High Ang-2 Research Articles

Elevated Proangiogenic Markers are Associated with Vascular Complications within Ghanaian Sickle Cell Disease Patients.

Sickle cell disease (SCD) is an inherited blood disorder that can result in vasculopathy and end organ damage. Angiogenesis has been implicated as a key contributing factor to vascular mediated tissue injury in SCD. The relative plasma levels of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and vascular endothelial growth factor (VEGF) greatly influence angiogenesis. Dysregulation of these growth factors, leading to a pro-angiogenic state in SCD patients, has been documented in the developed world but there is very little data in Africa. There is the need, therefore, for studies in Ghanaian SCD patients. The aim of this study was to assess plasma levels of Ang-1, Ang-2, and VEGF in homozygous (HbSS) SCD patients with or without complications and healthy controls (HbAA) in Ghana. The study was a case-control study involving 544 participants: 396 HbSS SCD patients and 148 HbAA healthy controls. The study was conducted at the Center for Clinical Genetics (Sickle Cell Clinic) and Accra Area Blood Centre for National Blood transfusion at the Korle-Bu Teaching Hospital, Accra, Ghana. The plasma levels of Ang-1, Ang-2, and VEGF of study participants were measured with a double sandwich enzyme-linked immunosorbent assay (ELISA) technique. Complete blood count (CBC) was measured with an autoanalyser. The mean plasma Ang-1, Ang-2, and VEGF were significantly higher in HbSS SCD patients with or without complications than healthy controls (p < 0.001). The Ang-2/Ang-1 ratio was significantly lower in the controls than the HbSS patients (p < 0.001). The Ang-2/Ang-1 ratio was higher in the HbSS patients with leg ulcers as compared with patients with other complications and healthy controls (p < 0.001). There were higher leucocyte counts in HbSS patients than healthy controls. Overall, there was elevated plasma levels of Ang-1, Ang-2, and VEGF in SCD patients. The higher Ang-2/Ang-1 plasma levels in patients with leg ulcers suggests a possible ongoing angiogenesis and response to inflammatory stimuli. The study provides a first report on plasma levels of angiopoietin-1, angiopoietin-2, and vascular endothelial growth factors in homozygous sickle cell disease patients in Ghana.

276 - Transcriptomal Evidence for Dysregulation of Angiogenic Factors and Inflammatory Markers after Allogeneic Hematopoietic Cell Transplantation

Abstract Introduction: Angiogenic factors (AF) are the prototypic growth factors required for wound healing, and are linked to the immune response after tissue injury. Angiogenesis and inflammation together influence recovery from organ damage after hematopoietic stem cell transplantation (HCT), as well as contribute to the risk for complications such as acute graft-versus-host disease (aGVHD). We have recently described alterations in circulating angiogenic factors occurring at the onset of aGVHD post-HCT (BBMT 2015 21(6):1029), with dysregulation in AF associated with tissue repair versus those reflective of tissue damage. We hypothesized that the AF-inflammatory signature identified in the plasma in severe aGVHD could be linked to the gene expression of circulating donor cells after HCT. We performed transcriptomal analysis of changes in the peripheral blood immune cells and correlated with plasma AF levels in order to understand if the circulating donor cells might contribute to the AF profile. Methods: Peripheral blood samples from 3 patients (uncomplicated without aGVHD, steroid responsive aGVHD, and steroid refractory aGVHD) were compared to a healthy stem cell donor. Total RNA was derived from whole blood samples, and samples were amplified and labeled using MessageAMP Premier RNA Amplification kit (Ambion). Microarray analyses were performed using Human PrimeView GeneChip arrays (Affymetrix). Plasma AF were quantified using a multiplex angiogenesis array (Millipore). Results: Patients were matched as able according to age, gender, conditioning intensity, donor characteristics, and disease (Table). All patients were 100% donor chimeras and without active infections at the time of blood draw. Compared to HCT donor, all HCT recipients had increased circulating levels of Angiopoietin-2 (Ang-2), endoglin, follistatin, placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) (Figure). However, in steroid refractory aGVHD, levels of epidermal growth factor (EGF) were markedly lower compared to donor, steroid-responsive, and uncomplicated HCT recipients (1.4 pg/ml vs 32.4, 137.1, and 204.8 pg/ml respectively), while levels of VEGF-D, Ang-2, leptin, and PlGF were highest in steroid refractory aGVHD. In microarray analysis (Figure), steroid refractory aGVHD was again associated with a lower gene expression of EGF, but higher expression of only VEGF-D and leptin. Gene expression of Ang-2 and PlGF was not higher in hematopoietic cells, despite higher plasma levels, in steroid-refractory aGVHD, leading to the hypothesis that these growth factors may be predominantly host-derived. Interestingly, we found higher transcript expression of IFN-gamma, comparing steroid refractory versus steroid responsive aGVHD, along with a lower transcript expression of inflammatory cytokines TNF, IL-6, and IL-1B. Conclusions: The plasma profile of steroid-refractory aGVHD (low EGF, and high VEGF-D, Ang-2, leptin, and PlGF) appears to be only partially explained by donor hematopoietic cell gene expression. Gene expression of Ang-2 and PlGF are lower in steroid-refractory aGVHD, suggesting that the plasma elevations in these factors might be predominantly host-derived. Ang-2 likely reflects host endothelial tissue damage as previously described, while the PlGF response to aGVHD requires further investigation. The elevation in several damage-associated factors in the plasma and transcriptome in a healthy HCT recipient may reflect normal adaptation of donor cells to a new host environment. These results are hypothesis generating and mechanistic studies aimed at understanding and manipulating the dysregulated system of AF and downstream signaling pathways are indicated, with the goal of ameliorating severe aGVHD and facilitating tissue repair, without excess immunosuppression. Download : Download high-res image (153KB) Download : Download full-size image Figure . Disclosures Holtan: Incyte: Other: One-time advisory board member.

