High Ang-2 Expression Research Articles

Ang-2 is a potential molecular marker for lymphatic metastasis and better response to bevacizumab therapy in ovarian cancer.

In ovarian cancer, there are two main routes of metastasis, namely intraperitoneal and retroperitoneal. Their biologic background is poorly understood. Identifying molecular markers involved might enable the development of tailored therapy regimens. Moreover, no reliable markers for response to anti-angiogenic treatment with bevacizumab are yet established. Angiopoietin-2 (Ang-2) is an angiogenic growth factor, involved in lymphatic activation and is associated with tumor progression. Here, we assessed the potential of Ang-2 as a molecular marker in metastasis and treatment of ovarian cancer. In our study, quantitative and qualitative protein Ang-2 expression in tumor tissue of ovarian cancer patients was analyzed by Western blot (n = 138) and immunohistochemistry (n = 58). Further, Ang-2 levels in blood samples were quantified in enzyme-linked immunosorbent assay (n = 38). Expression levels of different tumor spread patterns were evaluated, and survival analyses were made. We observed that Ang-2 expression is significantly higher in tumors with retroperitoneal dissemination (pT1a-pT3b, pN1) compared to those showing intraperitoneal tumor growth (pT3c, pN0). In addition, patients with high Ang-2 expression have significantly longer overall survival compared to patients with low Ang-2 expression. Patients with high Ang-2 expression benefit significantly from therapy with bevacizumab. All in all, Ang-2 may serve as a molecular marker for patients with tumors prone to spread to lymph nodes and for patients who might benefit from bevacizumab therapy.

DCE-MRI-based radiomics in predicting angiopoietin-2 expression in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the sixth most common cancer, and the third leading cause of cancer death worldwide. Studies have shown that increased angiopoietin-2 (Ang-2) expression relative to Ang-1 expression in tumors is associated with a poor prognosis.The purpose of this study was to investigate the efficacy of predicting Ang-2 expression in HCC by preoperative dynamic contrast‐enhanced magnetic resonance imaging (DCE-MRI)-based radiomics.MethodsThe data of 52 patients with HCC who underwent surgical resection in our hospital were retrospectively analyzed. Ang-2 expression in HCC was analyzed by immunohistochemistry. All patients underwent preoperative upper abdominal DCE-MRI and intravoxel incoherent motion diffusion-weighted imaging scans. Radiomics features were extracted from the early and late arterial and portal phases of axial DCE-MRI. Univariate analysis and least absolute shrinkage and selection operator (LASSO) was performed to select the optimal radiomics features for analysis. A logistic regression analysis was performed to establish a DCE-MRI radiomics model, clinic-radiologic (CR) model and combined model integrating the radiomics score with CR factors. The stability of each model was verified by 10-fold cross-validation. Receiver operating characteristic (ROC) curve analysis, calibration curve analysis and decision curve analysis (DCA) were employed to evaluate these models.ResultsAmong the 52 HCC patients, high Ang-2 expression was found in 30, and low Ang-2 expression was found in 22. The areas under the ROC curve (AUCs) for the radiomics model, CR model and combined model for predicting Ang-2 expression were 0.800, 0.874, and 0.933, respectively. The DeLong test showed that there was no significant difference in the AUC between the radiomics model and the CR model (p > 0.05) but that the AUC for the combined model was significantly greater than those for the other 2 models (p < 0.05). The DCA results showed that the combined model outperformed the other 2 models and had the highest net benefit.ConclusionThe DCE-MRI-based radiomics model has the potential to predict Ang-2 expression in HCC patients; the combined model integrating the radiomics score with CR factors can further improve the prediction performance.Graphical abstract

Angiopoietin-2 expression and its relationship with lymphangiogenesis and clinicopathological characteristics in cutaneous malignant melanoma.

