Substitution of C-terminus of VEGFR-2 with VEGFR-1 promotes VEGFR-1 activation and endothelial cell proliferation

VEGFR-1 is devoid of ligand-dependent tyrosine autophosphorylation and its activation is not associated with proliferation of endothelial cells. The molecular mechanism responsible for this characteristic of VEGFR-1 is not known. In this study, we show that VEGFR-1 is devoid of ligand-dependent downregulation and failed to stimulate intracellular calcium release, cell migration and angiogenesis in vitro. To understand the molecular mechanisms responsible for the poor tyrosine autophosphorylation of VEGFR-1, we have either deleted the carboxyl terminus of VEGFR-1 or exchanged it with the carboxyl terminus of VEGFR-2. The deletion of carboxyl terminus of VEGFR-1 did not reverse its defective ligand-dependent autophosphorylation. The carboxyl terminus-swapped VEGFR-1, however, displayed ligand-dependent autophosphorylation, downregulation and also conveyed strong mitogenic responses. Thus, the carboxyl tail of VEGFR-1 restrains the ligand-dependent kinase activation and downregulation of VEGFR-1 and its ability to convey the angiogenic responses in endothelial cells.     

Neuropilin-1 and VEGF correlate with somatostatin expression and microvessel density in ovarian tumours

Vascular endothelial growth factor (VEGF), is an angiogenic growth factor, expressed more highly in malignant than benign ovarian tumours. Neuropilin-1, which can act as a VEGF receptor has been shown to be associated with tumour angiogenesis in some cancer systems. Somatostatin (SST), a potentially anti-angiogenic factor, acts via somatostatin receptors that are expressed in ovarian cancer. We used immunohistochemistry to demonstrate expression of Neuropilin-1 in 63 malignant and 35 benign ovarian tumours and compared it to VEGF, Flt, Flk, SST expression and tumour microvessel density (MVD). Neuropilin-1 was expressed in 34/63 malignant and 22/35 benign lesions. VEGF, Flt, Flk and SST were expressed more highly in the epithelium of malignant and the vessels of benign lesions. VEGF expression correlated with SST expression in the epithelium (p<0.001) and the vessels (p<0.001), this co-expression was confirmed by dual immunostaining. The MVD for malignant lesions was higher than benign (p<0.001) and positively correlated to epithelial VEGF expression (p=0.001) and negatively correlated to vascular VEGF expression (p=0.025). These results show that Neuropilin-1 is expressed in ovarian tumours and also show that VEGF and SST are co-expressed in the same tissue compartments raising the intriguing possibility that SST may be important in angiogenesis in ovarian cancer.     

Effects of oestradiol and tamoxifen on VEGF, soluble VEGFR-1, and VEGFR-2 in breast cancer and endothelial cells

Angiogenesis is regulated by the balance between pro- and antiangiogenic factors. Vascular endothelial growth factor (VEGF), acting via the receptors VEGFR-1 and VEGFR-2, is a key mediator of tumour angiogenesis. The soluble form of the VEGF receptor-1 (sVEGFR-1) is an important negative regulator of VEGF-mediated angiogenesis. The majority of breast cancers are oestrogen dependent, but it is not fully understood how oestrogen and the antioestrogen, tamoxifen, affect the balance of angiogenic factors. Angiogenesis is a result of the interplay between cancer and endothelial cells, and sex steroids may exert effects on both cell types. In this study we show that oestradiol decreased secreted sVEGFR-1, increased secreted VEGF, and decreased the ratio of sVEGFR-1/VEGF in MCF-7 human breast cancer cells. The addition of tamoxifen opposed these effects. Moreover, human umbilical vein endothelial cells (HUVEC) incubated with supernatants from oestradiol-treated MCF-7 cells exhibited higher VEGFR-2 levels than controls. In vivo, MCF-7 tumours from oestradiol+tamoxifen-treated nude mice exhibited decreased tumour vasculature. Our results suggest that tamoxifen and oestradiol exert dual effects on the angiogenic environment in breast cancer by regulating cancer cell-secreted angiogenic ligands such as VEGF and sVEGFR-1 and by affecting VEGFR-2 expression of endothelial cells.     

