy was observed in all cases; in particular, there was an increased VEGF expression (post-chemotherapy VEGF - biopsy VEGF) in 11 cases/16. CD31 expression increased in 11 cases/16 and decreased in 3 cases after chemotherapy. The data relating to the change in staining following chemotherapy appear statistically significant for VEGF expression (p < 0,05), but not for CD31 (p > 0,05).ConclusionsEven if the study included few patients, these results confirm that VEGF and CD31 expression is affected by multidrug chemotherapy including HDMTX. The expression of angiogenic factors that increase microvessel density (MVD) can contribute to the penetration of chemotherapeutic drugs into the tumor in the adjuvant stage of treatment. So VEGF could have a paradoxical effect: it is associated with a poor outcome but it could be a potential target for anti-angiogenic therapy.

192. Cardiac-Specific and Hypoxia-Responsive Vascular Endothelial Growth Factor Expression in Ischemic Hearts

Expression of Vascular Endothelial Growth Factor and Vascular Endothelial Growth Factor Receptor-2 (KDR/Flk-1) in Ischemic Skeletal Muscle and Its Regeneration

e14672 Background: There is no suggested molecular indicator in identifying which patients will benefit from anti-angiogenic treatment in metastatic colorectal cancer. Over expression of vascular endothelial growth factor (VEGF) and Hypoxia Inducible Factor 1-alpha (HIF-1α) are associated with bad prognosis. In this study, VEGF and HIF-1α expression and their clinical significance are studied in tumor tissues of patients with colorectal cancer receiving treatment with bevacizumab. Methods: VEGF and HIF-1α were observed immunohistochemically in primary tumors of 53 patients. The expressions were separated by evaluating low and high of VEGF and HIF-1α expression. We evaluated whether expression of VEGF and HIF-1α can help to predict treatment response, progression free survival (PFS), and overall survival (OS). Results: Fifty-three patients were enrolled in the study. Median age was 55. VEGF was strongly expressed in 30 patients (57%) whilst low expression was observed in 23 of them (43%). When VEGF expression was evaluated in association with therapy response rates; the clinical benefit rate was 38% in the low expression group whereas it was 62% in high expression group. This difference was statistically significant (p=0.01). In the group with strong VEGF expression PFS was 10 months whereas it was 8 months in the low expression group (p=0.009). When evaluated for OS, 26 months versus 15 months was in favor of highly expressed VEGF group (p=0.03). Highly expressed HIF-1α was found in 29 patients (55%), on the other hand low expressed HIF-1α was detected in 24 (%45) patients. For clinical benefit rates, PFS and OS there was no difference between high and low expressed HIF-1α groups. Conclusions: It has been demonstrated that VEGF and HIF-1α expressions are associated with poor prognosis in several tumors, mainly colorectal carcinomas. With the better understanding of carciogenesis and angiogenesis at molecular level, especially VEGF and HIF-1α became target molecules of the therapy. According to results of our study, VEGF expression is a predictive factor in designating the metastatic colorectal cancer treatment.

Evidence indicates that cyclooxygenase (COX)-2-derived prostaglandins (PGs) contribute to tumor growth by inducing angiogenesis. We investigated the role of COX-2 in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). COX-2 and vascular endothelial growth factor (VEGF) expressions were examined by immunohistochemistry in 24 HBV-associated HCC. Tumor micro-vessel density (MVD) was assessed using CD34 immunohistochemistry. Hep3B HCC cell line, which carries integrated HBV genome, was stably transfected with human COX-2 cDNA. COX-2 and VEGF expressions were determined by Western blot while PG level was determined by ELISA. The effects of PGs on VEGF expression were also investigated. Expression of COX-2 and VEGF in HCC cells were observed in 19 (79%) and 16 (67%) cases, respectively. Well-differentiated HCC expressed COX-2 more strongly than less-differentiated HCC (p<0.001). COX-2 expression was found to correlate with VEGF expression and MVD (p=0.003 and 0.004, respectively). COX-2 overexpressing Hep3B clone had higher VEGF expression as compared to non-COX-2 expressing clone and parental cells. Treatment of the COX-2 overexpressing cells with a COX-2-selective inhibitor, NS-398 (10 microM), decreased PGE2 level and attenuated VEGF expression. Addition of PGE2 (10 microM) and the stable analog of PGI2, carbaprostacyclin (5 microM), to Hep3B cells also increased VEGF expression. Up-regulation of COX-2 correlates with VEGF expression and tumor angiogenesis in HBV-associated HCC. Moreover, COX-2 up-regulates VEGF expression in HCC cells, possibly via PGs production. Selective inhibition of COX-2 may block HCC associated angiogenesis and thus provides a rational approach for treatment of this malignancy.