Modulation of the angiogenesis response through Ha-ras control,placenta growth factor,and angiopoietin expression in mouse skin carcinogenesis

Tumor angiogenesis is governed by a complex balance of positive and negative angiogenic factors. Development of chemically-induced mouse skin tumors appears to be highly dependent on an early burst of neovascularization. We have previously shown that Ha-ras-driven vascular endothelial growth factor (VEGF) expression plays a pivotal role in this process. However, the status of other critical positive and negative angiogenic factors throughout skin tumorigenesis has not been studied to the same extent. In the present study, we show that another VEGF family member, placenta growth factor (PlGF), was highly upregulated at all tumor stages in a ras-dependent manner. The study of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), ligands of receptor tyrosine kinase 2 (Tie-2), showed that while stroma-derived Ang-2 was increased, epidermal Ang-1 expression was completely abolished at early papilloma formation. Studies using epidermal tumor cell lines suggest that the disappearance of Ang-1 also depends on ras activation, extending the plethora of events controlled by this oncogene in mouse skin carcinogenesis. Our results indicated that tumor development occurred in a strong angiogenesis-prone scenario in which PlGF and Ang-2 acted cooperatively with VEGF, whereas the negative or stabilizing effect of Ang-1 was abrogated. A time-course sequence of expression of angiogenic factors expressed throughout tumor growth, as well as the identification of key signaling molecules triggering the angiogenic response, may contribute to the development and testing of antiangiogenic therapeutic strategies with this in vivo tumor model.     

High-level expression of angiogenic factors is associated with advanced tumor stage in human neuroblastomas.

Angiogenesis is essential for tumor growth and metastasis and depends on the production of angiogenic factors by tumor cells. Neuroblastoma (NB) is a common pediatric tumor of neural crest origin, which is biologically and clinically heterogeneous. Increased tumor vascular index correlates with poor outcome of NB. To determine which angiogenic factors contribute to NB angiogenesis and thereby support tumor progression, we examined the expression of eight angiogenic factors [vascular endothelial growth factor (VEGF), VEGF-B, VEGF-C, basic fibroblast growth factor, angiopoietin (Ang)-1, Ang-2, transforming growth factor alpha, and platelet-derived growth factor (PDGF)] by semiquantitative RT-PCR in 37 NB primary tumors and in 22 NB cell lines. We also analyzed the relationship between angiogenic factor expression and clinicopathological factors as well as patient survival. All eight angiogenic factors examined were expressed at various levels in NB cell lines and tumors, suggesting their involvement in NB angiogenesis. The expression levels of most angiogenic factors were correlated with each other, suggesting their synergy in regulating the angiogenic process. Significantly higher expression levels of VEGF, VEGF-B, VEGF-C, basic fibroblast growth factor, Ang-2, transforming growth factor alpha, and PDGF-A (P < 0.0001-0.026) were found in advanced-stage tumors (stages 3 and 4) compared with low-stage tumors (stages 1, 2, and 4S). Expression of PDGF-A was significantly associated with patient survival (P = 0.04). The redundancy in angiogenic factor expression suggests that inhibition of VEGF bioactivity alone might not be a sufficient approach for antiangiogenic therapy of human NB.     

Two independent mechanisms essential for tumor angiogenesis: inhibition of human melanoma xenograft growth by interfering with either the vascular endothelial growth factor receptor pathway or the Tie-2 pathway.

Protein ligands and receptor tyrosine kinases that specifically regulate endothelial cell function are mainly involved in physiological as well as in disease-related angiogenesis. These ligand/receptor systems include the vascular endothelial growth factor (VEGF) and the angiopoietin (Ang) families, and their receptors, the VEGF receptor family and the tyrosine kinase with immunoglobulin-like and epidermal growth factor homology domains (Tie) family. In the present study, the contribution of these endothelium-specific ligand/receptor systems to tumor angiogenesis was evaluated. A375v human melanoma cells, which express at least the angiogenic growth factors VEGF, VEGF-C, and Ang-1, were stably transfected to overexpress the extracellular ligand-binding domains of the endothelium-specific receptor tyrosine kinases fms-like tyrosine kinase-1 (Flt-1), Flt-4, Tie-1, and Tie-2, respectively. In vitro proliferation and colony formation assays confirmed that expression of the extracellular receptor domains inhibited neither tumor cell mitogenesis nor the ability to produce anchorage-independent growth. Nude mouse xenografts revealed that interference with either the VEGF receptor pathway or the Tie-2 pathway resulted in a significant inhibition of tumor growth and tumor angiogenesis. In contrast, interference with the Flt-4 pathway or the Tie-1 pathway was without significant effect. Our results show that both the VEGF receptor pathway and the Tie-2 pathway are essential for A375v melanoma xenograft growth. The inhibition of the VEGF receptor pathway cannot be compensated by the Tie-2 pathway, nor vice versa. These findings suggest that the VEGF receptor pathway and the Tie-2 pathway have to be considered as two independent mediators essential for the process of in vivo angiogenesis.     