The aim of this study was to investigate angiopoietin-2 (Ang-2/ANGPT2) expression and its relationship with lymphangiogenesis and clinicopathological characteristics in cutaneous malignant melanoma (CMM). Gene expression differences between metastatic melanoma and melanoma in situ in 472 patients from the TCGA database were analyzed. The target gene Ang-2 was screened. A clinical study was conducted to analyze the correlation between Ang-2 expression in CMM and tumor-associated lymphangiogenesis. A total of 42 patients with primary CMM who underwent extended tumor resection procedures at the Affiliated Hospital of Jiangsu University were included in this study. Clinical data (gender, age, lymph node metastasis, Breslow thickness, and clinical stage) were collected. The expression levels of both Ang-2 and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) proteins were detected by immunohistochemistry (IHC). Lymphatic vascular density (LVD) was counted by using LYVE-1 to label lymphatic endothelial cells (LECs) in peritumoral and intratumoral areas per high-magnification field of view. Statistical analysis was performed using the Pearson correlation test and Student's t-test. Using the TCGA database, it was found that the gene expression level of Ang-2 in 368 cases of metastatic melanoma was significantly higher than that in 104 cases of melanoma in situ. Correlation analysis showed a significant relationship between Ang-2 and LYVE-1, and vascular endothelial growth factor receptor 3(VEGFR3) expression, respectively, in CMM. Moreover, the optimal cutoff value of survival analysis showed that high Ang-2 expression in CMM had a worse prognosis, based on data from the TCGA database. Our research showed that Ang-2 was more highly expressed in the group of patients with lymph node metastasis and in the group of stage 3C-4 patients than in the group of patients with no lymph node metastasis and in the group of stage 0-3B patients. Our research also showed that LVD in the group of patients with lymph node metastasis and in the group of stage 3C-4 patients was significantly higher than that in the group of no lymph node metastasis and in the group of stage 0-3B patients, respectively. Breslow thickness also correlated with Ang-2 expression and LVD. Ang-2 expression was not related to sex or age. Ang-2 expression was obviously correlated with LVD. An evaluation of Ang-2 expression and LVD can be used to predict the risk of tumor lymphatic metastasis and determine the prognosis of CMM. These results may also provide a new clinical treatment strategy for CMM.

The Effect of Bevacizumab and Propranolol on Nasal Polyposis.

Objective This study aims to evaluate the effects of bevacizumab and propranolol from the point of view of a possible antiangiogenic effect in a model of primary nasal polyp (NP) tissue culture. Methods NP samples of 21 patients and normal healthy nasal mucosa samples of 7 patients were cultured. Samples were divided into four groups as follows (healthy nasal mucosa, NP without any treatment, NP treated with propranolol, NP treated with bevacizumab). Cultured tissues were formalin fixed and paraffin embedded. Tissue sections and immunohistochemical VEGF-A, angiopoietin-1 (Ang-1), and angiopoietin-2 (Ang-2) expressions were evaluated. ELISA was also performed for each one of them. Results Both propranolol and bevacizumab significantly decreased the expressions of VEGF-A and Ang-1, and they significantly increased the expression of Ang-2 in comparison to the control NP group. In the healthy nasal mucosa group, no significant expression of VEGF-A was seen, a slight (+) Ang-1 expression, and a high (+++) Ang-2 expression were observed. Conclusion Bevacizumab and propranolol exert an antiangiogenic effect on NP tissues, mainly by decreasing VEGF-A and Ang-1 expression, increasing Ang-2 expression.

Role of angiopoietins in mesothelioma progression

Background and objectiveAnti-angiogenic treatment has been recently shown to be clinically beneficial for mesothelioma patients. Angiopoietins-1 and -2 are key regulators of tumor angiogenesis. Ang-1 is mainly known to promote angiogenesis and vessel stability, while Ang-2 could serve as an antagonist of Ang-1 causing vessel regression and destabilization or enhance angiogenesis in a context-dependent manner. We hypothesized that Ang-1 would promote and Ang2 would halt experimental mesothelioma by affecting tumor angiogenesis. MethodsTo examine the effects of angiopoietins in mesothelioma angiogenesis and in vivo growth we constructed Ang-1 or Ang-2 overexpressing AE17 and AB1 mesothelioma cells and implanted them in the respective syngeneic animals. We also explored the clinical relevance of our observations using the human tumoral mRNAseq data available in the TCGA database. Results and conclusionsAng-1 promotes mesothelioma angiogenesis and growth while the effect of Ang-2 is context-dependent. Low Ang-1 levels in human mesotheliomas are associated with the epitheloid subtype. Tumors of high Ang-1, or concurrent high Ang-2 and VEGF expression present high PECAM-1 and CDH5 expression, markers of vascularity and vascular stability, respectively. Our results highlight the importance of angiopoietins in mesothelioma pathophysiology and pave the way for the clinical development of novel anti-angiogenic strategies.

