T cell-specific loss of Pten leads to defects in central and peripheral tolerance

PTEN, a tumor suppressor gene, is essential for embryogenesis. We used the Cre-loxP system to generate a T cell-specific deletion of the Pten gene (Pten(flox/-) mice). All Pten(flox/-) mice develop CD4+ T cell lymphomas by 17 weeks. Pten(flox/-) mice show increased thymic cellularity due in part to a defect in thymic negative selection. Pten(flox/-) mice exhibit elevated levels of B cells and CD4+ T cells in the periphery, spontaneous activation of CD4+ T cells, autoantibody production, and hypergammaglobulinemia. Pten(flox/-) T cells hyperproliferate, are autoreactive, secrete increased levels of Th1/Th2 cytokines, resist apoptosis, and show increased phosphorylation of PKB/Akt and ERK. Peripheral tolerance to SEB is also impaired in Pten(flox/-) mice. PTEN is thus an important regulator of T cell homeostasis and self-tolerance.     

Cell Type-Specific Expression of Angiopoietin-1 and Angiopoietin-2 Suggests a Role in Glioblastoma Angiogenesis

Glioblastomas are highly vascular tumors which overexpress the angiogenesis factor vascular endothelial growth factor (VEGF). VEGF and its receptors, VEGF-R1 and VEGF-R2, have been shown to be necessary for embryonic angiogenesis as well as for tumor angiogenesis. Recently, the angiopoietin/Tie2 receptor system has been shown to exert functions in the cardiovascular system that are distinct from VEGF but are also critical for normal vascular development. To assess the potential role of Tie2 and its ligands angiopoietin-1 and angiopoietin-2 in tumor vascularization, we analyzed their expression pattern in human gliomas. Tie-2 was up-regulated in tumor endothelium compared to normal human brain tissue. We further observed cell type-specific up-regulation of the message for both angiopoietin-1 and angiopoietin-2 in gliomas. Whereas Ang-1 mRNA was expressed in tumor cells, Ang-2 mRNA was detected in endothelial cells of a subset of glioblastoma blood vessels. Small capillaries with few periendothelial support cells showed strong expression of Angiopoietin-2, whereas larger glioblastoma vessels with many periendothelial support cells showed little or no expression. Although the function of Tie2 and its ligands in tumor angiogenesis remains a subject of speculation, our findings are in agreement with a recently proposed hypothesis that in the presence of VEGF, local production of Ang-2 might promote angiogenesis.     

Ang-1/Tie2系统与病理性血管形成的关系

血管生成素1(Ang-1)是继血管内皮生长因子(VEGF)之后,人们发现的又一重要的促血管生成因子。血管生成素家族包括Ang-l、Ang-2、Ang-3、Ang-4四种分子。其共同的特异性受体为Tie-2。目前对Ang-1/Tie2系统参与新生血管形成、促进血管成熟、抑制血管渗漏及炎症的作用研究相对深入,在创伤后修复、缺血后再通、肿瘤、糖尿病并发症及子宫内膜异位等多种病理性血管形成过程中起重要作用。     

Angiopoietin-2 is implicated in the regulation of tumor angiogenesis

We addressed the effect of angiopoietin expression on tumor growth and metastasis. Overexpression of angiopoietin-2 (Ang-2) in Lewis lung carcinoma and TA3 mammary carcinoma cells inhibited their ability to form metastatic tumors and prolonged the survival of mice injected with the corresponding transfectants. In contrast, angiopoietin-1 (Ang-1) overexpression had no detectable effect on the ability of either tumor type to disseminate. Tumors derived from Ang-2-overexpressing cells displayed aberrant angiogenic vessels that took the form of vascular cords or aggregated vascular endothelial cells with few associated smooth muscle cells. These vascular cords or aggregates were accompanied by endothelial and tumor cell apoptosis, suggesting that an imbalance in Ang-2 expression with respect to Ang-1 and vascular endothelial growth factor may disrupt angiogenesis and tumor survival in vivo. Our observations suggest that Ang-2 may play an important role in regulating tumor angiogenesis.     

