Higher Ang-2 Levels Research Articles

Association of angiopoietin-2, C-reactive protein and markers of obesity and insulin resistance with survival outcome in colorectal cancer

Background:This study investigated the relationship of obesity, insulin resistance, inflammation and angiogenesis with cancer progression and survival in a colorectal cancer cohort.Methods:Clinical and pathological data, along with anthropometric and follow-up data, were collected from 344 consecutive colorectal cancer patients. Serum samples at diagnosis were analysed by immunoassay for adiponectin, C-reactive protein (CRP), vascular endothelial growth factor-A (VEGF-A), angiopoietin-2 (Ang-2), insulin-like growth factor-1 (IGF-1), insulin and C-peptide.Results:Serum Ang-2 and VEGF-A levels increased with tumour T stage (P=0.007 and P=0.025, respectively) and N stage (P=0.02 and P=0.03, respectively), and correlated with CRP levels (r=0.43, P<0.001 and r=0.23, P<0.001, respectively). Angiopoietin-2 correlated with C-peptide (r=0.14, P=0.007) and VEGF-A with IGF-1 in males (r=0.25, P=0.001). Kaplan–Meier analysis showed that patients with high serum levels of CRP and Ang-2 had significantly reduced survival (both P⩽0.001). After adjusting for tumour stage and age, Ang-2 remained a significant predictor of survival. The CRP levels were inversely associated with survival in American Joint Committee on Cancer stage II patients (P=0.038), suggesting that CRP could be used to support treatment decisions in this subgroup. Serum markers and anthropometric measures of obesity correlated with each other, but not with survival.Conclusion:Our study supports the concept that obesity-related inflammation, rather than obesity itself, is associated with colorectal cancer progression and survival. The study confirms serum Ang-2 as a predictive marker for outcome of colorectal cancer.

Preoperative angiopoietin-2 serum levels: a marker of malignant potential in ovarian neoplasms and poor prognosis in epithelial ovarian cancer.

The aims of the study were to explore the levels of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in patients with benign, borderline, or malignant epithelial ovarian tumors and to compare them to those of healthy controls. In addition, we aimed to study how Ang-1 and Ang-2 levels predict the clinical course and survival of patients with epithelial ovarian cancer. We enrolled 150 patients with ovarian neoplasms and 34 women with healthy ovaries in this study. Furthermore, we measured the levels of Ang-1 and Ang-2 in patients having an ovarian metastasis or another cancer (n = 29). Serum samples were collected preoperatively at the time of diagnosis, and Ang-1 and Ang-2 levels were measured with an enzyme-linked immunosorbent assay. Angiopoietin-1 and Ang-2 levels were significantly elevated in serum samples of patients with ovarian carcinoma compared with healthy controls (P = 0.0005 and P < 0.0005, respectively). In addition, Ang-2 levels were significantly higher in patients with ovarian carcinoma compared with patients with benign (P < 0.0005) or borderline ovarian tumors (P = 0.011). In receiver operating characteristic analysis, the area under the curve for serum Ang-2 (0.77) was greater than Ang-1 (0.60) but lower than for cancer antigen 125 (0.95) to differentiate ovarian cancer from healthy control. High serum levels of Ang-1 and Ang-2 were associated with primary residual tumor more than 1 cm after debulking surgery, and high Ang-2 levels correlated positively with an advanced tumor stage (P = 0.042). Elevated Ang-2 level (>2.7 ng/mL) was a significant predictor of poor overall and recurrence-free survival (P = 0.043 and P = 0.033, respectively) when assessing Kaplan-Meier curves by a log-rank test. Patients with ovarian cancer have higher serum levels of angiopoietins than patients with benign or borderline tumors reflecting the increased angiogenesis. These results also suggest that Ang-2 may serve as an angiogenic marker of decreased patient survival in ovarian cancer.