IL-8 associates with a pro-angiogenic and mesenchymal subtype in glioblastoma

Glioblastoma (GBM) is a highly aggressive brain tumor characterized by a high rate of vascularization. However, therapeutic targeting of the vasculature through anti-vascular endothelial growth factor (VEGF) treatment has been disappointing, for which Angiopoietin-2 (Ang-2) upregulation has partly been held accountable. In this study we therefore explored the interplay of Ang-2 and VEGFA and their effect on angiogenesis in GBM, especially in the context of molecular subclasses. In a large patient cohort we identified that especially combined high expression of Ang-2 and VEGFA predicted poor overall survival of GBM patients. The high expression of both factors was also associated with increased IL-8 expression in GBM tissues, but in vitro stimulation with Ang-2 and/or VEGFA did not indicate tumor or endothelial cell-specific IL-8 responses. Glioblastoma stem cells (GSCs) of the mesenchymal (MES) subtype showed dramatically higher expression of IL8 when compared to proneural (PN) GSCs. Secreted IL-8 derived from MES GSCs induced endothelial proliferation and tube formation, and the MES GBMs had increased counts of proliferating endothelial cells. Our results highlight a critical pro-angiogenic role of IL-8 in MES GBMs.

Expressions and Clinical Significances of Angiopoietin-1, Angiopoietin-2, and Tie-2 Receptor in Patients With Colorectal Cancer

PurposeAngiopoietin-1 (Ang-1) plays a crucial role in vascular and hematopoietic development, mainly through its cognate receptor, Tie-2. Increased levels of Ang-2 have been shown to be correlated with abnormal tumor angiogenesis in several malignancies. Hence, we estimated the increased expression of Ang-2 relative to Ang-1 in patients with colorectal cancer and correlated our finding with prognosis in order to investigate the relationships between the expressions of Ang-1/Ang-2/Tie-2 receptor and the clinical parameters or overall survival of such patients.MethodsWe retrospectively analyzed 114 tissue samples from patients with colorectal cancer by using immunohistochemistry (IHC) to examine Ang-1, Ang-2, and Tie-2 expressions and to investigate the relationship between those expressions and clinical parameters or overall survival of such patients. A Western blot analysis was used for Ang-2 expression.ResultsIHC staining showed a link between Ang-1 and Tie-2 (P = 0.018), as well as meaningful correlations between Ang-2 and Tie-2 receptor (P = 0.022) and between lymph-node metastasis and Ang-2 (P = 0.025). The stronger the IHC staining for Ang-2 expression was, the shorter the cumulative survival was (P = 0.016).ConclusionA relationship was found to exist between Ang-2 and Tie-2 expressions. The Ang-2 was correlated with lymph-node metastasis, and high expression of Ang-2 was indicative of poor overall survival. These findings suggest that Ang-2 is a useful prognostic marker in the management of patients with colorectal cancer. In addition, we suggest that Ang/Tie-2 signaling plays an important role in the progression of colorectal cancer.

Novel Angiogenesis Markers as Long-Term Prognostic Factors in Patients With Renal Cell Cancer

ObjectiveTo evaluate Ang-2 expression alone and in combination with expression of cell proliferation and cell survival markers (MIB-1 and Bcl-2) and angiogenesis markers (VEGFR3 and CD31), and the associations of these markers with renal cell cancer (RCC) in long-term survival. Patients and MethodsOur study included 224 patients with RCC who were treated before the availability of antiangiogenic agents between 1985 and 1995, at the Pirkanmaa Hospital District in Finland. All tumor samples were reclassified and reevaluated by an experienced uropathologist, and parallel tissue microarrays (TMA) were performed for immunohistochemical analysis. Kaplan-Meier's survival estimation method and Cox proportional hazards models were used for survival analysis. ResultsThe percentage of Ang-2 expression in the tumor area varied from 0.07 to 25.65. Ang-2 expression was significantly associated with the tumor grade and stage, as well as the MIB-1, Bcl-2, and VEGFR3 expression (P = .042, P = .019, P = .039, P = .013, and P = .005, respectively). The highest Ang-2 expression predicted better survival, P < .05. High Bcl-2 and low MIB-1 expression combined with Ang-2 expression was associated with better survival. Multivariate analysis showed poorer survival in patients with low Ang-2 or high MIB-1 expressions: HR 1.89, 95% CI 1.16 to 3.08, P = .010 and HR 2.20, 95% CI 1.36 to 3.54, P = .001, respectively. ConclusionsVery high Ang-2 expression was associated with better survival in patients with RCC. Ang-2 expression correlated with tumor stage and grade, but it was still an independent prognostic factor in a multivariate analysis.