Expressional regulation of angiopoietin-1 and -2 and the tie-1 and -2 receptor tyrosine kinases during cutaneous wound healing: a comparative study of normal and impaired repair

It has become evident that a closely regulated presence of vascular endothelial growth factor (VEGF) and angiopoietin (Ang) factors determines the fate of blood vessel formation during angiogenesis. As angiogenesis is central to a normal wound-healing process, we investigated the regulation of Ang-1 and -2 and the related tyrosine kinase with immunoglobulin and epidermal growth factor homology (Tie)-1 and -2 receptors during normal repair in Balb/c mice and diabetes-impaired wound healing conditions in genetically diabetic (db/db) mice. For both normal and impaired healing conditions, we observed a constitutive expression of Ang-1, which was paralleled by an increase of Ang-2 upon injury. Whereas the observed Ang-2 expression declines from Day 7 after injury in control mice, diabetic-impaired healing was characterized by still increasing amounts of Ang-2 at these time points. Furthermore, Tie-1 was strongly induced during repair with a prolonged expression in diabetic mice, whereas Tie-2 expression was constitutive during normal repair but completely absent in diabetes-impaired healing. The overexpression of Ang-2 in the presence of markedly reduced VEGF in wounds of diabetic mice was associated with a dramatic decrease in endothelial cell numbers compared with normal healing as assessed by analysis of the endothelium-specific markers CD31 and von Willebrand factor, whereas the lymphatic endothelium remained stable as determined by expression of VEGF receptor-3 (VEGFR-3/Flt-4).     

Expression of the angiopoietins and their receptor Tie2 in human renal clear cell carcinomas; regulation by the von Hippel-Lindau gene and hypoxia

Angiogenesis is essential for tumour growth and metastasis, and is co-ordinated by several classes of angiogenic factors. To determine the significance and regulation of the angiopoietin (Ang) pathway in highly vascular human renal cell carcinomas (RCCs), this study has investigated the expression of the Ang-1, Ang-2, Ang-4, and Tie2 genes in a series of normal (n = 26) and neoplastic (n = 45; clear cell n = 35, papillary n = 10) human kidney tissues, examined the pattern of Ang-2 and Tie2 protein expression, and correlated expression with clinicopathological variables. The effect of the von Hippel-Lindau (VHL) gene and hypoxia in the renal cell lines RCC786-0 and RCC4 has also been investigated. Ang-1, Ang-2 and Tie2, but not Ang-4 mRNA, were detected in normal and tumour samples. A significant increase in Ang-2 (p < 0.001) and a decrease in Tie2 receptor mRNA (p = 0.001) were observed, but no significant difference was observed in Ang-1 mRNA abundance between normal kidney and RCC (p = 0.37). Immunohistochemistry for Ang-2 showed strong expression in vascular endothelium and weak expression in tumour cells, whereas Tie2 was expressed exclusively on endothelium. Tie2 gene expression was positively correlated with Ang-2 expression in cancers (p = 0.001) and showed a borderline significant association with Ang-1 (p = 0.06), but there was no significant relationship between Ang-1 and Ang-2 (p = 0.69). No significant relationships were observed in clear cell carcinomas between Ang-1, Ang-2 and Tie2 mRNA abundance and patient sex, patient age, or tumour size (p > 0.05). However, there was significantly greater Ang-1 (p = 0.02), Ang-2 (p = 0.03), and Tie2 (p = 0.04) mRNA abundance in clear cell than in chromophil RCCs. Ang-2 gene expression was down-regulated by hypoxia in VHL wild-type RCC786-0 and RCC4 transfectants (p = 0.0002 and p = 0.04, respectively), mirroring the low expression in human tumour cells. These data suggest that it is endothelial induction of Ang-2 in tumours that regulates vessel stability and supports targeting Tie2 as an effective novel anti-angiogenic therapy in clear cell RCCs.     