Angiogenesis in cholangiocellular carcinoma: expression of vascular endothelial growth factor, angiopoietin-1/2, thrombospondin-1 and clinicopathological significance

Angiogenesis in cholangiocellular carcinoma (CCC) has rarely been investigated. The aim of this study was to determine the angiogenesis status of CCC and assess its relationship with angiogenic factors and clinicopathological characteristics. We examined 33 surgically resected CCC specimens. Tumor angiogenesis was assessed by microvessel density (MVD) using the anti-CD34 antibody, and the expression of VEGF, Ang-1, Ang-2, and TSP-1 was determined by immunohistochemistry. The mean (+/- SD) MVD was 87.2+/-52.6/mm2 (range, 0-229/mm2). A total of 75.6% cases were positive for VEGF expression, 36% for Ang-1, 57.6% for Ang-2 and 45.5% for TSP-1. VEGF and Ang-2 expression was associated with a significantly higher level of MVD (p=0.004 and 0.015, respectively). TSP-1 expression was associated with a significantly lower level of MVD (p=0.005) and a higher level of intrahepatic metastasis (46.7% vs. 5.6%, p=0.012). There was no significant correlation between VEGF, Ang-1, Ang-2, and TSP-1 expression and tumor size, capsule formation, infiltration of capsule, portal vein invasion, intrahepatic metastasis or CCC differentiation. There was no significant correlation between MVD levels, VEGF, Ang-1, Ang-2, and TSP-1 expression and postoperative survival. A considerable degree of angiogenesis, comparable to that of other solid tumors, was observed in CCC. VEGF and Ang-2 might play a proangiogenic role, and TSP-1 may play an inhibitory role in CCC. Although TSP-1 may increase intrahepatic CCC metastases, neither MVD levels nor the expression of VEGF, Ang-1, or Ang-2 was associated with clinicopathological factors and prognosis.     

Changes in plasma vascular endothelial growth factor, angiopoietins, and their receptors following surgery for breast cancer

BACKGROUND: Vascular endothelial growth factor (VEGF), a major angiogenic growth factor, is involved in the pathogenesis of cancer. Plasma VEGF is raised in breast cancer and falls after successful surgery. Less is known about angiopoietins 1 and 2 (Ang-1, Ang-2). All three growth factors act on cells via receptors; Flt-1 for VEGF and Tie-2 for the angiopoietins. Cancer is also marked by abnormalities in platelet activation (marked by soluble P selectin) and inflammation (interleukin-6 [IL6]). We hypothesised altered plasma Ang-1, Ang-2, Flt-1 and Tie-2 in breast cancer that would normalize after 3 and 12 months treatment (i.e., surgery plus chemo/radiotherapy). METHODS: Baseline venous blood was obtained from 40 women with breast cancer and 30 age-matched women with benign breast disease (BBD) also requiring surgery. Samples were taken again 3 months and 1 year later. Soluble P selectin, IL6, VEGF, Ang-1, Ang-2, Flt-1 and Tie-2 were measured in citrated plasma by ELISA. RESULTS: Women with breast cancer had raised VEGF (7-fold), Ang-1 (50% higher) and Tie-2 (2-fold), but lower Flt-1 (to 26%), compared to the BBD women that broadly correlated with markers of platelet activation and inflammation. A level of Tie-2 or VEGF >95th percentile of the BBD group correctly identified 68% and 52% of the women with breast cancer. After 3 months of treatment, VEGF and Ang-1 normalized (as did IL6 and soluble P selectin) but Tie-2 was significantly lower only after 1 year. There were no significant changes in the women with BBD. CONCLUSIONS: Treatment for breast cancer (surgery followed by chemotherapy and/or radiotherapy) is effective in reducing plasma VEGF, Tie-2 and Ang-1. These may be linked pathogenically with coagulation and inflammation.     

p73 Overexpression increases VEGF and reduces thrombospondin-1 production: implications for tumor angiogenesis.