Association of Angiopoietin-2 and Ki-67 Expression with Vascular Density and Sunitinib Response in Metastatic Renal Cell Carcinoma.

The Angiopoietin-2 (Ang2, Angpt2) growth factor is a context-dependent antagonist/agonist ligand of the endothelial Tie2 receptor tyrosine kinase and known to promote tumour angiogenesis and metastasis. Angiopoietin antagonists have been tested in clinical cancer trials in combination with VEGF-based anti-angiogenic therapy, including sunitinib, which is widely used as a first-line therapy for metastatic renal cell carcinoma (mRCC). However, little is known about Ang2 protein expression in human tumours and the correlation of tumour Ang2 expression with tumour vascularization, tumour cell proliferation and response to anti-angiogenic therapies. Here, we evaluated, using immunohistochemistry, the expression of Ang2, CD31 and the cell proliferation marker Ki-67 in the primary kidney cancer from 136 mRCC patients, who received first-line sunitinib after nephrectomy. Ang2 protein expression was restrained to RCC tumour vessels, and correlated with tumour vascularization and response to sunitinib. High pre-therapeutic Ang2 expression, and more strongly, combined high expression of both Ang2 and CD31, were associated with a high clinical benefit rate (CBR). Low cancer Ki-67 expression, but not Ang2 or CD31 expression, was associated with favourable progression-free (PFS) and overall survival (OS) as compared to patients with high Ki-67 expression (PFS 6.5 vs. 10.6 months, P = 0.009; OS, 15.7 vs. 28.5 months, P = 0.015). In summary, in this study to investigate endothelial Ang2 in mRCC patients treated with first-line sunitinib, high cancer Ang2 expression was associated with the CBR, but not PFS or OS, whereas low Ki-67 expression was significantly associated with long PFS and OS.

Abstract 4301: Endothelial angiopoietin-2 expression correlates with tumor angiogenesis and response to sunitinib in metastatic renal cell carcinoma

Abstract Background: Angiopoietin-2 (Ang2) is an endothelial cell derived growth factor, which promotes tumor angiogenesis, tumor growth and metastasis. Ang2 is a promising target for anti-angiogenic therapies, but its potential as a biomarker for anti-angiogenic therapies remains unknown. The purpose of this study was to evaluate expression of the Ang2-Tie2 signalling pathway components in metastatic RCC (mRCC) patients treated with sunitinib, a modern multitarget TKI. Materials and methods: We evaluated Ang2, CD31 and Ki67 expression in tumors of 136 patients with metastatic RCC (mRCC) treated with first-line sunitinib from formalin-fixed paraffin-embedded tumor samples using immunohistochemistry. Results: Ang2 was exclusively expressed in the endothelial cells of tumor blood vessels. Using a cutoff of ≥50 Ang2+ vessels/microscopic field, 38% of the samples had high Ang2 expression. High Ang2 expression correlated with high CD31+ vessel density (p = 0.002). 91% of patients with high Ang2 expression had partial response (PR) or stable disease (SD) as best response to sunitinib as compared with 76% of those with low Ang2 expression (P = 0.033). High CD31+ vessel density correlated with a high clinical benefit rate (P = 0.015), and with low levels of Ki-67+ tumor cells (P = 0.018). Combined high expression of both Ang2 and CD31 predicted even better clinical benefit rate with 100% of the patients with high Ang2 and CD31 expression (n = 25) achieving PR/SD as compared with 77% among the rest of the patients (n = 101; P = 0.007). Only one patient (8%, n = 12) with papillary RCC, but 49 (41%) patients with clear cell RCC had high Ang2 expression (P = 0.03). Conclusions: In this first study to investigate Ang2 expression and outcome in mRCC patients treated with sunitinib, high Ang2 expression in the tumor vasculature was predictive for sunitinib efficacy. Citation Format: Anita Lampinen, Juhana Rautiola, Tuomas Mirtti, Ari Ristimäki, Heikki Joensuu, Petri Bono, Pipsa Saharinen. Endothelial angiopoietin-2 expression correlates with tumor angiogenesis and response to sunitinib in metastatic renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4301. doi:10.1158/1538-7445.AM2015-4301

Correlation of endothelial angiopoietin-2 expression with tumor angiogenesis and response to sunitinib in metastatic renal cell carcinoma.