Expression of angiopoietin 1, 2 and their common receptor Tie2 in human gastric carcinoma: implication for angiogenesis

Angiogenesis, formation of new microvessels providing oxygen and nutrient supply, is essential for tumor growth. It is dependent on the production of angiogenic growth factors by tumor cells. Angiopoietin 1 (Ang-1) and 2 (Ang-2) and their common receptor, Tie2, are thought to be critical regulators of tumor angiogenesis. We examined expression of Ang-1, Ang-2, and their common receptor Tie2 mRNAs and proteins in gastric cancers using in situ hybridization and immunohistochemistry. We also investigated the relationship between their expression and differentiation of cancer cells, lymph node metastasis, tumor size, depth of cancer cell invasion, TNM staging and microvessel density (MVD). The expression of Ang-1, Ang-2, and Tie2 mRNA in cancer cells significantly correlated with the MVD (p<0.001, <0.001 and =0.019, respectively). Ang-1 and Tie2 positivity correlated with advanced gastric cancers (p<0.05) and larger cancers had higher positive rates of Ang-1, Ang-2, and Tie2 mRNA expression (p<0.001, =0.010 and =0.039, respectively). Significant positive correlations were also found between mRNA expression of Tie2 and those of Ang-1 and Ang-2 (p<0.01 and <0.001, respectively). These findings indicate that the expression of Ang-1 and Ang-2 is important for tumor angiogenesis, and suggest a possible role of autocrine/paracrine function of angiopoietin/Tie2 system in gastric cancer progression.     

Angiopoietins: a link between angiogenesis and inflammation

The angiopoietin (Ang)-Tie ligand-receptor system has a key regulatory role in regulating vascular integrity and quiescence. Besides its role in angiogenesis, it is an important regulator in numerous diseases including inflammation. Ang-1-mediated Tie2 activation is required to maintain the quiescent resting state of the endothelium. Agonistic Ang-1 functions are antagonized by Ang-2, which is believed to inhibit Ang-1-Tie2 signaling. Ang-2 destabilizes the quiescent endothelium and primes it to respond to exogenous stimuli, thereby facilitating the activities of inflammatory (tumor necrosis factor and interleukin-1) and angiogenic (vascular endothelial growth factor) cytokines. Intriguingly, Ang-2 is expressed weakly by the resting endothelium but becomes strongly upregulated following endothelial activation. Moreover, endothelial cells store Ang-2 in Weibel-Palade bodies from where it can be made available quickly following stimulation, suggesting a role of Ang-2 in controlling rapid vascular adaptive processes. This suggests that Ang-2 is the dynamic regulator of the Ang-Tie2 axis, thereby functioning as a built-in switch controlling the transition of the resting quiescent endothelium towards the activated responsive endothelium.     

Direct actions of angiopoietin-1 on human endothelium: evidence for network stabilization, cell survival, and interaction with other angiogenic growth factors

Angiopoietin-1 (Ang-1) is a recently described angiogenic protein that activates the endothelial Tie 2 receptor. Disruption of the Ang-1 gene shows that it has an indispensable role in blood vessel development, but it is not clear what specific effects, if any, Ang-1 has on endothelial cell (EC) phenotypes. Here, we show that Ang-1 dose-dependently stabilizes HUVEC network organization for up to 48 hours; this action of Ang-1 is dependent on Tie-2 receptor activation, because a soluble form of the Tie2-, but not the Tie1-receptor, completely blocks the effects of Ang-1. Moreover, we show that Ang-1 potentiates the actions of other angiogenic growth factors. Ang-1 markedly increases the survival of vascular networks (up to 96 hours) exposed to either vascular endothelial growth factor or endothelial cell growth supplement, a form of acidic fibroblast growth factor. In addition, Ang-1 prevents apoptotic death in HUVEC triggered by withdrawal of endothelial cell growth supplement. Collectively, these data are consistent with the idea that Ang-1 directly acts on human EC and interacts with other angiogenic molecules to stabilize vascular structures by promoting the survival of differentiated ECs.     

Xanthatin,a novel potent inhibitor of VEGFR2 signaling,inhibits angiogenesis and tumor growth in breast cancer cells

Anti-angiogenesis targeting vascular endothelial growth factor receptor 2 (VEGFR2) has emerged as an important tool for cancer treatment. In this study, we described a novel VEGFR2 inhibitor, xanthatin, which inhibits tumor angiogenesis and growth. The biochemical profiles of xanthatin were investigated using kinase assay, migration assay, tube formation, Matrigel plug assay, western blot, immunofluorescence and human tumor xenograft model. Xanthatin significantly inhibited growth, migration and tube formation of human umbilical vascular endothelial cell as well as inhibited vascular endothelial growth factor (VEGF)-stimulated angiogenesis. In addition, it inhibited VEGF-induced phosphorylation of VEGFR2 and its downstream signaling regulator. Moreover, xanthatin directly inhibit proliferation of breast cancer cells MDA-MB-231. Oral administration of xanthatin could markedly inhibit human tumor xenograft growth and decreased microvessel densities (MVD) in tumor sections. Taken together, these preclinical evaluations suggest that xanthatin inhibits angiogenesis and may be a promising anticancer drug candidate.     