Endothelial Activation and Blood-Brain Barrier Disruption in Neurotoxicity after CD19 CAR-T Cell Immunotherapy

CD19 chimeric antigen receptor (CAR)-modified T cell therapy has produced impressive results in patients (pts) with CD19+B cell malignancies; however, treatment can be complicated by neurologic adverse events (AEs). The presentation and pathogenesis of neurotoxicity (NT) are incompletely understood.We report a clinical and pathologic study evaluating NT in 133 B-ALL, NHL and CLL pts treated with lymphodepletion chemotherapy and CD19 CAR-T cells (NCT01865617). Neurologic AEs were observed in 53 of 133 pts (40%; 19% grade [gr] 1-2; 16% gr 3; 2% gr 4; 3% gr 5). Most neurologic AEs were reversible. Delirium, headache, and language disturbances were the most frequently observed neurologic AEs, presenting in 66%, 55%, and 34% of pts with NT, respectively. Multivariable analysis showed that higher burden of malignant B cells in marrow, lymphodepletion with cyclophosphamide and fludarabine, and higher infused CAR-T cell dose were associated with increased risk of NT and cytokine release syndrome (CRS).The severity of NT correlated with higher peak concentrations of cytokines that may activate endothelial cells (EC), such as IL-6, IFN-γ, and TNF-α. Pts who developed gr ≥3 NT had more severe CRS with higher CAR-T cell counts in blood and evidence of vascular dysfunction. In severe NT, hypofibrinogenemia with elevated PT, aPTT, and d-dimer were consistent with EC activation. We confirmed in vivo EC activation during NT by demonstrating high concentrations of serum angiopoietin-2 (Ang-2) and von Willebrand Factor (VWF), which are released from Weibel-Palade bodies on EC activation. In vitro, serum from pts with NT induced activation and VWF release from human umbilical vein ECs (HUVECs), which suggests that the finding of a reduced fraction of high molecular weight VWF multimers in vivo during gr ≥4 NT results from consumption on platelets and sequestration on activated EC. Consistent with this explanation, we found reduced activity of the VWF-cleaving protease ADAMTS13 relative to VWF levels and more severe thrombocytopenia in pts with gr ≥4 NT.We considered that cytokine-induced EC activation might alter the integrity of the blood-brain barrier (BBB) during NT. During severe NT, CSF analyses demonstrated increased permeability of the BBB to protein and leukocytes, including CAR-T cells. The gradient between serum and CSF cytokines observed before lymphodepletion was lost during acute NT, consistent with inability of the BBB to shield the CNS from high concentrations of plasma cytokines. Brain vascular pericytes (BVP) play a critical role in vascular and BBB support, and when exposed to high TNF-α or IFN-γ concentrations BVP exhibited stress (increased cleaved caspase-3 and reduced PDGFRβ) and secreted IL-6 and VEGF, further promoting EC activation and BBB permeability.Neuropathologic examination of the brain of a patient with fatal NT demonstrated disrupted endothelium by CD31 immunohistochemistry and EC activation was confirmed by intravascular VWF binding and CD61+ platelet microthrombi. Further evidence of breach of the BBB included red blood cell extravasation from multiple vessels, vascular lesions with karyorrhexis, perivascular CD8+ T cell infiltration, and fibrinoid vessel wall necrosis. CAR-T cells were detected in the CNS.We investigated strategies to reduce the risks of severe NT. Logistic probability curves demonstrated that reduction in CAR-T cell dose to reduce the peak in vivo CAR-T cell blood count was associated with reduced risk of NT, but that the narrow therapeutic index of this approach would lead to loss of anti-tumor efficacy. To maintain CAR-T cell peak counts in blood, we developed a strategy to identify pts