Ki-67、VEGF在胃肠问质瘤中表达及与MVD的相关性

目的：探讨Ki-67和VEGF在胃肠间质瘤中的表达及与临床病理因素的关系，VEGF、微血管密度（MVD）和细胞增殖之间的相关性。方法：采用免疫组化S—P法检测44例胃肠间质瘤组织中Ki-67、VEGF表达及计数MVD值和Ki-67PI。结果：VEGF、Ki-67在GIST组织中阳性表达分别为77．3％、63．6％。VEGF、Ki-67表达在不同大小的肿块之间有统计学差异（P〈0．01）；MVD值和Ki-67PI在VEGF阳性和阴性组的比较有统计学差异（P〈0．01）；VEGF、MVD和Ki-67PI之间呈显著正相关（相关系数分别为0．26、0．44和0．84，P〈0．01）。结论：VEGF促进GIST组织中的新生血管形成，为肿瘤组织提供了丰富的血液和营养，并使细胞增殖活性增强，促进肿瘤细胞的增殖、肿瘤的生长、发展和转移；Ki-67PI为GIST的预后判断提供了比较客观的依据。     

Ki-67、VEGF在胃肠间质瘤中表达及与MVD的相关性

目的:探讨Ki-67和VEGF在胃肠间质瘤中的表达及与临床病理因素的关系,VEGF、微血管密度(MVD)和细胞增殖之间的相关性。方法:采用免疫组化S-P法检测44例胃肠间质瘤组织中Ki-67、VEGF表达及计数MVD值和Ki-67PI。结果:VEGF、Ki-67在GIST组织中阳性表达分别为77.3%、63.6%,VEGF、Ki-67表达在不同大小的肿块之间有统计学差异(P<0.01);MVD值和Ki-67PI在VEGF阳性和阴性组的比较有统计学差异(P<0.01);VEGF、MVD和Ki-67PI之间呈显著正相关(相关系数分别为0.26、0.44和0.84,P<0.01)。结论:VEGF促进GIST组织中的新生血管形成,为肿瘤组织提供了丰富的血液和营养,并使细胞增殖活性增强,促进肿瘤细胞的增殖、肿瘤的生长、发展和转移;Ki-67PI为GIST的预后判断提供了比较客观的依据。     

Increased microvessel density in mucinous compared with malignant serous and benign tumours of the ovary.

Microvessel density of benign, borderline and malignant ovarian tumours was studied immunohistochemically using antibodies to the endothelial cell markers CD31, CD34 and factor VIII-related antigen. Microvessel density was compared in tumours of different histological subtype, stage and patient outcome. CD31-immunostained sections were examined and regions of high and average microvessel density were selected. Identical regions were located on CD34- and factor VIII-related antigen-immunostained serial sections and microvessel counts obtained and converted to vessels mm(-2). CD31 and CD34 immunostaining revealed increased microvessel density in both the high and average vessel density regions of mucinous (222.4 +/- 24.8; 79.9 +/- 8.5) compared with serous (105.4 +/- 20.7; 33.3 +/- 6.8) and benign (84.4 +/- 19.4; 20.4 +/- 4.4) tumours (P < 0.001). CD31 and CD34 immunostaining also revealed increased microvessel density in early-stage mucinous tumours (234.6 +/- 28.2; 87.8 +/- 9.2) compared with that observed in both early- (72.8 +/- 15; 12.9 +/- 2.4) and late- (115.6 +/- 26.5; 29.8 +/- 8.5) stage serous tumours (P < 0.001). No differences in microvessel density in samples from patients with differing outcomes were observed (P > 0.05). Reduced factor VIII-related antigen compared with CD31 and CD34 immunostaining was observed in both borderline and malignant mucinous and serous tumours (P < 0.02) but not in benign tumours (P > 0.05). Our results contradict the putative association between increased microvessel density and poor prognosis and suggest that the level and control of angiogenesis may differ between ovarian tumour types.     