Angiopoietin-2 Interferes with Anti-VEGFR2–Induced Vessel Normalization and Survival Benefit in Mice Bearing Gliomas

In brain tumors, cerebral edema is a significant source of morbidity and mortality. Recent studies have shown that inhibition of vascular endothelial growth factor (VEGF) signaling induces transient vascular normalization and reduces cerebral edema, resulting in a modest survival benefit in glioblastoma patients. During anti-VEGF treatment, circulating levels of angiopoietin (Ang)-2 remained high after an initial minor reduction. It is not known, however, whether Ang-2 can modulate anti-VEGF treatment of glioblastoma. Here, we used an orthotopic glioma model to test the hypothesis that Ang-2 is an additional target for improving the efficacy of current anti-VEGF therapies in glioma patients. To recapitulate high levels of Ang-2 in glioblastoma patients during anti-VEGF treatment, Ang-2 was ectopically expressed in U87 glioma cells. Animal survival and tumor growth were assessed to determine the effects of Ang-2 and anti-VEGF receptor 2 (VEGFR2) treatment. We also monitored morphologic and functional vascular changes using multiphoton laser scanning microscopy and immunohistochemistry. Ectopic expression of Ang-2 had no effect on vascular permeability, tumor growth, or survival, although it resulted in higher vascular density, with dilated vessels and reduced mural cell coverage. On the other hand, when combined with anti-VEGFR2 treatment, Ang-2 destabilized vessels without affecting vessel regression and compromised the survival benefit of VEGFR2 inhibition by increasing vascular permeability. VEGFR2 inhibition normalized tumor vasculature whereas ectopic expression of Ang-2 diminished the beneficial effects of VEGFR2 blockade by inhibiting vessel normalization. Cancer treatment regimens combining anti-VEGF and anti-Ang-2 agents may be an effective strategy to improve the efficacy of current anti-VEGF therapies.

Angiopoietin-2 Plasma Dosage Predicts Time to First Treatment (TTFT) and Overall Survival (OS) in Chronic Lymphocytic Leukemia.

Abstract 1260▪▪This icon denotes an abstract that is clinically relevant.Angiopoietin-2 (Ang2) is a 75 kDa secreted glycoprotein able to bind to the receptor tyrosine kinase Tie-2 and detectable especially in vascular remodeling sites. High Ang2 levels are observed in highly vascularized tumors and may play a role in tumor angiogenesis. We have recently demonstrated that Ang2 protein secreted by CLL cells increases in vivo and in vitro angiogenesis and that, in a small CLL cohort, higher Ang2 mRNA expression is associated with advanced stage disease and shorter progression-free survival. In order to further investigate potential prognostic role of Ang2 in CLL, we measured Ang2 plasma levels using an ELISA-based method, in a large multicentric series: 42(13.3%) of 316 analyzed patients had previously received treatment and an additional 53(16.8%) required therapy during the study course, with a median TTFT of 40 months. Ang2 dosage ranged from 972 to 17281 pg/ml (median, 2061 pg/ml). The best cut-off point, generated by ROC analysis and Youden's index (state variable, treating), was 2460 pg/ml and divided our cohort in two subsets (high Ang2 and low Ang2) composed by 100 (31.6%) and 216 (68.4%) patients, respectively. The median TTFT resulted significantly shorter (P<.001) in the high Ang2 subgroup (77.5 months) than in the low one (179.2 months). Cox univariate analysis identified Ang2 ≥ 2460 pg/ml as a predictor of reduced TTFT (HR 2.437; 95%CI 1.621 - 3.664, P<.001) as well as advanced Binet stage, unmutated IGHV status, high CD38, ZAP-70 and CD49d expression, intermediate/high cytogenetic risk and high β2 microglobulin (P<.001 in all instances). Multivariate analysis confirmed that high Ang2 levels were an independent prognosticator for TTFT (HR 1.739; 95%CI 1.059 – 2.857; P=.029) together with inter/high FISH risk (P<.001) and unmutated IGHV status (P= .002). Comparing OS between high Ang2 and low Ang2 subgroup, we found that 26% of high Ang2 patients were dead at 10-years from diagnosis, in contrast with 7% of low Ang2 ones (P=.002). In univariate analysis, Ang2 ≥ 2460 pg/ml resulted to be a predictor of poor OS (HR 3.566; 95%CI 1.496 – 8.499; P=.004) as well as most of the other known unfavorable prognosticators. Significant association was found between high Ang2 plasma level and Binet stages B-C (P<.001), high β2 microglobulin (P<.001), unmutated IGHV status (P<.001), high CD38 and ZAP-70 expression (P<.001 and P=.003, respectively) and inter/high cytogenetic risk (P=.005). However, a relevant percentage of patients showed high Ang2 levels in the presence of favorable markers and vice versa: in this cases, defined as discordant, Ang2 helps to refine prognosis identifying CLL subgroup with precocious need for treatment and reduced survival despite characterized by favorable prognostic factors. In order to evaluate if Ang2 dosage could be modulated by treatment, we compare Ang2 level of plasma samples collected before (n=53) and after (n=42) the first therapy and we did not find any statistical difference (P=.6). Finally, we studied two serial plasma samples collected from 36 patients (median interval, 19 months; range, 3-30 months) and we did not find significant changes in Ang2 level between the two measurements, both for the 7 patients treated inside this interval (P=.612) and for the 29 cases untreated (P=.347). These data first demonstrate the prognostic role of Ang2 plasma level for TTFT and OS in CLL, and shows how Ang2 contributes to refine the prognostic assessment of CLL. The measurement of serial plasma samples demonstrates that Ang2 levels are quite stable during the disease course and are not modulated by current treatments: this suggests that higher Ang2 secretion could be a peculiar biological characteristic of more aggressive CLL, already present in the first disease steps. Our study implies a simple ELISA method, with a high reproducibility and reliability of results, that could be used both for clinical practice and for further studies, in order to better understand CLL pathogenesis and identify new target therapies. Since several molecules targeting microenvironment and angiogenesis are developing in clinical trials for CLL, it could be very interesting to evaluate if they are able to modulate Ang2 secretion and modify the disease clinical aggressiveness. DisclosuresNo relevant conflicts of interest to declare.