early after CAR-T cell infusion who might be at risk of subsequent severe NT and could be candidates for early intervention. Classification tree modeling demonstrated that in the first 36 hours after CAR-T cell infusion pts with fever ≥38.9°C, serum IL-6 ≥16 pg/mL, and MCP-1 ≥1343.5 pg/mL were at high risk of subsequent gr ≥4 NT (sensitivity 100%; specificity 94%). We also investigated whether pts with pre-existing endothelial activation were at higher risk of NT. Before lymphodepletion, pts who developed gr ≥4 NT had higher Ang-2:Ang-1 ratios than those with gr ≤3 NT, indicating that endothelial activation before lymphodepletion or CAR-T cell infusion may be a risk factor for NT that identifies pts who would benefit from a modified treatment regimen. DisclosuresTurtle:Adaptive Biotechnologies: Other: Advisory board; Bluebird Bio: Other: Advisory board; Gilead Sciences: Other: Advisory board; Precision Biosciences: Other: Advisory board; Celgene: Other: Advisory board; Juno Therapeutics: Other: Advisory board, Patents & Royalties, Research Funding. Liles:Juno Therapeutics: Consultancy, Other: Advisory board. Li:Juno Therapeutics: Employment, Equity Ownership. Yeung:Gilead: Research Funding. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Maloney:Celgene: Other: Advisory board; Kite Pharmaceuticals: Other: Advisory board; Juno Theraapeutics: Other: Advisory board, Patents & Royalties, Research Funding; Roche/Genetech: Other: Advisory board.

Prospective evaluation of the factors predicting the prognosis of advanced hepatocellular carcinoma (HCC) patients treated with sorafenib.

e15674 Background: Several factors including pro-angiogenic cytokines have been reported as predictive markers for the treatment effect of sorafenib in patients with HCC; however, many of them were determined based on the results of retrospective studies. Methods: In this prospective cohort study (UMIN000009771), we recruited advanced HCC patients who were treated with sorafenib, and periodically measured serum levels of eight pro-angiogenic cytokines, which were risk factors for the prognosis reported by us and others. The effect of the cytokine expressions before and at 2 (4) weeks after starting sorafenib and at PD, on progression free survival (PFS), overall survival (OS), and post progression survival (PPS) were evaluated. The effect of early appearance of adverse events within 30 days, which were reported as other risk factors, was also evaluated. Results: Eighty patients were enrolled. Median PFS and OS were 3.1 and 11.7 months, respectively. There was no statistically significant variable that could predict PFS, which included patients’ characteristics, cytokines before the therapy, and early onset of adverse events. However, PFS of the patients who showed the decrease of platelet-derived growth factor after 2 weeks (HR2.78, 95%CI 1.35-6.21, P=0.005) or vascular endothelial growth factor after 4 weeks (HR3.31, 95%CI 1.59-6.83, P=0.001) compared to the values before sorafenib treatment was short. OS was short in patients with poor performance status, large tumor size (&gt;30mm), large tumor number (&gt;5), high des-gamma-carboxyprothrombin (DCP, &gt;100mAU/mL), high hepatocyte growth factor, and high angiopoietin-2 (Ang-2) before therapy, in addition to no hand foot syndrome within 30 days. Multivariate analysis with these factors revealed that high Ang-2 (HR2.25, 95%CI 1.08-4.86, p=0.029) and high DCP (HR3.55, 95%CI 1.25-12.7, p=0.014) were the independent risk factors for OS. High Ang-2 at PD could also be a marker of short PPS. Conclusions: The prediction of PFS, OS and PPS is possible by measuring serum pro-angiogenic cytokines at appropriate timing. Ang-2 is a key cytokine for predicting the prognosis of HCC patients treated with sorafenib.