461 Background: The endothelial growth factor angiopoietin-2 (Ang2) is a target for novel anti-angiogenic therapies, but its potential as a biomarker remains unknown. Methods: We evaluated Ang2, CD31 and Ki67 expression in tumors of 136 patients with metastatic RCC (mRCC) treated with first-line sunitinib from tumor samples. Results: Ang2 was exclusively expressed in the endothelial cells of tumor blood vessels. Using a cutoff of ≥50 Ang2+ vessels/microscopic field, 38% of the samples had high Ang2 expression. High Ang2 expression correlated with high CD31+ vessel density (p=0.002). 91% of patients with high Ang2 expression had partial response (PR) or stable disease (SD) as best response to sunitinib compared to 76% of those with low Ang2 expression (p=0.033). High CD31+ vessel density correlated with high clinical benefit rate (p=0.015), and with low levels of Ki-67+ tumor cells (p=0.018). Combined high expression of both Ang2 and CD31 predicted a better clinical benefit rate with 100% of the patients with high Ang2 and CD31 expression (n=25) achieving PR/SD compared to 77% among the rest of the patients (n=101; p=0.007). Only one (8%, n=12) patient with papillary RCC had high Ang2 expression, whereas 49 (41%, n = 120) patients with clear cell RCC had high Ang2 expression (p=0.03). Conclusions: In this first study to investigate Ang2 expression and outcome in mRCC patients treated with an angiogenesis inhibitor, high Ang2 expression in the tumor vasculature was predictive for sunitinib efficacy.

Identification of Novel Molecular Markers for Prognosis Estimation of Acute Myeloid Leukemia: Over-Expression of PDCD7, FIS1 and Ang2 May Indicate Poor Prognosis in Pretreatment Patients with Acute Myeloid Leukemia

Numerous factors impact on the prognosis of acute myeloid leukemia (AML), among which molecular genetic abnormalities are developed increasingly, however, accurate prediction for newly diagnosed AML patients remains unsatisfied. For further improving the prognosis evaluation system, we investigated the transcripts levels of PDCD7, FIS1, FAM3A, CA6, APP, KLRF1, ATCAY, GGT5 and Ang2 in 97 AML patients and 30 non-malignant controls, and validated using the published microarray data from 225 cytogenetically normal AML (CN-AML) patients treated according to the German AMLCG-1999 protocol. Real-time quantitative polymerase chain reaction and western blot were carried out, and clinical data were collected and analyzed. High Ang2 and FIS1 expression discriminated the CR rate of AML patients (62.5% versus 82.9% for Ang2, P = 0.011; 61.4% versus 82.2% for FIS1, P = 0.029). In CN-AML, patients with high FIS1 expression were more likely to be resistant to two courses of induction (P = 0.035). Overall survival (OS) and relapse-free survival (RFS) were shorter in CN-AML patients with high PDCD7 expression (P<0.001; P = 0.006), and PDCD7 was revealed to be an independent risk factor for OS in CN-AML (P = 0.004). In the analysis of published data from 225 CN-AML patients, PDCD7 remained independently predicting OS in CN-AML (P = 0.039). As a conclusion, Ang2 and FIS1 seem related to decreased CR rate of AML patients, and PDCD7 is associated with shorter OS and RFS in CN-AML. Hence, PDCD7, Ang2 and FIS1 may indicate a more aggressive form and poor prognosis of AML.

Expression of Angiopoietin-2 and Vascular Endothelial Growth Factor Receptor-3 Correlates with Lymphangiogenesis and Angiogenesis and Affects Survival of Oral Squamous Cell Carcinoma

