The angiopoietin/Tie2 system has been identified as the second vascular-specific receptor tyrosine kinase system controlling vessel assembly, maturation, and quiescence.

Genetic experiments (loss-of-function and gain-of-function) have established the role of Angiopoietin/Tie ligand/receptor tyrosine kinase system as a regulator of vessel maturation and quiescence. Angiopoietin-2 (Ang-2) acts on Tie2-expressing resting endothelial cells as an antagonistic ligand to negatively interfere with the vessel stabilizing effects of constitutive Ang-1/Tie-2 signaling. Ang-2 thereby controls the vascular response to inflammation-inducing as well as angiogenesis-inducing cytokines. This study was aimed at assessing the role of Ang-2 as an autocrine (i.e. endothelial-derived) regulator of rapid vascular responses (within minutes) caused by permeability-inducing agents. Employing two independent in vivo assays to quantitatively assess vascular leakage (tracheal microsphere assay, 1–5 min and Miles assay, 20 min), the immediate vascular response to histamine, bradykinin and VEGF was analyzed in Ang-2-deficient (Ang-2−/−) mice. In comparison to the wild type control mice, the Ang2−/− mice demonstrated a significantly attenuated response. The Ang-2−/− phenotype was rescued by systemic administration (paracrine) of an adenovirus encoding Ang-2. Furthermore, cytokine-induced intracellular calcium influx was impaired in Ang-2−/− endothelioma cells, consistent with reduced phospholipase activation in vivo. Additionally, recombinant human Ang-2 (rhAng-2) alone was unable to induce vascular leakage. In summary, we report here in a definite genetic setting that Ang-2 is critical for multiple vascular permeability-inducing cytokines.

No abstract

Suppression of neo‐angiogenesis is a clinically used anti‐tumor strategy with new targets such as angiopoietin‐2 (Ang2) being proposed. However, the functions of Ang2 in vascular remodeling, inflammation and tumor growth are not consistent. We examined effect of depletion of host Ang2 on liver colony formation using Ang2 deficient (Ang2−/−) mice. Surprisingly, the metastatic colonies formed in Ang2−/− mice were larger than those in the wild type. These colonies had greater vascular density with more pericyte coverage than the vessels in liver colonies in the wild type. Liver VEGF concentration in both genotypes was equivalent, and thus, the differences appeared VEGF independent. However, after colony formation, the serum concentration of granulocyte‐colony stimulating factor (G‐CSF) and CXCL1 in Ang2−/− mice was 12 and 6 times greater than after colony formation in wild type. Increase of these two cytokines was associated with two times greater numbers of neutrophils recruited to the liver. Two times more Tie2+/CD11b+/CD31− cells were present in the tumors in Ang2−/− than in the wild type livers. These results suggest that the depletion of host Ang2 induced compensatory VEGF‐independent angiogenic mechanisms and thus enhanced liver metastatic colony growth and colony vascularity. They further indicate organotypic differences in response to tumor metastasis. In contrast, Ang2 deficiency inhibited tumor growth during metastatic colony formation in the lung, consistent with the reports of decreased pulmonary seeding of tumor cells after pharmacological inhibition of Ang2. Further studies are thus required to assess the effects of pharmacological Ang2 blockade for cancer patients particularly in the liver.

For growth and survival in metastasis, tumor colonies must develop new intratumoral vasculature for supply of nutrients and oxygen. Since angiopoietin-2 (Ang2) resulted in a burst of angiogenesis in the glioma model, we examined the effects of host Ang2 on colony growth and neo-angiogenesis during the liver colonization. Liver colonization was induced through the intrasplenic injection of MC38-GFP (murin colorectal carcinoma cells) into two genotype mice of the ang2−/− mice and the wild type mice. When colonies were established, the livers were isolated and analyzed using flow cytometry, immunoflorescent histochemistry and ELISA. In addition, the blood was collected from vena cava for measurement of MCP-1 at 14 days. Flow cytometry analyses of whole livers showed that the tumor cell population in the ang2−/− mice was approximately 2 times larger than in the wild type mice. Immunoflorescent histochemistry of the liver tissue using antibodies for CD31, CD34, CD146, VE-cadherin and NG2 showed that intratumoral vascular density and vascular maturity in the ang2−/− mice were approximately 2 times higher than the wild type mice. Intratumoral vessels were significantly positive for CD146 and CD34, markers more frequently associated with neo-angiogenesis, but much less for CD31. There was no significant difference in VEGF level which is known to mediate vascular response in combination with Ang2. CD11b positive cells and MCP-1 concentration in the livers were equivalent in two genotypes. Notably, there were approximately 10 times more Tie2+/CD11b+ cells in the ang2−/− mice than the wild type mice. CD11c+/CD45+ cell populations were slightly different in two genotypes. These results suggested that host Ang2 would inhibit tumor growth and neo-angiogenesis in the liver, and that the inhibition is correlated with the decreased recruitment of TEMs, one of myeloid cells, into the liver. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2847. doi:10.1158/1538-7445.AM2011-2847

Supplementary Figures 1-3 from Host-Derived Angiopoietin-2 Affects Early Stages of Tumor Development and Vessel Maturation but Is Dispensable for Later Stages of Tumor Growth

Supplementary Figures 1-3 from Host-Derived Angiopoietin-2 Affects Early Stages of Tumor Development and Vessel Maturation but Is Dispensable for Later Stages of Tumor Growth

<div>Abstract<p>The angiopoietin/Tie2 system has been identified as the second vascular-specific receptor tyrosine kinase system controlling vessel assembly, maturation, and quiescence. Angiopoietin-2 (Ang-2) is prominently up-regulated in the host-derived vasculature of most tumors, making it an attractive candidate for antiangiogenic intervention. Yet, the net outcome of Ang-2 functions on tumor angiogenesis is believed to be contextual depending on the local cytokine milieu. Correspondingly, Ang-2 manipulatory therapies have been shown to exert protumorigenic as well as antitumorigenic effects. To clarify the role of Ang-2 for angiogenesis and tumor growth in a definite genetic experimental setting, the present study was aimed at comparatively studying the growth of different tumors in wild-type and Ang-2–deficient mice. Lewis lung carcinomas, MT-ret melanomas, and B16F10 melanomas all grew slower in Ang-2–deficient mice. Yet, tumor growth in wild-type and Ang-2–deficient mice dissociated during early stages of tumor development, whereas tumor growth rates during later stages of primary tumor progression were similar. Analysis of the intratumoral vascular architecture revealed no major differences in microvessel density and perfusion characteristics. However, diameters of intratumoral microvessels were smaller in tumors grown in Ang-2–deficient mice, and the vasculature had an altered pattern of pericyte recruitment and maturation. Ang-2–deficient tumor vessels had higher pericyte coverage indices. Recruited pericytes were desmin and NG2 positive and predominately α-smooth muscle actin negative, indicative of a more mature pericyte phenotype. Collectively, the experiments define the role of Ang-2 during tumor angiogenesis and establish a better rationale for combination therapies involving Ang-2 manipulatory therapies. [Cancer Res 2009;69(4):1324–33]</p></div>