Ang-2 polymorphisms in relation to outcome in advanced HCC patients receiving sorafenib.

e15666 Background: Angiopoietin-2 (Ang-2) is a crucial angiogenic factor. By binding to its receptor Tie2, Ang-2 cooperates with the VEGF pathway to maintain normal physiological functions. In the presence of VEGF, Ang-2 destabilizes blood vessels and promotes vascular sprouting. In cancers, Ang-2 is linked to not only angiogenesis but also invasive and metastatic phenotypes. Although sorafenib exerts no significant activity against Tie2, the predictive value of Ang-2 has been explored in 2 studies. Llovet et al conducted a large biomarker study based on SHARP study. The authors found that a high baseline plasma Ang-2 level was an independent factor for poorer OS but not for reduced sorafenib efficacy. Polymorphism analysis seems to have more advantages than protein or gene expression analysis. Gene expression analysis is performed on biological material collected at a specific time in the natural history of the disease. It is also subject to the influence of a number of laboratory biases. Conversely, polymorphism analysis can be performed at any time during the course of the disease, is not substantially influenced by laboratory biases and is less expensive. In our study we analysed the role of ANG-2 polymorphisms in relation to clinical outcome in patients with hepatocellular carcinoma treated with sorafenib. Methods: We analyzed 135 patients with hepatocellular carcinoma treated with sorafenib. Peripheral blood samples or FFPE tumor tissues were available for DNA extraction and genotyping analysis. Nine Ang-2 polymorphisms were analyzed by direct sequencing or Real Time PCR method. Results: With regard to Ang4 rs55633437 was observed that patients carrying the allele GG were associated with a better PFS and OS. The variants GG were associated with a median OS of 16.9 months vs 6.5 months of variants GT and TT (p = 0.016). The variants GT and TT were associated with a median PFS of 2.94 months vs 4.67 months of variants GG (p = 0.03). These data were confirmed by multivariate analysis Conclusions: Ang4 rs55633437 could represent prognostic markers in patients with advanced hepatocellular carcinoma treated with sorafenib.

Ang-2 polymorphisms in relation to outcome in advanced HCC patients receiving sorafenib

Background: Sorafenib represents the standard care for advanced hepatocellular carcinoma. Angiopoietin-2 (Ang-2) is a crucial angiogenic factor. By binding to its receptor Tie2, Ang-2 cooperates with the VEGF pathway to maintain normal physiological functions. In the presence of VEGF, Ang-2 destabilizes blood vessels and promotes vascular sprouting. In cancers, Ang-2 is linked to not only angiogenesis but also invasive and metastatic phenotypes. Although sorafenib exerts no significant activity against Tie2, the predictive value of Ang-2 has been explored in 2 studies. Llovet et al conducted a large biomarker study based on SHARP study. The authors found that a high baseline plasma Ang-2 level was an independent factor for poorer OS but not for reduced sorafenib efficacy. Conversely, in a small retrospective study, a high serum Ang-2 level was associated with a lower DCR and poorer PFS. The actual role of Ang-2 in predicting sorafenib efficacy warrants further investigations. Polymorphism analysis seems to have more advantages than protein or gene expression analysis. Gene expression analysis is performed on biological material collected at a specific time in the natural history of the disease. It is also subject to the influence of a number of laboratory biases. In our study we analysed the role of ANG-2 polymorphisms in relation to clinical outcome in patients with hepatocellular carcinoma treated with sorafenib.