Vascular apoptosis and involution in gliomas precede neovascularization: a novel concept for glioma growth and angiogenesis

Vascular changes in gliomas were analyzed by implanting fluorescent-labeled glioma 261 cells in the brains of 28 mice. Seven animals were killed each week for 4 weeks. We investigated the expression of angiopoietin-2 (Ang-2) by in situ hybridization and compared it with the distribution of apoptotic cells identified by DNA strand breaks (using the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling [TUNEL] method) and transmission electron microscopy (TEM). As early as 1 week after implantation, tumor cells accumulated around vessels, which expressed Ang-2 and were TUNEL negative. TEM showed tumor cells adjacent to the vascular cells "lifting up" the normal astrocytic feet processes away from the endothelial cells and disrupting normal pericytic cuffing. After 2 weeks the number of perivascular glioma cells had increased. No increase in the number of blood vessels was detected at this time. Vascular cells remained positive for Ang-2 and rare ones were TUNEL positive. TEM showed closely packed proliferating perivascular tumor cells. After 3 weeks, there was vascular involution with scant zones of tumor necrosis. Ang-2 was still detected in vascular cells, but now numerous vascular cells were TUNEL positive. In addition, TEM showed apoptotic vascular cells. After 4 weeks, there were extensive areas of tumor necrosis with pseudopalisading and adjacent angiogenesis. Ang-2 was detected in vascular cells at the edge of the tumors in the invaded brain and in vessels surrounded by tumor cells. At both 3 and 4 weeks, most of the TUNEL-positive tumor cells lacked morphological features characteristic of apoptosis and displayed features consistent with necrotic cell death as determined by TEM. Only rare tumor cells appeared truly apoptotic. In contrast, the TUNEL-positive endothelial cells and pericytes were round and shrunken, with condensed nuclear chromatin by TEM, suggesting that vascular cells were undergoing an apoptotic cell death. These results suggest that vascular cell apoptosis and involution preceded tumor necrosis and that angiogenesis is a later event in tumor progression in experimental gliomas. Moreover, Ang-2 is detected prior to the onset of apoptosis in vascular cells and could be linked to vascular involution.     

Angiopoietin switching regulates angiogenesis and progression of human hepatocellular carcinoma

Aim: Angiopoietin 1 (Ang-1) and its antagonist, angiopoietin 2 (Ang-2), are novel ligands that regulate the Tie2 receptor. The Ang-2 gene is upregulated in the hypervascular type of human hepatocellular carcinoma (HCC). To gain a better understanding of the role of the Ang-Tie2 system in HCC the expression of these genes was investigated in a series of human HCCs. METHODS: The expression of the angiopoietin and Tie2 proteins was investigated in nine normal liver tissues and 52 surgically resected HCCs. In addition, the effects of hypoxic stimuli on Ang-1, Ang-2, vascular endothelial growth factor (VEGF), and erythropoietin (EPO) expression was investigated in Hep3B cells. RESULTS: Ang-1, rather than Ang-2, was more frequently expressed in the normal liver. Ang-1 was expressed in 68% of HCCs, whereas Ang-2 was expressed in 81%, and was significantly higher in poorly differentiated HCCs characterised by high vascularity (p = 0.02), and in tumours with a peliotic change (p = 0.02). Strong expression of Tie2 was seen in tumour vessels in accordance with Ang-2 expression. In Hep3B cells, hypoxic stimuli upregulated VEGF and EPO, but not Ang-1 or Ang-2. CONCLUSIONS: These data support the evidence that the reversal of Ang-1 and Ang-2 expression plays an important role in the angiogenic and dedifferentiation processes in HCC. The hypoxic stimuli were not responsible for Ang-2 upregulation, unlike that of VEGF, in human HCC cells.     