Tumor neovascularization is controlled by a balance between positive and negative effectors, whose production can be regulated by oncogenes and tumor suppressor genes. The aim of this study was to investigate whether the angiogenic potential of tumors could also be controlled by p73, a gene homologous to the tumor suppressor p53, whose involvement in tumor angiogenesis is known. We have studied the production of proangiogenic (VEGF, FGF-2, PIGF and PDGF) and antiangiogenic (TSP-1) factors in two p73 overexpressing clones obtained from the human ovarian carcinoma cells A2780. TSP-1 was downregulated in both clones compared to mock transfected cells, both at mRNA and protein level. Conversely, both clones showed an increased production of VEGF mRNA and protein. For both TSP-1 and VEGF, regulation of expression was partially due to modulation of the promoter activity, and was dependent on p53 status. Production of the other angiogenic factors FGF-2, PIGF and PDGF-B was also increased in p73 overexpressing clones. The two clones were more angiogenic than parental cells, as shown in vitro by their increased chemotactic activity for endothelial cells, and in vivo by the generation of more vascularized tumors. These findings suggest a potential role of p73 in tumor angiogenesis.     

Marrow angiogenesis-associated factors as prognostic biomarkers in patients with acute myelogenous leukaemia

Bone marrow (BM) neoangiogenesis plays an important role in acute myelogenous leukaemia (AML), and depends on the interplay of members of the vascular endothelial growth factor (VEGF) and angiopoietin (Ang) families. We determined the marrow levels of seven molecules associated with angiogenesis in 52 AML patients before chemotherapy and 20 healthy controls: VEGF-A, VEGF/PlGF, VEGF-C, VEGF-D, Ang-1, Ang-2, and Tie-2. All the molecules were quantified using enzyme-linked immunosorbent assay (ELISA). Comparing to normal controls, the marrow levels of VEGF/PlGF, Ang-2, and Tie-2 were significantly higher, and those of VEGF-C and Ang-1 were significantly lower in the AML patients (P<0.001). A total of 31 patients were further subjected to survival analysis. Patients with lower Tie-2 (<26 ng ml(-1)) and Ang-2 levels (<4500 pg ml(-1)) displayed a survival advantage (P=0.037 and 0.042, respectively), same as patients with higher VEGF/PlGF (> or =1 pg ml(-1)) and VEGF-D levels (> or =350 pg ml(-1)) (P=0.020 and 0.016, respectively). An angio-index ((Ang-2 x Tie-2)/(VEGF/PlGF x VEGF-D)) was established and multivariate Cox regression analysis revealed that patients with higher angio-index values (> or =50) displayed poor prognosis (hazard ratio 5.91, 95% confidence interval 1.99-17.56; P=0.001). The angio-index is closely associated with the clinical outcome of AML patients and may be valuable in disease prognosis.     

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.     

VEGF、VEGF-C和VEGF-D在甲状腺乳头状癌组织中的表达及意义

背景与目的:研究表明血管内皮生长因子C和D(vascularendothelialgrowthfactor-Cand-D,VEGF-C,VEGF-D)与淋巴管的生成有关。局部淋巴结转移是甲状腺乳头状癌的特征,本研究的目是探讨血管内皮生长因子及其C和D(VEGF、VEGF-C和VEGF-D)蛋白在甲状腺乳头状癌的表达及其意义。方法:应用免疫组织化学SP法检测115例甲状腺乳头状癌和20例结节性甲状腺肿中VEGF、VEGF-C和VEGF-D蛋白的表达。结果:甲状腺乳头状癌VEGF、VEGF-C和VEGF-D的阳性率分别为79.1%、87.0%和72.2%。结节性甲状腺肿组织中VEGF、VEGF-C和VEGF-D的阳性率分别为30.0%、15.0%和20.0%。甲状腺乳头状癌VEGF、VEGF-C和VEGF-D的阳性率较结节性甲状腺肿明显增高。VEGF的表达与甲状腺癌的大小有关。VEGF、VEGF-C和VEGF-D在淋巴结转移组和非转移组的阳性率分别为84.7%、93.2%、83.1%和73.2%、80.4%、60.7%。VEGF-C和VEGF-D的阳性表达与甲状腺乳头状癌的淋巴结转移显著相关(P<0.05;P<0.01)。结论:VEGF与甲状腺乳头状癌的生长有关。VEGF-C和VEGF-D与淋巴结转移密切相关,提示甲状腺乳头状癌标本VEGF-C和VEGF-D的检测可作为预测肿瘤转移的指标之一。     