BackgroundBoth Ang-2 and VEGFR-3 are major regulators of angiogenesis and lymphangiogenesis, respectively, and thus may affect prognosis of OSCC. We sought to determine the associations between Ang-2 and VEGFR-3 expression and survival of OSCC.MethodsAng-2 and VEGFR-3 expression was determined immunohistochemically in tumor tissues from 112 patients with OSCC; OSCC-adjacent noncancerous oral tissue from 85 OSCC patients; and normal oral mucosa from 37 cancer-free individuals. A log-rank test and Cox proportional hazard models were used to compare survival among different groups with expression of Ang-2 and VEGFR-3.ResultsAng-2 and VEGFR-3 expression was upregulated in OSCC compared to nontumor tissue (all P<0.05). High Ang-2 expression positively correlated with microvessel density (MVD) (P<0.01), and high VEGFR-3 expression positively correlated with lymph node metastasis (P<0.01) and lymphatic vessel density (LVD) (P<0.01). The patients with high expression of Ang-2 alone or in combination with VEGFR-3 had a significantly worse survival than in patients with low expression of Ang-2 or any other co-expression status (all P<0.05), respectively. Furthermore, multivariable analysis showed that patients with high expression of Ang-2 alone or in combination with VEGFR-3 had a significantly increased risk of death compared with those with low expression of Ang-2 or any other co-expression status (HR, 2.7, 95% CI, 1.1–6.2 and 5.0, 1.3–15.4, respectively).ConclusionsThese results suggest that increased expression in tumors of Ang-2 may individually, or in combination with VEGFR-3, predict poor prognosis of OSCC.

Expression of angiopoietin-2 and prognosis in hepatocellular carcinoma

Objective To study the relationship between the expression of angiopoietins-2 (Ang-2) and prognosis in hepatocellular carcinoma (HCC) after hepatic resection.Methods Sixty patients with primary liver cancer were retrospectively analyzed and the expression of Ang-2 in carcinoma tissues were assayed by immunohistochemical staining.The interrelationships among the expression of Ang-2,clinicopathological factors and the grade of differentiation of HCC cells were studied.The relationship between the expressions of Ang-2 and survival time was analysed.Results The expression rate of Ang-2 was 70％.Ang-2 staining had no correlation with clinicopathological factors (P ＜ 0.05),but it had a significant correlation with the grade of differentiation of HCC cells (x2 =5.424,P =0.025).Ang-2 of moderately and poorly differentiated HCC were higher (66.7％) than those of well-differentiated HCC.The disease-free survival rates of patients with high Ang-2 expression and low Ang-2 expression were 50％ (1-year),33.3％ (3-year),16.7％ (5-year) and 89.6％ (1-year),50.0％ (3-year),27.1％ (5-year),respectively.The disease-free survival of patients with high Ang-2 expression were significantly less than those of patients with low Ang-2 expression (x2 =6.477,P =0.021).Conclusion The expressions of Ang-2 are correlated with the grade of differentiation of HCC cells,but are not correlate with clinicopathological factors.The survivals of patients with Ang-2 high expression are lower than those of low expression.Ang-2 is a significant predictive factor for recurrence after hepatic resection in HCC patients. Key words: Angiopoietin-2 ; Carcinoma, hepatocellular; Immunohistochemistry

The promotion of angiogenesis by growth factors integrated with ECM proteins through coiled-coil structures

An appropriate method to bind extracellular matrix (ECM) proteins and growth factors using advanced protein engineering techniques has the potential to enhance cell proliferation and differentiation for tissue regeneration and repair. In this study we developed a method to co-immobilize non-covalently an ECM protein to three different types of growth factors: basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and single-chain vascular endothelial growth factor (scVEGF121) through a coiled-coil structure formed by helixA/helixB in order to promote angiogenesis. The designed ECM was established by fusing two repeats of elastin-derived unit (APGVGV)12, cell-adhesive sequence (RGD), laminin-derived IKVAV sequence and collagen-binding domain (CBD) to obtain CBDEREI2. HelixA was fused to each growth factor and helixB to the engineered ECM. Human umbilical vein endothelial cells (HUVECs) were cultured on engineered ECM and growth factors connected through the coiled-coil formation between helixA and helixB. Cell proliferation and capillary tube-like formation were monitored. Moreover, the differentiated cells with high expression of Ang-2 suggested the ECM remodeling. Our approach of non-covalent coupling method should provide a protein-release control system as a new contribution in biomaterial for tissue engineering field.