Effects of Eicosapentaenoic Acid on Endothelial Cell-Derived Microparticles, Angiopoietins and Adiponectin in Patients with Type 2 Diabetes

The aim of this study was to evaluate the significance of endothelial cell-derived microparticles (EDMP), angiopoietin-2 (Ang-2) and adiponectin in hyperlipidemic patients with and without type 2 diabetes mellitus, and to compare the two for the effects of eicosapentaenoic acid (EPA) on these markers. One hundred and twenty-six hyperlipidemic patients with and without type 2 diabetes mellitus received EPA 1,800 mg daily, and 50 of the patients were non-diabetic. EDMP and Ang-2 levels prior to treatment were higher in diabetic patients than in non-diabetic patients, whereas adiponectin levels were lower in diabetics. When diabetic patients were classified into two groups on the basis of Ang-2 levels, the levels of all markers remained unchanged in those without a high Ang-2 level after EPA treatment. In contrast, all markers except for adiponectin were decreased significantly in diabetic patients with high Ang-2 levels after 6 months of EPA treatment. These diabetic patients with high Ang-2 levels displayed a more significant increase in adiponectin levels after EPA treatment than those who did not. These results suggest that EPA possesses an adiponectin-dependent anti-atherosclerotic effect and may be beneficial for the prevention of vascular complications in diabetic patients with high Ang-2 levels.

Angiopoietin‐2 levels in the hepatic vein as a useful predictor of tumor invasiveness and prognosis in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is characteristically a hypervascular tumor and its progression is known to be closely related to angiogenesis. In this study, we investigated angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) levels in the hepatic vein draining from HCC, as well as in the peripheral vein, to evaluate their relation to clinicopathological features and prognosis. To obtain hepatic venous blood samples, a catheter was placed into the main branch of the hepatic vein draining from HCC in 21 patients. The Ang-2 and VEGF levels in both the hepatic and peripheral veins were investigated. Furthermore, Ang-2 mRNA expression in surgically resected HCC was evaluated by quantitative reverse-transcription polymerase chain reaction (RT-PCR), as well as microvessel density (MVD) by CD34 immunostaining. Ang-2 levels in the hepatic vein significantly correlated with Ang-2 mRNA expression in HCC, but Ang-2 levels in the peripheral vein did not correlate. Furthermore, a significant correlation was found between hepatic venous Ang-2 and MVD levels, whereas there was no significant correlation with hepatic venous VEGF levels. When hepatic venous Ang-2 levels were compared with clinicopathological features, a significant relationship was found between high Ang-2 levels and portal vein invasion. The survival for patients in the high hepatic venous Ang-2 group was significantly poorer when compared with the low group. Preoperative hepatic venous Ang-2 levels may be a good predictor for portal vein invasion and also prognosis in patients with HCC.