Immunohistochemical study of VEGF,angiopoietin 2 and their receptors in the neovascularization following microinjection of C6 glioma cells into rat brain

BACKGROUND: Recent papers suggest that two angiogenic factors (angiopoietin 2 and vascular endothelial growth factor) cooperate in tumoral angiogenesis to support the growing tumor. The purpose of the present work was to demonstrate the existence of such cooperation in a longitudinal study of a brain tumor model during tumor growth by means of immunohistochemistry. MATERIALS AND METHODS: The study was performed on 31 rats bearing C6 glioma. At different stages of tumor growth, the histological aspects were described and sections were immunostained for VEGF, Ang-2 and their receptors VEGFR-1, VEGFR-2 and Tie-2. Immunostaining was semi-quantitatively analyzed and the localization of immunostained cells was reported. RESULTS: Ang-2 and Tie-2 were detected in the endothelial cells of vessels surrounded by tumor cells, occuring early in our study, with immunostaining taking place from day 4 to day 24. Immunostaining with VEGF (on tumoral cells) and VEGFR-2 (on endothelial cells) was present after 8 days of tumor growth. A clear increase of vessel density can be observed at the periphery of the tumors after 16 days of tumor growth. At that time, areas of necrosis were present in the tumor with concomitant VEGF and Ang-2 expression. CONCLUSION: The present study demonstrated cooperation between the early effect of Ang-2 and the secondary effect of VEGF to elaborate new vessels in a longitudinal study of experimental brain tumors. This study is favorable to the new model of tumor angiogenesis, with successive vessel cooption, regression and growth mediated by angiopoietins and VEGF.     

Angiopoietins in angiogenesis

Tie-1 and Tie-2 tyrosine kinase receptors are expressed specifically on vascular endothelial cells and on a certain subtype of macrophages implicated in angiogenesis, thus, they have been a major focus of angiogenesis research. Tie-1 and Tie-2 are essential for vascular maturation during developmental, physiological and pathological angiogenesis. Angiopoietin 1–4 (Ang-1–4) have been identified as bona fide ligands of the Tie-2 receptor, while Tie-1 remains an orphan receptor which is able to heterodimerize with Tie-2 and to modulate Tie-2 signal transduction. The most exhaustively studied angiopoietins are Ang-1 and Ang-2. Ang-1 is a critical player in vessel maturation and it mediates migration, adhesion and survival of endothelial cells. Ang-2 disrupts the connections between the endothelium and perivascular cells and promotes cell death and vascular regression. Yet, in conjunction with VEGF, Ang-2 promotes neo-vascularization. Hence, angiopoietins exert crucial roles in the angiogenic switch during tumor progression, and increased expression of Ang-2 relative to Ang-1 in tumors correlates with poor prognosis. Its central role in the regulation of physiological and pathological angiogenesis makes the angiopoietin/Tie signaling pathway a therapeutically attractive target for the treatment of vascular disease and cancer.     

The angiogenic and prognostic implications of VEGF, Ang-1, Ang-2, and MMP-9 for hepatocellular carcinoma with background of hepatitis B virus

The objective of this study was to investigate the expressions of angiogenic factors and elucidate their angiogenic and prognostic roles in hepatocellular carcinoma (HCC) with background of hepatitis B virus (HBV). We evaluated microvessel density (MVD) of HCC, and investigated immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietins (Ang-1 and Ang-2), and matrix metalloproteinases-9 (MMP-9) in 67 specimens of surgically resected HCC, which were all positive for hepatitis B surface antigen. We investigated the relationship between their expressions and clinicopathological factors or prognosis. The microvessel density (MVD) of tumor tissue and surrounding normal liver tissue was 93.1 ± 43.8/mm² and 30.4 ± 14.8/mm², respectively. The MVD of well-differentiated HCC was significantly less than that of poorly differentiated HCC. MVD was positively correlated with VEGF and Ang-2 expression (P = 0.0023 and 0.0265, respectively). There was less tumor recurrence in low Ang-2 and low MMP-9 group than high Ang-2 and/or high MMP-9 group (P = 0.002). In Cox regression model, portal vein thrombus and intrahepatic metastasis was the risk factors of tumor recurrence (P = 0.003 and 0.001, respectively). Our study showed that the expression of VEGF and Ang-2 were positively correlated with MVD. Ang-2 expression and/or MMP-9 expression might be a significant predictive factor for recurrence after resection in HCC patients with the background of HBV.     