Targeting the Tie2/Tek receptor in astrocytomas

Tie2 is an endothelial cell-specific receptor tyrosine kinase, whose activation is positively and negatively modulated by angiopoietin-1 and angiopoietin-2, respectively. Angiopoietin-mediated modulation of Tie2 activation contributes to normal vessel development and stability, however, its role in tumor angiogenesis is not well known. We investigated the role of Tie2 activation in malignant astrocytomas, a common and highly vascularized primary human brain tumor. We found that Tie2 expression and activation increases with increasing malignancy grade of astrocytomas. Inhibition of Tie2, using a kinase-deficient Tie2 construct, decreases growth of malignant human astrocytoma subcutaneous and intracranial xenografts. Tie2 inactivation disrupted the tumor vascularity, with a decrease in microvascular density, increased presence of abnormally dilated vessels, and loss of interaction between endothelial cells and surrounding smooth muscle cells, all collectively resulting in increased tumor cell apoptosis. Overall, these findings strongly suggest that Tie2 activation contributes significantly to astrocytoma tumor angiogenesis and growth. We postulate that targeting Tie2 activation, either independently or in conjunction with other anti-angiogenic therapies, such as against vascular endothelial growth factor, is of potential clinical interest.     

Genetic analysis of Pten and Tsc2 functional interactions in the mouse reveals asymmetrical haploinsufficiency in tumor suppression

The role of tumor suppressor haploinsufficiency in oncogenesis is still poorly understood. The PTEN and TSC2 tumor suppressors function to antagonize mTOR (mammalian target of rapamycin) activation by Akt; hence, compound heterozygous inactivation of Pten and Tsc2 in the mouse may in principle exacerbate the tumor phenotypes observed in the single mutants in a reciprocal manner. In contrast, we found that while Tsc2 heterozygosity unmasks Pten haploinsufficiency in growth and tumor suppression, tumorigenesis in Tsc2+/- mutants is surprisingly not accelerated by Pten heterozygosity, even though mTOR activation is cooperatively enhanced by compound Pten/Tsc2 heterozygosity. We show that the wild-type alleles of both Pten and Tsc2 are retained in prostate tumors from both Pten+/- and Pten+/-Tsc2+/- mice, whereas TSC-related tumor lesions are invariably associated with Tsc2 loss of heterozygosity (LOH) in both Tsc2+/- and Pten+/-Tsc2+/- mice. These findings demonstrate that inactivation of TSC2 is epistatic to PTEN in the control of tumor initiation and progression and, importantly, that both Pten and Tsc2 are haploinsufficient for suppression of tumorigenesis initiated by Pten heterozygosity, while neither Pten nor Tsc2 is haploinsufficient for repression of carcinogenesis arising from Tsc2 heterozygosity, providing a rationale for the differential cancer susceptibility of the two human conditions associated with PTEN or TSC2 heterozygous mutations.     

Myeloid cells functioning in tumor vascularization as a novel therapeutic target

Angiogenesis, the sprouting of new blood vessels to sustain growth, is an important new target in solid tumor therapy. Initial studies focused on the role of the tumor cell in promoting angiogenesis; yet more recent work has demonstrated that host cells in the tumor microenvironment also play a critical role in tumor vascularization. Additionally, vasculogenesis in which new blood vessels develop from vascular progenitor cells also contributes to tumor growth. Recent studies propose a central role for cells of the myeloid lineage in triggering vessel growth by releasing angiogenic factors and perhaps by incorporating directly into nascent blood vessels. We will review studies that support a critical role for myeloid cells in neovascularization, with a focus on cells that express various monocytic/dendritic cell markers, including vascular leukocytes (VLCs), Tie2+ monocytes, and vascular endothelial growth factor receptor 2 (VEGFR2)+ monocytes, among others. The evidence that these myeloid cells represent bona fide therapeutic targets for solid tumors will be reviewed. Finally, we will address some controversies and challenges in the field with a focus on future directions.     