The detection and clinical significance of serum angiopoietin-2 levels in non-small cell lung cancer

Objective To investigate serum angiopoietin-2(Ang-2)levels in non-small cell lung cancer(NSCLC)and its clinical significance.Methods Patients with NSCLC(n=98)and healthycontrols (n=30)were recruited into this study.Serum Ang-2 levels were measured using enzyme-linked immunosorbent assays(ELISA).The results were compared between patients with ifferent tumor stages,metastasis status,and postoperative recurrence 1 year after surgery.Results Patients with NSCLC had higher Ang-2 levels[median 3 230.5 ng/L,range(1 231.6～4 612.5)ng/L ]than the control group [median 831.7 ng/L,range(592.4-1 701.1)ng/L,U=15,P<0.01].Serum Ang-2 levels in stage Ⅲpatients[median 2 311.7 ng/Lrange(1 512.9～3 530.7)ng/L]were significantly higher than stage I [median 1 593.6 ng/L,range(1 235.2～2 763.1)ng/L,H=27.66,P<0.01]and stage Ⅱ patients [median 1 581.4 ng/L,range(1 231.6～2 852.3)ng/L,H=27.66,P<0.01],but significantly lower than stage Ⅳ patients[median 3 808.2 ng/L,range(2 235.8～4 612.5)ng/L,H=65.6P<0.01].Serum Ang-2 levels were higher in patients with distant metastasis or postoperative recurrence than those without them.Conclusion Serum Ang-2 level in patients with NSCLC could be a useful marker for metastasis and postoperative recurrence. Key words: Lung neoplasms; Carcinoma,non-small-cell tung; Angiopoietin-2; Enzyme-linked immunosorbent assay

Angiopoietin-2, Marker and Mediator of Endothelial Activation With Prognostic Significance Early After Trauma?

To measure plasma levels of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and vascular endothelial growth factor (VEGF) early after trauma and to determine their clinical significance. Angiopoietins and VEGF play a central role in the physiology and pathophysiology of endothelial cells. Ang-2 has recently been shown to have pathogenetic significance in sepsis and acute lung injury. Little is known about the role of angiopoietins and VEGF early after trauma. Blood specimens from consecutive major trauma patients were obtained immediately upon arrival in the emergency department and plasma samples assayed for Ang-1, Ang-2, VEGF, markers of endothelial activation, protein C pathway, fibrinolytic system, and complement. Base deficit was used as a measure of tissue hypoperfusion. Data were collected prospectively. Blood samples were obtained from 208 adult trauma patients within 30 minutes after injury before any significant fluid resuscitation. Plasma levels of Ang-2, but not Ang-1 and VEGF were increased and correlated independently with severity of injury and tissue hypoperfusion. Furthermore, plasma levels of Ang-2 correlated with markers of endothelial activation, coagulation abnormalities, and activation of the complement cascade and were associated with worse clinical outcome. Ang-2 is released early after trauma with the degree proportional to both injury severity and systemic hypoperfusion. High levels of Ang-2 were associated with an activated endothelium, coagulation abnormalities, complement activation, and worse clinical outcome. These data indicate that Ang-2 is a marker and possibly a direct mediator of endothelial activation and dysfunction after severe trauma.

VEGF-A, VEGF-C, and CD31 Expression by Leukemia Cells Are Prognostic Factors for Overall Survival in Acute Myeloid Leukemia (AML).