Enhanced expression of Tie2, its ligand angiopoietin-1, vascular endothelial growth factor, and CD31 in human non-small cell lung carcinomas.

Expression of angiogenesis-associated genes was compared in 32 primary non-small cell lung carcinoma samples (14 adenocarcinomas, 17 squamous cell carcinomas, and 1 large cell carcinoma) and paired adjacent noncancerous lung tissues using a multiprobe RNase protection assay. Levels of Tie2, angiopoietin (Ang)-1, vascular endothelial growth factor (VEGF), and CD31 mRNAs were higher in cancers than in adjacent noncancerous tissues, in contrast to the fms-like tyrosine kinase (Flt)-1, Flt-4, Tie1, thrombin receptor, endoglin, and VEGF-C, for which no differences were evident. Overexpression did not seem to differ with histological type and pathological stage. Significant positive correlations were found between mRNA expression of Ang-1 and those of Tie2 and CD31, and that of VEGF and those of Flt-1 and CD31. These findings suggest that Ang-1 and VEGF are important angiogenic factors in human non-small cell lung carcinomas.     

Interleukin-1beta regulates angiopoietin-1 expression in human endothelial cells

Angiopoietin (Ang)-1 is an important regulator of endothelial cell (EC) survival and stabilization. Ang-1 exerts its biological effects by binding to the EC-specific tyrosine kinase receptor Tie-2, and initiates intracellular signaling in ECs. However, regulatory mechanisms for endothelial Ang-1 expression have not been completely elucidated. In this study, we investigated the effects of angiogenic cytokines and growth factors on Ang-1 expression in human umbilical vein ECs (HUVECs). Northern blot analysis was performed after HUVECs were exposed to interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, platelet-derived growth factor-BB, insulin-like growth factor-1, or vascular endothelial growth factor (VEGF). Both IL-1beta and tumor necrosis factor-alpha caused marked down-regulation of Ang-1 mRNA levels at 4 h with a further decrease observed at 24 h. Using signaling inhibitors, we identified the P38 pathway as the pathway that mediates IL-1beta down-regulation of Ang-1. Furthermore, treatment of cells with IL-1beta indirectly (via down-regulation of Ang-1) led to a decrease in Tie-2 autophosphorylation levels in HUVECs. We previously demonstrated that IL-1beta regulates VEGF expression in tumor cells. This observation was confirmed in ECs in the present study. Because pericytes play a role in regulating EC function, we also determined whether IL-1beta would also down-regulate Ang-1 in human vascular smooth muscle cells. Similar to our findings in HUVECs, we found that IL-1beta decreased Ang-1 expression in human vascular smooth muscle cells. Direct effects of IL-1beta on angiogenesis were investigated by use of an in vivo Gelfoam angiogenesis assay in which IL-1beta produced a significant increase in vessel counts (P = 0.0189). These results suggest that IL-1beta indirectly regulates angiogenesis by modulating the expression of Ang-1. IL-1beta may trigger a proangiogenic response by decreasing Ang-1 levels in ECs and pericytes and up-regulating VEGF in ECs and tumor cells.     

Expression of endothelial cell-associated molecules in AML cells.

Recently, it has been clarified that interaction between hematopoietic cells and endothelial cells is important in normal hematopoiesis and leukemogenesis. In this study, we examined the relationship between AML cells and endothelial cells by analyzing the expression profile of angiogenic factors, angiopoietin-1 (Ang-1), Ang-2, Tie-2 (a receptor for angiopoietins) and vascular endothelial growth factor (VEGF). Our results demonstrated that CD7(+)AML expressed Ang-2 mRNA frequently and integrin-family adhesion molecules (CD11c and CD18) intensively, suggesting the close correlation with endothelial cells. On the other hand, in t(8;21) AML cells, expression of Ang-2 was infrequent and expression of integrin-family adhesion molecules (CD11b, CD11c and CD18) was weak, suggesting the sparse association with endothelial cells. As for CD7(+)AML cells, despite the frequent and intense expression of endothelial cell-associated molecules (such as Ang-2, CD11c and CD18), intensity of Tie-2 expression was quite low (P < 0.05). Ang-2 expressed in CD7(+)AML cells is not considered to act in an autocrine fashion, but to work on endothelial cells to "feed" leukemic cells. Although Ang-2 is recognized as a natural antagonist for Tie-2, our data presented here suggested the alternative role of Ang-2 in the relationship between endothelial cells and leukemia cells, at least in a subset of leukemia such as CD7(+)AML. These results were supported by the study using AML cell lines, KG-1 (CD7 negative) and its subline KG-1a (CD7 positive); KG-1 had mRNA expression profile of Ang-1(+)Ang-2(-)Tie-2(+), while KG-1a showed Ang-1(+)Ang-2(+)Tie-2(-). These difference in the expression profile of angiogenic factors between CD7(+)AML and t(8;21)AML may explain the characteristic morphological features of these leukemias (CD7(+)AML as blastic type and t(8;21)AML as differentiative type).     