184P - Clinical Significance of Angiopoietin-2 Serum Levels in Non-Small Cell Lung Cancer

ABSTRACT Introduction Tumor vasculature is a very promising target in NSCLC, with VEGF inhibitors as part of standard care in some patients. Angiopoietin-2 (Ang-2) plays critical roles in angiogenesis in concert with VEGF. High tumor Ang-2 expression has negative prognostic implications in lung cancer patients, especially when VEGF expression is high. The aim of this study was the evaluation of Ang-2 and VEGF serum levels in NSCLC and tumor stages and its comparison with healthy controls. Patients and methods The study included 30 controls, 145 NSCLC patients, (48 epidermoid, 73 adenocarcinomas, 19 undiferenciated NSCLC, 4 large cells and 1 mixed carcinoma), divided in 13 early and 132 advanced stages). Ang-2 and VEGF levels were evaluated in subject samples with R&D Quantikine ELISA Kit, according to manufacturer's instructions. Results Ang-2 levels were significantly different in the control and case groups (Mann-Whitney test, p = 0.003). Mean concentration values were 2855.50 pmol/ml (SE 268.64) vs 4164.90 pmol/ml (SE 200.56), respectively. Regarding stage analysis, Ang-2 levels were also statistically different according to tumor stage (Mann-Whitney test, p = 0.016): mean 2824.64 pmol/ml (standard error 472.82) and 4202.57 pmol/ml (203.05) for early and advanced stages, respectively. VEGF levels were significantly different in the control and case groups (Mann-Whitney test, p = 0.001). Mean concentration values were 312.63 pmol/ml (standard error 59.62) vs 675.51 pmol/ml (standard error 55.30), respectively. Regarding stage analysis, there were no statistically significant differences between VEGF levels and tumor stage (Man-Whitney test, p = 0.197). Conclusions Angiogenesis inhibitors have obvious, yet modest, antitumor activity in NSCLC. Ang-2 seems to be a promising target of such inhibitors and its serum levels may be useful to predict which patients will benefit more with this type of treatment, surpassing the need to obtain tumor tissue samples to assess its levels of expression. Disclosure All authors have declared no conflicts of interest.

Abnormal regulation of neo-vascularisation in deep partial thickness scalds in rats with diabetes mellitus

Objective This study observed the degree of neo-vascularisation and differential expression of angiogenesis growth factors and their receptor in deep partial-thickness scald wound with diabetes mellitus. Methods Sixty Sprague-Dawley rats were randomised into a control group and an STZ-induced diabetic group, inflicted with partial-thickness scalding of 20% total body surface area (20% TBSA) on the back. Wound specimens were harvested immediately after scald and on 1, 3, 7, 10, 14 and 21 post-scald days (PSDs) to observe histological changes, and wound healing rates were calculated. The degree of neo-vascularisation in wound (labelled with blue microsphere) and the quantity of vascular endothelial cells (labelled with red CD31) were also measured by double-labelling immunofluorescence. Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Tie-2, vascular endothelial growth factor (VEGF) and Flt-1 messenger RNA (mRNA) were analysed by real-time RT-PCR. Ang-1, Ang-2 and VEGF protein expressions were measured by Western blotting. Results Wound healing was markedly impaired in diabetic rats. The diabetic rats show inhibited vascularity in the wound edge at every time point (the quantitation of vascularity was 60.0 ± 3.0 in the control group and 12.0 ± 1.4 in the diabetic group, p < 0.01 on day 7). Although neo-vascularisation in the number of endothelial cells was not significantly different compared with the normal group, part of new vascular endothelial cells did not form the vascular function. After injury, expression of Ang-2 mRNA and protein were increased in both groups, and the normal group showed decreases on day 7, 14 and 21, whereas the diabetic group showed significant increases. Although the expression VEGF and its receptors before injury was higher than the normal group, the level at 1, 3 and 7 days after injury was significantly lower than that 14 days, and that at 21 days after injury was significantly higher than the normal group. Conclusion Vascular endothelial cells can proliferate actively in the diabetic wound with deep partial-thickness burns, but it is still poor in blood supply due to lack of functional capillaries. The mechanism may be related to sustained abnormal high expression of Ang-2 and down-regulated VEGF.