INTRODUCTION: Bone marrow angiogenesis has increasingly been recognized as important for leukemogenesis. Identification of vascular endothelial growth factor (VEGF) mediated autocrine and paracrine loops in subsets of acute myeloid leukemia (AML) support multiple mechanisms of action for these factors. We examined whether expression of pro-angiogenic growth factors VEGF-A, VEGF-C, and angiopoietin-2 (Ang-2) correlated with marrow neovascularization (CD31 staining) and overall survival of AML patients (pts).PATIENTS AND METHODS: Expression of VEGF-A, VEGF-C, and Ang-2 was determined in leukemia bone marrow samples at diagnosis by quantitative real-time reverse transcription polymerase chain reaction (Q-PCR) using 18s RNA controls. Expression was then analyzed for fold-differences versus normal bone marrow control samples (n=10). VEGF-A and VEGF-C protein expression in leukemia cells and CD31 protein expression in microvessels and leukemia cells was evaluated by immunohistochemistry (IHC) in the corresponding bone marrow biopsies from the same pts. A cohort of 96 AML pts (46 male, 50 female) was examined with a median age of 67.5 years (range 21–96 years). Half (n=46) achieved an initial complete remission (CR) on standard induction chemotherapy regimens. Median overall survival was 9.3 months. No pts had recurrent cytogenetic abnormalities, and 76 had normal karyotype.RESULTS: Leukemia blast VEGF-A (median 1.77 fold increase, range 0.06–23.62) and Ang-2 (median 6.9 fold increase, range 0.07–3100) expression by Q-PCR were consistently higher than normal bone marrow controls in contrast to VEGF-C that was lower than normal controls (median 0.51, range 0.04–27.93). Using a cut-off of 1.5 fold to denote growth factor overexpression by Q-PCR, we found that prolonged pt survival correlated with low VEGF-C levels (mean 609 ± 92 n=78 vs. high VEGF-C 235 ± 46 days, n=18, p =0.056) and high Ang-2 levels (mean 581± 91, n=77 vs. low Ang-2 366 ± 98 days, n=19 vs. p=0.26) but was not statistically significant. No difference in survival was noted in low vs. high VEGF-A expressing samples (mean 545 + 171, n=26, vs. 511 + 105, n=70, p=0.96). No correlation between CD31+ microvessels and survival was noted. By IHC, most leukemic marrow biopsies were positive for VEGF-A (n=60) and negative for VEGF-C (n=87) and CD31 (n=78). Univariate analysis of overall survival using Cox proportional hazard model demonstrated that age (p=0.0008), achievement of CR (p<0.0001), karyotype (normal vs. complex/other, p=0.0210), and VEGF-A (p=0.0272), VEGF-C (p=0.0255), and CD31 positive IHC expression by AML blasts (p=0.0152) were independent prognostic factors. Multivariate analysis showed that the only factors of statistical significance were achievement of CR (hazard ratio 4.09, 95% CI 2.28–7.33, p <0.0001) and VEGF-C expression by IHC (hazard ratio 0.08, 95% CI 0.01-0.81).CONCLUSIONS: These results show that expression of VEGF-A, VEGF-C, and CD31 (in AML blasts) by IHC in bone marrow biopsies represent prognostic factors for overall survival in AML. The importance of VEGF-C mediated angiogenic processes as an independent prognostic factor in AML outcome suggests that targeted inhibition of this factor may be of future therapeutic benefit in subsets of AML pts.

Release of angiopoietin-1 by primary human acute myelogenous leukemia cells is associated with mutations of nucleophosmin, increased by bone marrow stromal cells and possibly antagonized by high systemic angiopoietin-2 levels

The balance between proangiogenic Angiopoietin-1 (Ang-1) and the antagonistic Ang-2 is important both for leukemogenesis and chemosensitivity in human acute myelogenous leukemia (AML). We examined the release of Ang-1 and Ang-2 by AML cells cultured alone and in cocultures with stromal cells. Detectable Ang-1 release from AML cells was observed for most patients (62/91), whereas Ang-2 release was detected only for a minority (23/91). Coculture of AML and stromal cells led to increased Ang-1 levels. Furthermore, the role of the angiopoietin system was investigated by characterizing whether the differences in angiopoietin expression in AML patients can be related to nucleophosmin (NPM1) mutations. We compared the gene expression profiles of AML cells derived from 19 patients with FLT3 mutations and normal cytogenetics with and without NPM1 mutations and observed increased expression of Ang-1 in patients with NPM1 mutations. Finally, we found significantly higher Ang-2 levels in serum of AML patients compared with healthy controls. Our results suggest that AML cells are a major source of Ang-1 in leukemic bone marrow, especially in patients with NPM1 mutations, but the local levels are also influenced by stromal cells. Local Ang-2 release from AML cells is less common, but high systemic levels of Ang-2 may affect bone marrow angioregulation.