Endometrial tumorigenesis in Pten(+/-) mice is independent of coexistence of estrogen and estrogen receptor α

Numerous studies support the role for mutations in the phosphatase and tensin homologue (PTEN) tumor suppressor gene and unopposed estrogen stimulation in the pathogenesis of uterine endometrioid carcinoma. However, the relation between PTEN signaling and estrogen/estrogen receptor in endometrial tumorigenesis remains unresolved. We used genetically engineered mice as a model to address this relation. Mice with a single deleted Pten allele (Pten(+/-)) spontaneously develop complex atypical hyperplasia and ~20% develop endometrial cancer. To determine the effect of removing endogenous estrogen, we performed oophorectomies on Pten(+/-) mice. Although there was a reduction in the number and severity of hyperplastic lesions, the endometrial phenotype persisted, suggesting that Pten mutation, independent of estrogen, can initiate the development of complex atypical hyperplasia. To recapitulate the situation in women with unopposed estrogen, we implanted 17β-estradiol pellets in adult female Pten heterozygous mice, resulting in increased carcinoma incidence. Because studies have shown that estrogen largely acts on the endometrium via estrogen receptor ERα, we generated Pten(+/-)ERα(-/-) mice. Strikingly, 88.9% of Pten(+/-)ERα(-/-) mice developed endometrial hyperplasia/carcinoma. Furthermore, Pten(+/-)ERα(-/-) mice showed a higher incidence of in situ and invasive carcinoma, suggesting that endometrial tumorigenesis can progress in the absence of ERα. Thus, the relation between Pten alterations and estrogen signaling in the development of endometrial carcinoma is complex; the results presented herein have important implications for the treatment of endometrial hyperplasia and carcinoma in women.     

Angiogenetic axis angiopoietins/Tie2 and VEGF in familial breast cancer

Angiogenesis leads to the formation of blood vessels from pre-existing ones, allowing tumor growth. Vascular endothelial growth factor (VEGF) and Angiopoietins (Ang-1, Ang-2) have a pivotal role in tumor angiogenesis but few data regarding their role in hereditary breast cancer are available. The aim of the present study was to analyze Ang-1, Ang-2, tyrosine-protein kinase receptor Tie2 and VEGF expression and their correlation in a cohort of familial and sporadic breast cancers in order to verify whether the presence of germline mutations in BRCA may have a role in tumor microenvironment regulation. Tumor samples from a cohort of 41 patients with a first diagnosis and a family history of breast cancer and 19 patients with sporadic breast cancers were enrolled. The expression of Tie2, Ang-1, Ang-2 and VEGF were analyzed by quantitative real-time PCR. Patients harboring BRCA mutations had higher levels of Ang-1 (P=0.05), Ang-2 (P=0.02) and VEGF (P=0.04) mRNA compared with those without BRCA mutations (BRCAX). The same was observed in triple-negative breast cancer (TNBC). Moreover, a positive correlation between Ang-2 and VEGF was found in both the familial breast cancer group (BRCA carriers: r=0.83; P<0.0001 and BRCAX: r=0.58; P=0.008) and in TNBC (r=0.62; P=0.007). The higher levels of Ang-1, Ang-2 and VEGF mRNA found in BRCA carriers and TNBCs suggest that they could be attractive angiogenic therapeutic targets in these breast cancers.     

Effective clearance of intracellular Leishmania major in vivo requires Pten in macrophages

Leishmaniases are a major international public health problem, and macrophages are crucial for host resistance to this parasite. To determine if phosphatase and tensin homologue deleted on chromosome ten (Pten), a negative regulator of the PI3K pathway, plays a role in macrophage-mediated resistance to Leishmania, we generated C57BL/6 mice lacking Pten specifically in macrophages (LysMCrePten(flox/flox) mice). Examination of lesions resulting from Leishmania major infection showed that LysMCrePten(flox/flox) mice were more susceptible to the parasite than wild-type (WT) mice in the early phase of the infection, but were eventually able to eliminate the pathogen. In vitro Pten-deficient macrophages showed a reduced ability to kill parasites in response to IFN-gamma treatment, possibly because the mutant cells exhibited decreased TNF secretion that correlated with reductions in inducible nitric oxide synthase expression and nitric oxide production. In response to various TLR ligands, Pten-deficient macrophages produced less TNF and IL-12 but more IL-10 than WT cells. However, analysis of cells in the lymph nodes draining L. major inoculation sites indicated that both LysMCrePten(flox/flox) and WT mice developed normal Th1 responses following L. major infection, in line with the ability of LysMCrePten(flox/flox) mice to eventually eliminate the parasite. Our results indicate that the efficient clearance of intracellular parasites requires Pten in macrophages.