Angiopoietin pathway gene expression associated with poor breast cancer survival.

Angiogenesis is one of the hallmarks of cancer and is essential for cancer progression and metastasis. However, clinical trials with vascular endothelial growth factor (VEGF) pathway inhibitors have failed to show overall survival benefit in breast cancer. Targeted therapy against the angiopoietin pathway, a downstream angiogenesis cascade, could be effective in breast cancer. This study investigates the association of angiopoietin pathway gene expression with breast cancer survival using a "big data" approach employing RNA sequencing data from The Cancer Genome Atlas (TCGA). A total of 888 patients with adequate gene expression, disease-free survival (DFS), and overall survival (OS) data were selected for analysis. DFS and OS were calculated for patients with high and low expression of angiopoietin and VEGF pathway genes using TCGA data. Gene-specific thresholds to dichotomize patients into high and low expression were determined and survival plots were generated. The TCGA cohort was representative of national breast cancer patients with respect to stage, pathology, and survival. High Ang2 gene expression was associated with not only decreased DFS (p=0.05), but also decreased OS (p<0.05). High co-expression of Ang2 and its receptor Tie2 was associated with both decreased DFS and OS (p<0.05). There was strong correlation between angiopoietin and VEGF pathway genes. While high expression of VEGFA alone was not associated with survival, high co-expression with Ang2 was associated with decreased OS. This study validates TCGA as a representative database providing genomic data and survival outcomes in breast cancer. Our TCGA data support the angiopoietin pathway as a key mediator in the pathologic angiogenic switch in breast cancer.

Vasopressin in Septic Shock; Assessment of Sepsis Biomarkers: A Randomized, Controlled Trial.

Background and Aims:Vasopressin (VP) in sepsis apart from vasoconstrictive effect may have some immunomodulatory effects. The aim of this study was to evaluate the effect of VP on different aspect of sepsis by measuring of sepsis biomarkers.Materials and Methods:In this trial, a total number of 42 septic shock patients were included. The first group received norepinephrine (NE) infusion to reach the target mean arterial pressure (MAP) of ≥ 65 mm Hg and the second group received arginine vasopressin (AVP) infusion in addition to NE. Serum lactate, C-reactive protein (CRP), interleukin-6 (IL-6), IL-10, pentraxin 3 (PTX3), angiopoietin 1 and 2 (Ang 1 and 2) levels were assessed.Results:Level of IL-6 and IL-10 decreased, but there was no significant difference between the two groups after 48 h. CRP and PTX3 levels were not also significantly different between groups. Although Angs were not statistically different, there was a trend toward higher Ang-1 in and lower Ang 2 in AVP group after 24 and 48 h. In addition, lactate level did not differ between NE and AVP groups. There was no interaction between VP and hydrocortisone use on IL-6, IL-10, and PTX3, but a significant statistical interaction on Ang 1 and Ang 2 were observed.Conclusions:Although analysis of sepsis biomarkers showed no significant difference between two groups, no immunomodulatory effect for VP alone, subgroup analysis of hydrocortisone used in this study showed that the combination of glucocorticoids and AVP had a significant effect on Angs level which eventually causes less endothelial permeability and higher MAP in this group of patients.

Angiopoietin-2, its soluble receptor Tie-2, and metabolic syndrome components in a population-based sample.

Since angiopoietin-2 (Ang-2) levels strongly correlate with cardiovascular mortality and subclinical cardiovascular disease, it was hypothesized that levels of Ang-2 and its soluble receptor (sTie-2) were associated with the metabolic syndrome (MetS) and individual MetS components. Within the population-based Study of Health in Pomerania, two sets of analyses were performed. First, Ang-2 and sTie-2 were related to the prevalence of MetS and its components cross-sectionally (n = 3,205). Second, the association between baseline Ang-2 and sTie-2 and incident MetS or longitudinal changes in its components in 1,295 individuals was investigated. High Ang-2 levels (90th percentile), compared with low Ang-2 levels (10th percentile), were positively associated with MetS (OR: 1.78) and with the following MetS criteria: increased triglycerides, lower HDL cholesterol, and higher non-fasting glucose. Furthermore, high sTie-2 levels (90th percentile), compared with low levels (10th percentile), were positively related to MetS (OR: 1.58) and most of its components. However, Ang-2 and sTie-2 levels were not associated with incident MetS or longitudinal change in components of MetS. Ang-2 and sTie-2 levels were cross-sectionally associated with MetS and several of its components. However, Ang-2 and sTie-2 levels were not associated with incident MetS or changes in individual MetS components during follow-up.