Expression of angiopoietins and vascular endothelial growth factors and their clinical significance in acute myeloid leukemia

Angiogenic factors play an essential role in normal and pathologic angiogenesis, but their clinical role in acute myeloid leukemia (AML) remains unclear. We investigated the expression of Ang-1, Ang-2, Tie2, VEGF-A, and VEGF-C genes in bone marrow (BM) mononuclear cells by real-time quantitative PCR (RQ-PCR) in a cohort of 126 patients with newly diagnosed de novo AML and normal marrow donors. Here we show that high pre-treatment levels of Ang-2 in the BM indicate an unfavorable prognosis in AML. Only karyotype (hazard ratio 2.19, 95% CI 1.25–3.42, P = 0.005) and expression of Ang-2 (hazard ratio 2.05, 95% CI 1.20–3.52, P = 0.009), but not other angiogenic factors, were independent prognostic factors for overall survival by multivariate analysis. The prognostic significance of Ang-2 expression was more obvious in the subgroup of patients with intermediate-risk cytogenetics. Subgroup analysis showed that Ang-2 expression had prognostic impact on patients with low (but not high) Ang-1 or Tie2 levels, and on patients with high (but not low) VEGF-A or VEGF-C levels.

Circulating angiopoietin-2 is a strong prognostic factor in acute myeloid leukemia

Angiogenesis plays an important role in solid tumors and hematologic malignancies. The angiopoietins act as essential regulators in this process. We investigated the impact of circulating angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and soluble Tie2 (sTie2) on overall survival in patients with acute myeloid leukemia (AML). Ang-1, Ang-2 and sTie2 were measured in plasma samples from 68 AML patients and 11 controls using enzyme-linked immunosorbent assay. Circulating levels of Ang-2 and sTie2 (median (range): 1098.0 (361.4-4147.6) pg/ml and 3.40 (1.21-10.00) ng/ml, respectively) were significantly elevated in AML patients as compared to controls (307.9 (199.7-1225.0) pg/ml and 2.88 (1.71-3.29) ng/ml; P<0.001 and P=0.014). In a univariate Cox proportional hazards model, higher levels of Ang-2 and sTie2 were predictive of poor survival. In multivariate analyses, Ang-2 and cytogenetics proved to be independent prognostic factors, with a relative risk of 4.07 (95% confidence interval (CI) 1.88-8.81) and 2.70 (95% CI 1.25-5.81), respectively. The 3-year survival rate for AML patients with Ang-2 levels>/=1495.6 pg/ml was only 14.7% compared to 64.7% for those with Ang-2 levels<1495.6 pg/ml. These data provide evidence that circulating Ang-2 represents an independent prognostic factor in AML and may be used as a prognostic tool in the risk-adapted management of AML.

Systemic Overexpression of Angiopoietin-2 Promotes Tumor Microvessel Regression and Inhibits Angiogenesis and Tumor Growth

Angiopoietin-2 (Ang-2) is a conditional antagonist and agonist for the endothelium-specific Tie-2 receptor. Although endogenous Ang-2 cooperates with vascular endothelial growth factor (VEGF) to protect tumor endothelial cells, the effect on tumor vasculature of high levels of exogenous Ang-2 with different levels of VEGF has not been studied in detail. Here, we report that systemic overexpression of Ang-2 leads to unexpected massive tumor vessel regression within 24 h, even without concomitant inhibition of VEGF. By impairing pericyte coverage of the tumor vasculature, Ang-2 destabilizes the tumor vascular bed while improving perfusion in surviving tumor vessels. Ang-2 overexpression transiently exacerbates tumor hypoxia without affecting ATP levels. Although sustained systemic Ang-2 overexpression does not affect tumor hypoxia and proliferation, it significantly inhibits tumor angiogenesis, promotes tumor apoptosis, and suppresses tumor growth. The similar antitumoral, antiangiogenic efficacy of systemic overexpression of Ang-2, soluble VEGF receptor-1, and the combination of both suggests that concomitant VEGF inhibition is not required for Ang-2-induced tumor vessel regression and growth delay. This study shows the important roles of Ang-2-induced pericyte dropout during tumor vessel regression. It also reveals that elevated Ang-2 levels have profound pleiotropic effects on tumor vessel structure, perfusion, oxygenation, and apoptosis.

Expression of Angiopoietins and Their Receptor Tie2 in the Bone Marrow of Patients with Acute Myeloid Leukemia.