Quantification of microvessel density in human tumours

The current use of tumour microvascular density measurements in clinical practice is limited by the methods employed to identify and quantify the microvessels. The aim of this study was to assess tumour vascularity with three of the most commonly used endothelial markers and with different pre-treatment methods to determine which combination results in the best visualisation of endothelial cells. Serial sections of colorectal carcinoma were treated with microwave oven irradiation, pressure cooking or proteolytic digestion prior to immunostaining with von Willebrand factor (vWf), CD31 or CD34. The sensitivity of staining was enhanced by microwave pre-treatment for CD31 and CD34, and trypsinization for vWf. There was a statistically significant difference between microvascular counts obtained using antibodies for CD34 and vWf (p=0.004), and CD34 and CD31 (p=0.007), but not for CD31 and vWf (p=0.775). We conclude that the use of CD34 antibody on microwaved tissues is the most sensitive method to visualize and quantify vascular density in tumours.     

Human small cell lung cancer cells express functional VEGF receptors, VEGFR-2 and VEGFR-3

Studies have suggested that the vascular endothelial growth factors (VEGFs)/VEGF receptors (VEGF-Rs) system plays an important role in tumour growth and metastasis. We conducted the present study to clarify whether small cell lung cancer (SCLC) cells express functional VEGF-Rs and VEGFs, and their biological significance in the SCLC progression. We examined expression of VEGF and VEGF-C, and their receptors, VEGFR-2 and VEGFR-3, in five SCLC cell lines, NCI-H82, H209, H510, H526 and H660, by Western blotting. We evaluated whether hypoxic conditions up-regulate these protein expressions. We also examined whether VEGF addition and VEGF-D addition cause phosphorylation of the mitogen-activated protein kinase (MAPK) as well as VEGFR-2 and VEGFR-3. Further, we investigated whether VEGF addition and VEGF-D addition induced the proliferation and migration of the SCLC cells. VEGF, VEGF-C, VEGFR-2 and VEGFR-3 were detectable by Western blotting in all five SCLC cell lines,. The VEGF-Rs and VEGFs expression levels were increased by an incubation under hypoxic conditions in NCI-H82. VEGF addition and VEGF-D addition caused phosphorylation of MAPK as well as the VEGF-Rs themselves, and induced proliferation and migration of the SCLC cells. These results suggested potential of VEGF signal-pathway inhibitors as anti-cancer agents in SCLC treatment disturbing growth and migration of the cancer cells.     

Intra-tumoural microvessel density in human solid tumours

Over the last decade assessment of angiogenesis has emerged as a potentially useful biological prognostic and predictive factor in human solid tumours. With the development of highly specific endothelial markers that can be assessed in histological archival specimens, several quantitative studies have been performed in various solid tumours. The majority of published studies have shown a positive correlation between intra-tumoural microvessel density, a measure of tumour angiogenesis, and prognosis in solid tumours. A minority of studies have not demonstrated an association and this may be attributed to significant differences in the methodologies employed for sample selection, immunostaining techniques, vessel counting and statistical analysis, although a number of biological differences may account for the discrepancy. In this review we evaluate the quantification of angiogenesis by immunohistochemistry, the relationship between tumour vascularity and metastasis, and the clinicopathological studies correlating intra-tumoral microvessel density with prognosis and response to anti-cancer therapy. In view of the extensive nature of this retrospective body of data, comparative studies are needed to identify the optimum technique and endothelial antigens (activated or pan-endothelial antigens) but subsequently prospective studies that allocate treatment on the basis of microvessel density are required.     