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.

Impact of angiopoietin-1 and -2 on clinical course of idiopathic pulmonary fibrosis

BackgroundAngiopoietin (Ang) -1 and -2 are glycoproteins that play roles in vascular development, angiogenesis, and lung vascular permeability. Although the serum concentrations of Ang-1 and -2 have been evaluated in patients with sepsis, those in patients with idiopathic pulmonary fibrosis (IPF) have received less attention. ObjectiveTo elucidate the clinical significance of Ang-1 and -2 in patients with IPF. MethodsSeventy-five patients with IPF were retrospectively studied. Serum concentrations of Ang-1 and -2 at diagnosis of IPF were measured by enzyme-linked immunosorbent assay. The relationships of the Ang-1 and -2 concentrations with pulmonary function test results, high-resolution computed tomography findings, histologic findings, occurrence of acute exacerbation of IPF (AE-IPF), and prognosis were evaluated. ResultsThe median patient age was 68 year-old and the median observation period was 44 months. IPF patients with high Ang-2 concentrations showed a significantly lower forced vital capacity (FVC) (2.28 vs. 2.69 L, respectively; p = 0.047) and lower percent diffusion lung capacity for carbon monoxide (%DLCO) (61.4 vs. 81.4%, respectively; p = 0.015) than patients with low Ang-2 concentrations. Serum Ang-2 concentrations were negatively correlated with %DLCO (r = −0.375, p = 0.021) and the change in %FVC in 12 months (r = −0.348, p = 0.043). The Ang-2 concentration was significantly higher in several patients with AE-IPF than in patients in stable condition (p = 0.011). Finally, patients with high Ang-2 concentrations showed a significantly worse prognosis than those with low Ang-2 concentrations (log-rank p = 0.039). Multivariate analysis showed that the serum Ang-2 concentration, but not the Ang-1 concentration, was a significant prognostic factor in patients with IPF (hazard ratio 1.439, p = 0.028). ConclusionIncreases in the serum Ang-2 concentration were associated with disease progression and poor prognosis in patients with IPF.

Correlation between plasma angiopoietin-1, angiopoietin-2 and matrix metalloproteinase-2 in coronary heart disease.

IntroductionAngiopoietin-2 (Ang-2) plays a critical role in inducing tumor cell infiltration, and this invasive phenotype is caused by up-regulation of matrix metalloproteinase (MMP)-2. The relationship between Ang-2 and MMP-2 in atherosclerosis has not been reported yet. The aim is to measure the plasma concentrations of Ang-1, Ang-2 and MMP-2 and assess the correlation between the concentrations of these factors in coronary heart disease (CHD) patients.Material and methodsThe testing was done in a cross-sectional study. We prospectively enrolled 42 individuals with acute myocardial infarction, 42 individuals with unstable angina pectoris, 42 individuals with stable angina pectoris and 45 healthy control subjects. Concentrations of Ang-1, Ang-2 and MMP-2 were measured using the enzyme-linked immunosorbent assay (ELISA) method. Spearman’s rank correlation was calculated to evaluate the relationships between MMP-2 and Ang-1, and MMP-2 and Ang-2 in patients with CHD.ResultsPatients with acute myocardial infarction and unstable angina pectoris had higher Ang-2 and MMP-2 levels compared with stable angina patients and healthy control subjects (p < 0.05), while concentrations of Ang-1 were not statistically different between the groups. Spearman’s rank correlation showed that Ang-2 levels positively correlated with MMP-2 in patients with CHD (r = 0.679, p < 0.001).ConclusionsPlasma Ang-2 and MMP-2 levels but not Ang-1 levels were increased in patients with CHD. Ang-1 correlated weakly with MMP-2, whereas the Ang-2 and MMP-2 correlation was strong in patients with CHD. Ang-2 may play a role in atherosclerosis, and have an interaction with MMP-2.