HER2 signaling modulates the equilibrium between pro- and antiangiogenic factors via distinct pathways: implications for HER2-targeted antibody therapy

We determined the impact of HER2 signaling on two proangiogenic factors, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), and on an antiangiogenic factor, thrombospondin-1 (TSP-1). Re-expression of HER2 in MCF-7 and T-47D breast cancer cells that endogenously express low levels of HER2 resulted in elevated expression of VEGF and IL-8 and decreased expression of TSP-1. Inhibition of HER2 with a humanized anti-HER2 antibody (trastuzumab, or Herceptin) or a retrovirus-mediated small interfering RNA against HER2 (siHER2) decreased VEGF and IL-8 expression, but increased TSP-1 expression in BT474 breast cancer cells that express high levels of HER2. These in vitro results were further evaluated by treatment of BT474 xenografts in immunosuppressed mice with trastuzumab. Trastuzumab inhibited growth of BT474 xenografts and decreased microvascular density associated with downregulation of VEGF and IL-8 and with upregulation of TSP-1 expression. Inhibiting the PI3K-AKT pathway decreased VEGF and IL-8 expression. AKT1 overexpession increased VEGF and IL-8 expression, but did not increase TSP-1 expression. A p38 kinase inhibitor, SB203580, instead blocked TSP-1 expression and a p38 activator, MKK6, increased TSP-1 expression. Trastuzumab stimulated sustained p38 activation and SB203580 attenuated the TSP-1 upregulation induced by trastuzumab. HER2 signaling therefore influences the equilibrium between pro- and antiangiogenic factors via distinct signaling pathways. Trastuzumab inhibits angiogenesis and tumor growth, at least in part, through activation of the HER2-p38-TSP-1 pathway and inhibition of the HER2-PI3K-AKT-VEGF/IL-8 pathway.     

Clinical significance of angiopoietin-2 expression at the deepest invasive tumor site of advanced colorectal carcinoma

Angiopoietin (Ang)-2 and vascular endothelial growth factor (VEGF) are thought to be critical regulators in tumor angiogenesis. We investigated the clinical significance of Ang-2 expression at the deepest invasive tumor site under the influence of VEGF expression in relation to angiogenesis, invasive/metastatic potential, and prognosis of advanced colorectal carcinoma (CRC). One hundred and fifty-two patients who underwent surgical resection for advanced CRC were enrolled in this study. Ang-2 and VEGF expression were examined immunohistochemically. Tumor microvessel density (MVD) was examined by immunohistochemical staining against CD34. Ang-2 and VEGF were expressed at the deepest invasive tumor site in 90 (59.2%) and 64 (42.1%) of 152 lesions, respectively. Patients with Ang-2 expression at the deepest invasive tumor site showed significantly (p<0.01) more frequent poorly histologic grade (71.9%), lymphatic involvement (65.9%), venous involvement (69.1%), lymph node metastasis (75.0%), liver metastasis (80.0%) and advanced disease (Dukes' stage C, 70.2%; stage D, 80.0%) than patients without Ang-2 expression. MVD was not significantly up-regulated by Ang-2 expression alone at the deepest invasive tumor site but was significantly up-regulated by VEGF expression at the deepest invasive tumor site under the positive-Ang-2 condition. In patients treated by curative surgery, patients with tumors showing both positive-Ang-2 and positive-VEGF condition at the deepest invasive tumor site had significantly poorer prognoses than patients with tumors under other conditions. Multivariate analysis with logistic regression for 5-year survival in cases of curative surgery showed that lymph node metastasis, VEGF expression and Ang-2 expression were significant factors to predict poor prognosis. Our results suggest that Ang-2 expression in collaboration with VEGF expression at the deepest invasive tumor site may result in tumor angiogenesis and that these angiogenic factors at the deepest invasive tumor site may be correlated with invasive/malignant potential and prognosis of advanced CRC.