Association between intratumoral free and total VEGF, soluble VEGFR-1, VEGFR-2 and prognosis in breast cancer

Vascular endothelial growth factor (VEGF) receptors consist of three cell-membrane type receptors (VEGFR-1, VEGFR-2 and VEGFR-3), and soluble form of VEGFR-1 (sVEGFR-1), an intrinsic negative counterpart of the VEGF. In this study, we measured intratumoral protein levels of free and total VEGF, VEGFR-2 and sVEGFR-1 from 202 primary breast cancer tissues and examined their prognostic values. A significant inverse correlation was found between free or total VEGF and oestrogen receptor (ER) status (P=0.042 and 0.032, respectively). A univariate analysis showed that low sVEGFR-1 and high total VEGF were significantly associated with poor prognosis in disease-free survival (DFS) and overall survival (OS). The ratio of sVEGFR-1 to total VEGF was a strong prognostic indicator (DFS: P=0.008; OS: P=0.0002). A multivariate analysis confirmed the independent prognostic values of total VEGF and the ratio of sVEGFR-1 to total VEGF. In subgroup analysis, total VEGF was a significant prognostic indicator for ER-positive tumours but not for ER-negative tumours, whereas sVEGFR-1 was significant for ER-negative tumours but not for ER-positive tumours. In conclusion, the intratumoral sVEGFR-1 level, VEGF level and the ratio of sVEGFR-1 to total VEGF are potent prognostic indicators of primary breast cancer, and might be relevant to ER status.     

Microvessel density, endothelial cell proliferation and tumour cell proliferation in human colorectal adenocarcinomas*

BACKGROUND: Thymidine incorporation studies performed in animal rumour models,revealed major differences in endothelial cell proliferationwhen tumour tissue was compared with normal tissue. The fractionof proliferating endothelial cells is reported to be increasedby a factor of 30 to 40 in tumour tissue. PATIENTS AND METHODS: To make it possible to analyze the endothelial cell proliferationin human tumours, an immuno-histochemical double staining techniquecomprising CD31, an endothelial cell marker, and Ki-67, a proliferationmarker, was developed. Endothelial cell proliferation was analysedin 21 primary human colorectal adenocarcinomas and in the adjacentmucosa. RESULTS: Proliferating endothelial cells were found throughout the entirecarcinoma. The mean overall endothelial cell labeling index(ECU) was 9.9% (range, 5.4–18.0), and the labeling indexof endothelial cells in areas of intense neovascularisationwas even higher. Mean ECLI in the vascular hot spots was 21.0%(range, 6.8–35.0), and the mean tumour cell labeling index(TCLI) in the maximally Ki-67 immunostained areas was 78.3%(range 47.0–89.7). In 14 of 21 carcinomas, these areaswere predominantly found at the luminal margin of the tumour,as were the vascular hot spots. A significant positive correlationwas found between tumour vascularity, measured in the vascularhot spots, and tumour cell proliferation, measured in the maximallyKi-67 immu-nostained areas (p < 0.05). To analyse this relationin more detail, microvessel density (MVD), TCLI and ECLI weredetermined per x400 microscopic field by scanning in sequencefrom the luminal tumour margin to the invasive tumour base.In all tumours, the pattern of the MVD per x 400 field, fromthe luminal margin to the tumour base, was similar to that ofthe TCLI and ECLI. CONCLUSIONS: These findings confirm that the fraction of cycling endothelialcells is higher in human colorectal carcinoma than in the adjacentmucosa which suggests that endothelial cells are proliferatingin most of the individual capillaries in tumour tissue. Regionaldifferences in MVD correlate with differences in tumour cellproliferation in these tumours. angiogenesis, colorectal tumours, endothelial cell proliferation, tumour cell proliferation, microvessel density     

Nonapical and cytoplasmic expression of interleukin-8, CXCR1, and CXCR2 correlates with cell proliferation and microvessel density in prostate cancer.