The Role of Angiogenesis Factor in Bladder Tumors

The study designed to detect Ang-1 expression as angiogenesis factor in bladder tumor tissues, and to clarifythe relationship between Ang-1 and clinicopathological grade of bladder cancer. In this study, the expression of Ang-1 in tumor cells was investigated in (30) patients with transitional cell carcinoma of bladder (TCC) and (20 )patients with inflammatory lesions of the bladder (BIL) as acontrol group from the histopathology laboratories ofAl-Hussain Teaching Hospital and private laboratories in Thi-Qar Province,Iraq . The tissue sections wereanalyzed immunohistochemically (IHC) to detection of Ang-1. The results showed a high positiveimmunohistochemical Ang-1 expression of TCC tissues than in BT tissues (76.7 % versus 40%; p≤ 0.01).Furthermore, the significantly highest level of Ang-1 positive cells was detected in the groups with the highestclinical grade (p≤ 0.05). Ang-1 antigen expression could be indicator of aggressive behavior of bladder cancer

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

High Angiopoietin-1 levels predict a good functional outcome within 72 h of an aneurysmal subarachnoid hemorrhage: A prospective study from a single center

BackgroundThe consequences of aneurysmal subarachnoid hemorrhage (aSAH) are lifelong and fatal. Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2) play an important role in the regulation of vascular structure and function. Our study examined the association between angiopoietin levels and functional outcomes among aSAH patients. MethodsWe enrolled 37 aSAH patients and 39 controls (matched on age and sex) at the Department of Neurosurgery in Tianjin Medical University General Hospital. Serum Ang-1, Ang-2, and Tie-2 levels were collected at 8, 24, and 72hours post-hemorrhage. After a 3-month follow-up period, patient outcomes were evaluated using the Glasgow Outcome Score (GOS). Logistic regression examined the association between angiopoietin levels and outcomes (good [GOS: 4–5] vs. poor [GOS: 1–3]). ResultsaSAH patients had higher levels of Ang-1 at 8hours post-hemorrhage compared to controls. Among aSAH patients, Ang-1 levels at 8, 24, and 72hours post-hemorrhage were higher among patients with a good outcome. Compared to patients with low Ang-1 levels, high Ang-1 levels at 72hours post-hemorrhage were associated with a good outcome. ConclusionsHigh Ang-1 levels were associated with a good functional outcome after aSAH. Abnormal angiopoietin levels may disrupt the blood–brain barrier and contribute to functional outcomes in aSAH patients.

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.

Angiopoietin-2 in Bone Marrow milieu promotes Multiple Myeloma-associated angiogenesis

Angiopoietin-2 (Ang-2) is involved in angiogenesis in both solid and hematological malignancies. In Multiple Myeloma (MM), serum Ang-2 correlates with disease progression and response to therapy. To address the patho-physiologic role of Ang-2 in MM associated angiogenesis, we used sera from patients with active MM, which contained significantly higher levels of the molecule, compared to those from patients with smoldering MM and Monoclonal Gammopathy of Undetermined Significance. MM Bone Marrow (BM) sera with high Ang-2 concentration specifically contributed to endothelial cell (EC) activation, while Ang-1 containing sera maintained EC stabilization. The functional dichotomy of Ang-1 and Ang-2 was confirmed by the triggering of distinctive signaling pathways down-stream the common Tie-2 receptor, i.e., the Akt or the ERK- phosphorylation pathway. Notably, Ang-2 but not VEGF serum levels correlated with BM micro-vessel density, further underscoring the key role of Ang-2 in angiogenesis. Western Blot, RT-PCR and immunocytochemistry identified MMEC as the major source of Ang-2, at variance with MM cells and CD14+ BM monocytes. These data suggest that Ang-2 produced in the BM milieu may contribute to MM angiogenesis and suggest that the molecule can be further exploited both as angiogenesis biomarker and as a potential therapeutic target.