Angiopoietin-1 (Ang-1) and its natural antagonist Angiopoietin-2 (Ang-2), both ligands for the receptor tyrosine kinase Tie2, are known to play an essential role in normal and pathological angiogenesis. However, the importance of angiopoietin signaling in the pathophysiology of hematologic neoplasias such as acute myeloid leukemia (AML) remains to be elucidated. We investigated the expression of Ang-1, Ang-2 and Tie2 by immunohistochemical analyses in bone marrow biopsies of 64 adult patients with newly diagnosed AML and correlated angiogenic factor expression with clinicopathological variables and long-term survival. Expression of Ang-2 was significantly increased in the bone marrow of AML patients (median [interquartile ranges]: 4.7 [3.3 – 5.7] AU [arbitrary units]) as compared with 16 control patients (1.5 [1.5 – 1.8] AU; P < 0.0001). In contrast, Ang-1 expression levels in AML patients did not differ from those found in controls. Thus, we observed a reversal of the Ang-1 and Ang-2 expression balance in the neoplastic bone marrow (Ang-2:Ang-1 ratio: 1.73) as compared with normal bone marrow (0.51; P < 0.0001). Furthermore, the angiopoietin receptor Tie2 was significantly overexpressed in leukemic blasts (3.8 [2.8 – 4.9] AU vs. 1.8 [1.6 – 2.3] AU; P < 0.0001). Patients expressing high levels of Ang-2 showed significantly longer overall survival (OS) than those with low Ang-2 levels (52.7 vs. 14.7 months; P = 0.039). The impact of Ang-2 expression on OS was especially evident in AML patients simultaneously expressing low levels of Ang-1 (P = 0.0298). Multivariate Cox regression analysis revealed karyotype and Ang-2 expression as independent prognostic factors for OS (hazard ratio [CI]: 3.06 [1.39 – 6.70] and 0.31 [0.14 – 0.69], respectively; P < 0.01). In conclusion, these data provide evidence that the alteration of angiopoietin balance in favor of Ang-2 may play a critical role in the pathophysiology of AML. Furthermore, high pre-therapeutic bone marrow Ang-2 levels indicate a favorable prognosis in polychemotherapy treated AML by a yet unknown mechanism.

Angiopoietin-2 Expression in B-Cell Chronic Lymphocytic Leukemia: Association with Clinical Outcome and Immunoglobulin Heavy-Chain Mutational Status.

B-cell Chronic Lymphocytic Leukemia (B-CLL) follows an extremely variable clinical course. For some patients CLL is an indolent disease that never progresses to the point of requiring therapy and these patients have a survival time similar to age-matched controls. On the contrary, other patients experience rapidly deteriorating blood count and organomegaly which requires prompt treatment. The overall survival (OS) times range from months to decades. B-CLL patients can be divided into two subgroups on the basis of the presence or absence of somatic mutations in the specific immunoglobulin heavy-chain variable region (IgVH) genes used by leukemic cells. Patients with unmutated IgVH genes usually have an advanced stage and an unfavourable cytogenetic features, require therapy and have a short survival. The biological reasons of the different behaviour of Ig-mutated and Ig-unmutated leukemic clones have not been fully elucidated yet. Angiogenesis is a very complex network which is closely regulated by the orchestrating functions of many angiogenic factors. The balance of cellular expression of all these angiogenic factors determines vascular stabilization or angiogenic remodelling and sprouting or vessel regression. Increasing evidence shows that neovascularization plays a role in the biology of chronic lymphocytic leukemia. The goal of this study was to evaluate the angiogenic status of Ig-mutated and Ig-unmutated CLL in the attempt to identify a possible role of angiogenesis in the adverse clinical outcome of Ig-unmutated CLL patients. So, we first performed a large scale gene-expression analysis on 29 B-CLL patients using microarrays comprising about 20,000 probes and 208 angiogenesis-related genes. We identified 64 up-regulated genes in Ig-unmutated CLL relative to Ig-mutated CLL. Among them, we found angiopoietin-2 (Ang-2) as one of the highest differentially expressed gene (p=3.02x10−6). Then, we evaluated the Ang-2 expression both at transcript and protein level in a wide cohort of CLL, in normal controls and in other haematological malignancies. The data showed an extremely wide range of Ang-2 expression in B-CLL: Ang-2 high-expressing cases were characterized by advanced Binet stage (p=0.032) and significantly shorter progression-free survival than Ang-2 low-expressing subset (median, 16 vs. 146 months) (p=0.006). Moreover, there was a strong correlation between the IgVH mutational status and the Ang-2 gene expression level (p<0.0001). Ig-mutated CLL exhibited very low Ang-2 expression, absolutely similar to the levels measured in healthy controls (median, 0.27 vs. 0.32, p=0.959). On the contrary, Ig-unmutated CLL expressed up to one hundred-fold higher levels of Ang-2 than normal controls(median, 38.61 vs. 0.32, p=0.002). Moreover, Ang-2 was up-regulated in all investigated CML, ALL and AML samples. We observed extremely high levels of expression in CML and ALL (median Ang-2 mRNA, 3948.96 and 190.00, respectively), whereas moderately high levels of Ang-2 were found in AML patients (median, 16.21). These data suggest that increased angiogenesis due to high Ang-2 expression may be involved in adverse clinical outcome of Ig-unmutated CLL patients.