PURPOSE: We characterized interleukin-8 (IL-8) and IL-8 receptor expression (CXCR1 and CXCR2) in prostate cancer to address their significance to this disease. EXPERIMENTAL DESIGN: Immunohistochemistry was conducted on 40 cases of human prostate biopsy containing histologically normal and neoplastic tissue, excised from patients with locally confined or invasive androgen-dependent prostate cancer, and 10 cases of transurethral resection of the prostate material from patients with androgen-independent disease. RESULTS: Weak to moderate IL-8 expression was strictly localized to the apical membrane of normal prostate epithelium. In contrast, membranous expression of IL-8, CXCR1, and CXCR2 was nonapical in cancer cells of Gleason pattern 3 and 4, whereas circumferential expression was present in Gleason pattern 5 and androgen-independent prostate cancer. Each of IL-8, CXCR1, and CXCR2 were also increasingly localized to the cytoplasm of cancer cells in correlation with advancing stage of disease. Cytoplasmic expression (but not apical membrane expression) of IL-8 in Gleason pattern 3 and 4 cancer correlated with Ki-67 expression (R = 0.79; P < 0.001), cyclin D1 expression (R = 0.79; P < 0.001), and microvessel density (R = 0.81; P < 0.001). In vitro studies on androgen-independent PC3 cells confirmed the mitogenic activity of IL-8, increasing the rate of cell proliferation through activation of both CXCR1 and CXCR2 receptors. CONCLUSIONS: We propose that the concurrent increase in IL-8 and IL-8 receptor expression in human prostate cancer induces autocrine signaling that may be functionally significant in initiating and promoting the progression of prostate cancer by underpinning cell proliferation and angiogenesis.     

Prognostic significance of microvessel density and vascular endothelial growth factor (VEGF) expression in non-Hodgkin's lymphoma

Angiogenesis has a major role in the pathogenesis of malignancies. Studies involving the role of angiogenesis have been most commonly performed in solid tumors. However, studies related to hemapoietic neoplasia and angiogenesis are relatively limited. We investigated the role of angiogenesis in non-Hodgkin's lymphomas (NHLs) and its relation with clinical and histopathologic prognostic indicators. In this respect, angiogenesis markers were evaluated in 71 patients with NHL and these were compared with other prognostic indicators including age, gender, histological grade, stage, extranodal involvement and survival. Microvessel density (MVD) using Factor VIII monoclonal antibody and vascular endothelial growth factor (VEGF) using monoclonal antibody for VEGF expression were studied in paraffin-embedded tissue samples. We did not find a significant relationship between MVD and patient characteristics including age, gender, stage, histological grade, nodal status, international prognostic index (IPI), and response to treatment. MVD was found to be greater in cases with B symptoms compared to those without B symptoms (14.6±5.7 and 11.4±5.3, respectively, p=0.002). No significant relationship was found between VEGF and age, gender, stage, histological grade, IPI, and overall survival. The complete and partial response rate to therapy was significantly higher in VEGF-negative patients than in the VEGF-positive patients (p=0.003). In conclusion, there appears to be a role for angiogenesis and angiogenic factors in NHLs. The combination of anti-angiogenic drugs with conventional anti-neoplastic treatment will probably be used in the future. Larger series of patients are needed to determine the prognostic value of angiogenesis in NHL.     

Prognostic relevance of microvessel density in colorectal tumours.

The importance of angiogenesis as a prognostic marker has been examined in 111 colorectal cancer patients with a minimum follow-up of 5 years. Tumour sections were immunostained with pan-endothelial antibody to CD31. Microvessels were identified and counted in 5 separate areas of highest vascularity (). Analysis of the data showed that the survival was not significantly affected by tumour site, size, grade, patients' age or gender. However, a statistically significant correlation was found between microvessel density (MVD) and survival: patients with an increased number of microvessels survived longer than those with a low number of microvessels (p=0.0007). Therefore, paradoxically, unlike other tumour types, in colorectal cancer MVD appears to be an indicator of good prognosis. The reasons that MVD correlates with good or poor prognosis are likely to vary in different tumours. For instance a frequent difficult issue in colon cancer is the presence of ulceration and adjacent severe inflammation which by itself can increase vascularity. Furthermore, overall prognosis will also depend on other factors, such as oncogenes, extracellular matrix components, adhesion molecules, growth factors, degree of apoptosis and the mode of metastatic spread.     