Abstract 4492: Blockade of angiopoietin-2 or Tie2 is equally effective at inhibiting tumor growth and reducing tumor vessel density in most human tumor xenograft models

Abstract Angiopoietin-1 (Ang1) and -2 (Ang2) regulate angiogenesis via the endothelial cell-specific receptor tyrosine kinase Tie2. Blocking Ang2 binding to Tie2 decreases vessel density and inhibits tumor growth in various human xenograft models. However, not all tumors respond to Ang2 blockade, which could be due to high Ang1 levels acting as another ligand for Tie2, to Ang2 activities independent of Tie2, or to a weak role for Ang-2/Tie2 signaling in these tumors. To elucidate if Ang1 plays an integral role in tumor angiogenesis and growth, we compared the effects of blocking Ang2 or Tie2 in various tumor xenografts with differing levels of Ang1 expression. We found that in six out of seven tumor models, antibodies that specifically bind Ang2 (REGN910, 10 mg/kg 2x/wk) or that bind Tie2 and block binding of both Ang1 and Ang2 (REGN1376, 10 mg/kg 2x/wk) were equally efficacious at inhibiting tumor growth and decreasing vessel density. In only one tumor model (Calu-6), blockade of Tie2 with REGN1376 was more effective than blockade of Ang2 (REGN910) at reducing tumor growth and decreasing tumor vessel density. Although Calu-6 tumors express high Ang1 levels, other tumors tested (Lox, LS174T) have comparable or higher Ang1 levels and respond similarly to Ang2 and Tie2 blockade, thus the levels of Ang1 do not account for the differential response of Calu-6 tumors. Taken together, our data suggest that Ang2 is the dominant ligand for Tie2 in the microenvironment of most tumors, and further, that the effects of Ang2 are mediated primarily via Tie2. These findings support the approach to specifically block the binding of Ang2 to Tie2 as an anti-angiogenic therapy. Citation Format: Alexander P. Adler, Christopher Daly, Asma A. Parveen, Thomas Nevins, Jing Shan, Jeanette Fairhurst, Tammy Huang, Joel Martin, Nicholas Papadopoulos, George D. Yancopoulos, Gavin Thurston, Gavin Thurston, Alexandra Eichten. Blockade of angiopoietin-2 or Tie2 is equally effective at inhibiting tumor growth and reducing tumor vessel density in most human tumor xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4492. doi:10.1158/1538-7445.AM2014-4492

Serum angiopoietin-2 and soluble VEGFR-2 levels predict malignancy of ovarian neoplasm and poor prognosis in epithelial ovarian cancer.

BackgroundThe aim of the study was to explore the serum levels of eight angiogenesis biomarkers in patients with benign, borderline or malignant epithelial ovarian neoplasms and to compare them to those of healthy controls. In addition, we aimed to study how those biomarkers predict the clinical course and survival of patients with epithelial ovarian cancer.MethodsWe enrolled 132 patients with ovarian neoplasms and 32 unaffected women in this study. Serum samples were collected preoperatively at the time of diagnosis and the levels of angiogenesis biomarkers were measured with an ELISA.ResultsLevels of Ang-1, Ang-2, VEGF, VEGF-D, VEGF/sVEGFR-2 and Ang-2/ sVEGFR-2 ratios were elevated whereas sVEGFR-2 was lower in patients with ovarian carcinoma than in women with normal ovaries, benign and/or borderline ovarian neoplasms. In ROC analysis, the area under the curve for serum Ang-2/sVEGFR-2 ratio (0.76) was greater than Ang-2 (0.75) and VEGF (0.65) but lower than for CA 125 (0.90) to differentiate ovarian cancer from benign or borderline ovarian tumors. In ovarian cancer high Ang-2/sVEGFR-2 ratio was associated with the presence of ascites, high stage and grade of ovarian cancer, with the size of primary residual tumor >1 cm and with recurrence of disease. Elevated Ang-2, VEGF, VEGF/sVEGFR-2, Ang-2/VEGF and Ang-2/sVEGFR-2 ratios and low level of sVEGFR-2 were significant predictors of poor overall survival (OS) and recurrence free survival (RFS) in univariate survival analyses.ConclusionsOvarian cancer patients had elevated levels of angiogenesis related growth factors in circulation reflecting increased angiogenesis and poor prognosis. The serum level of Ang-2 predicted most accurately poor OS and Ang-2/sVEGFR-2 ratio malignancy of ovarian neoplasms and short RFS.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-696) contains supplementary material, which is available to authorized users.