Prognostic Impacts of Angiopoietins in NSCLC Tumor Cells and Stroma: VEGF-A Impact Is Strongly Associated with Ang-2

IntroductionAngiopoietins and their receptor Tie-2 are, in concert with VEGF-A, key mediators in angiogenesis. This study evaluates the prognostic impact of all known human angiopoietins (Ang-1, Ang-2 and Ang-4) and their receptor Tie-2, as well as their relation to the prognostic expression of VEGF-A.Methods335 unselected stage I-IIIA NSCLC-patients were included and tissue samples of respective tumor cells and stroma were collected in tissue microarrays (TMAs). Immunohistochemistry (IHC) was used to semiquantitatively evaluate the expression of markers in duplicate tumor and stroma cores.Principal FindingsIn univariate analyses, low tumor cell expression of Ang-4 (P = 0.046) and low stromal expressions of Ang-4 (P = 0.009) and Ang-2 (P = 0.017) were individually associated with a poor survival. In the multivariate analysis, low stromal Ang-2 (HR 1.88; CI 95% 1.15-3.08) and Ang-4 (HR 1.47, CI 95% 1.02–2.11, P = 0.04) expressions were independently associated with a poor prognosis. In patients with high tumor cell expression of Ang-2, a concomitantly high tumor VEGF-A expression mediated a dramatic survival reduction (P<0.001). In the multivariate analysis of patients with high Ang-2 expression, high tumor VEGF-A expression appeared an independent poor prognosticator (HR 6.43; CI 95% 2.46–16.8; P<0.001).ConclusionsIn tumor cells, only Ang-4 expression has prognostic impact in NSCLC. In tumor stroma, Ang-4 and Ang-2 are independently associated with survival. The prognostic impact of tumor cell VEGF-A in NSCLC appears strongly associated with a concomitantly high tumor cell expression of Ang-2.

Angiopoietin-2 mRNA expression is increased in chronic lymphocytic leukemia patients with poor prognostic features

Several studies have demonstrated the potential prognostic importance of angiogenesis in chronic lymphocytic leukemia (CLL). Elevated expression of angiopoietin-2 (Ang-2), an angiogenic cytokine, was recently reported in CLL. However, data regarding prognostic significance of Ang-2 in CLL are limited. Therefore, we quantitated Ang-2 mRNA in purified mononuclear cells of 33 untreated CLL patients and compared the transcript levels to traditional as well as modern prognostic factors in patients with CLL (clinical stage, disease course, IgVH mutation status, CD38, and ZAP-70 expression). Elevated Ang-2 mRNA concentrations were detected in 12 cases; 21 patients had very low or undetectable levels of Ang-2 transcript. There was significant association between high Ang-2 mRNA levels and unmutated IgVH genes (n=27, P=0·010) and with CD38 expression (n=32, P=0·011), but not with ZAP-70 expression (n=32, P=0·784), Rai stage (n=33, P=0·305) or stable versus progressive clinical course (n=33, P=0·443). There was a trend towards shorter progression-free survival in patients with high Ang-2 expression; however, it did not reach statistical significance (P=0·090). Our pilot data show that Ang-2 mRNA is differentially expressed in patients with CLL and its increased expression appears to be associated with poor prognostic features. Further studies are needed to confirm the results in a larger patient cohort.

Angiopoietin-2 mRNA Expression Is Elevated in Chronic Lymphocytic Leukemia Patients with Poor Prognostic Features

Several studies have demonstrated potential prognostic importance of angiogenesis in chronic lymphocytic leukemia (CLL). Elevated expression of angiogenic activator angiopoietin-2 (Ang-2) was recently reported in CLL but data regarding prognostic significance of Ang-2 in CLL is limited. Therefore, we evaluated potential relationship between Ang-2 expression and other prognostic factors in patients with CLL. Ang-2 mRNA was quantified by real-time quantitative PCR in purified mononuclear cells of 33 untreated CLL patients. Ang-2 transcript levels were further analyzed with regard to clinical stage, disease course, IgVH mutation status, CD38 and ZAP-70 expression. Elevated Ang-2 mRNA was detected in 12 cases; twenty-one patients had very low or undetectable levels of Ang-2 transcript. High Ang-2 mRNA levels were significantly associated with unmutated IgVH genes (p=0.010) and high CD38 expression (p=0.011) but not with ZAP-70 expression (p=0.784), Rai stage (p=0.305) or stable vs. progressive clinical course (p=0.443). Interestingly, there was a trend towards shorter progression-free survival in patients with high Ang-2 expression, but it did not reach statistical significance (median 15 months vs. not reached; p=0.090). In conclusion, our data show that Ang-2 mRNA is differentially expressed in patients with CLL and its increased expression appears to be associated with poor prognostic features.