大肠癌组织c-Met和VEGF表达及其与微血管密度关系的初步研究

目的:探讨大肠癌组织c-Met和血管内皮生长因子(VEGF)的表达以及两者与肿瘤血管生成的关系及其临床病理学意义。方法:选取经病理确诊的51例大肠腺癌手术切除组织标本,免疫组化SP法检测c-Met和VEGF的表达及微血管密度(MVD)。结果:大肠癌组织中c-Met和VEGF的阳性表达率分别为72.55%(37/51)和52.94%(27/51);与Dukes分期密切相关,P<0.05。大肠癌组织c-Met、VEGF均为阳性时MVD值为37.94±7.53,单一阳性时分别为30.67±1.45和23.82±7.45,均为阴性时MVD值最小,为13.64±5.33。大肠癌组织中c-Met和VEGF的表达呈显著正相关,rs=0.614,P<0.05。结论:c-Met和VEGF在大肠癌发生、发展和转移过程中起重要作用,并与肿瘤血管生成相关。     

Vascular endothelial growth factor (VEGF) in breast cancer: comparison of plasma, serum, and tissue VEGF and microvessel density and effects of tamoxifen

The assessment of angiogenesis in breast cancer is of importance as a key indicator of survival and response to therapy. Circulating vascular endothelial growth factor (VEGF) measurements may provide a less subjective analysis than microvessel density (MVD) or immunohistochemical analysis of VEGF expression; however, most studies have used serum, which is now known to largely reflect platelet-derived VEGF concentrations. This study examined for the first time both plasma (VEGFp) and serum (VEGFs) VEGF concentrations in 201 blood samples from pre- and postmenopausal healthy controls and from patients with benign breast disease, localized breast cancer, breast cancer in remission, or metastatic breast cancer and related these to other clinicopathological markers. VEGFp but not VEGFs concentrations of patients with localized disease were significantly elevated compared with normal controls (P = 0.016). Patients with metastatic disease had higher VEGFp and VEGFs levels than normal controls (P < 0.001, P = 0.044 respectively), and higher VEGFp, but not VEGFs, than patients with benign disease (P = 0.009) and patients with localized disease (P = 0.004). However, the highest VEGFp and VEGFs concentrations were seen in patients in remission compared with normal controls (P < 0.001 and P = 0.008, respectively). VEGFp concentrations in patients in remission were also higher than in patients with benign disease (P = 0.01) or patients with localized disease (P = 0.005). Tamoxifen treatment was significantly associated with higher circulating and platelet-derived VEGF levels. Circulating VEGF did not correlate with any clinicopathological factor, including MVD or VEGF expression. VEGF expression was significantly correlated with estrogen receptor status and inversely correlated with tumor grade. MVD correlated with tumor size. Tamoxifen-induced increases in VEGF may be important in clinical prognosis or associated pathologies.     

Vascular endothelial growth factor/KDR activated microvessel density versus CD31 standard microvessel density in non-small cell lung cancer

Vascular endothelial growth factor (VEGF) is an important angiogenic factor, linked to poor outcome in human malignancies including non-small cell lung carcinoma (NSCLC). We used the 11B5 monoclonal antibody recognizing the VEGF/KDR complex (R. A. Brekken et al., Cancer Res., 58: 1952-1959, 1998) to assess the VEGF expression in cancer cells and the VEGF/KDR activated microvessel density (aMVD) in early operable NSCLC. The JC70 anti-CD31 monoclonal antibody was used to assess the standard MVD (sMVD). The aMVD was significantly higher in the invading front of the tumors and in the normal lung adjacent to the tumors as compared with normal lung distant to the tumor or to inner tumor areas (P < 0.0002). The sMVD was higher in the normal lung and decreased from the invading front to inner tumor areas (P < 0.0001). However, the vascular activation (aMVD:sMVD) was 4-6 times higher in the tumor areas as compared with lung from normal individuals (36-58% versus 9%; P < 0.0001). Fibroblast 11B5 reactivity, noted in 25% of cases, correlated with high aMVD and sMVD in the inner tumor areas. Multivariate analysis showed that aMVD was the most potent and independent prognostic factor (P = 0.001; t-ratio, 3.28). It is concluded that intense VEGF/KDR angiogenic pathway activation is a tumor-specific feature in more than 50% of NSCLC cases and is associated with poor postoperative outcome. Clinical trials involving targeting of the VEGF/KDR-positive vasculature with specific antibodies, such as 11B5, are, therefore, encouraged.