In utero angiopoietin-2 gene delivery remodels placental blood vessel phenotype: a murine model for studying placental angiogenesis

Angiopoietin (Ang)-2, the natural antagonist of the Ang1/Tie2 receptor is a complex regulator of blood vessel plasticity that plays a pivotal role in both vessel sprouting [in the presence of vascular endothelial growth factor (VEGF)-A] and vessel regression (in the absence of VEGF-A). Based on the spatial and temporal expression of Ang2 throughout human gestation, we recently suggested that the Ang2 may play a pivotal role in placental angiogenesis. Further, to examine this tenet we have developed a novel murine model system in which in utero Ang2 gene delivery via a non-replicating adenoviral expression vector has the potential to manipulate the blood vessel phenotype in vivo during pregnancy. Ang2 overexpression selectively and rapidly remodels the labyrinth perivascular extracellular matrix, subsequently promoting plasticity of the maternal and fetal vessels, which appear honeycombed due to a 2-fold increase in blood vessel luminal area. High levels of Ang2 impair endothelial cell adhesiveness, leading to vascular leakiness with perivascular oedema, which increases placental weight. These observations suggest that the Ang2 overexpression may play a key role in placental vascular remodelling. Furthermore, we suggest a novel new model to study the pathobiology of placental vascularization and the effect of placental blood vessels on fetal phenotype.

Different effects of angiopoietin‐2 in different vascular beds in the eye: new vessels are most sensitive

In this study, we used double transgenic mice with inducible expression of angiopoietin-2 (Ang2) to investigate the role of Ang2 in the retinal and choroidal circulations and in three models of ocular neovascularization (NV). Mice with induced expression of Ang2 ubiquitously, or specifically in the retina, survived and appeared grossly normal. They also had normal-appearing retinal and choroidal circulations, demonstrating that high levels of Ang2 did not induce regression of mature retinal or choroidal vessels. When Ang2 expression was induced soon after birth, there was increased density of the deep capillary bed on postnatal day (P) 11 that returned to normal by P18, the time that retinal vascular development is usually completed. In mice with ischemic retinopathy, induction of Ang2 during the ischemic period resulted in a significant increase in retinal NV, but induction of Ang2 at a later time point when ischemia (and vascular endothelial growth factor [VEGF]) was less, hastened regression of NV. In triple transgenic mice that coexpressed VEGF and Ang2, the increased expression of Ang2 inhibited VEGF-induced NV in the retina. Increased expression of Ang2 also resulted in regression of choroidal neovascularization. These data suggest that ocular neovascularization, but not mature retinal or choroidal vessels, is sensitive to Ang2; a high Ang2/VEGF ratio promotes regression, while high Ang2 in the setting of hypoxia and/or concomitantly high Ang2 and VEGF stimulate neovascularization.

Angiopoietin/tie-2 as mediators of angiogenesis: a role in congestive heart failure?

Angiogenic factors, in particular vascular endothelial growth factor (VEGF) and the angiopoietins, Ang-1 and -2, have recently generated significant interest, especially in oncology. The process of angiogenesis is also thought to occur in response to ischaemic conditions, which lie at the core of cardiovascular disease states such as coronary artery disease and congestive heart failure. However, current data do not conclusively show evidence of angiogenesis per se in these conditions, despite (for example) the presence of high levels of VEGF and Ang-2. High levels of these angiogenic factors in heart disease also have not translated into clinically significant new vessel formation, as in accelerated cancer growth or proliferative retinopathy. Indeed, we would hypothesize that these angiogenic markers--especially the angiopoietins--do not necessarily translate into new vessel formation in congestive heart failure (CHF), but may well reflect disturbances of endothelial integrity in CHF.