Expression of aquaporine-4 in central nervous system tumors

Cerebral edema is associated with common brain tumors. Aquaporine-4 (AQP4) is a member of the water channel protein family, which is thought to be a major factor regulating cerebral edema. To elucidate the characterization of the expression of AQP4 and the relationship of the expression of VEGF, we investigated the expression of AQP4 in tumors of the central nervous system immunohistochemically. Brain tumors and nontumorous cerebral tissue for control were evaluated by immunohistochemical staining using anti-AQP4, VEGF, CD34, and MIB-1. In tumor cells, only glial tumor cells showed a positive reaction for AQP4. The reactivities for immunostaining increased according to WHO grades. Reactive glial cells in edematous tissue also showed positive reactions. Although endothelial cells were negative and/or weakly positive for AQP4, the positive relationship suggested the expression of VEGF in endothelial cells in neovasculature and that of AQP 4 in tumor cells. APQ4 expression increased in human astrocytic tumors and edematous cerebral tissue. Upregulation of APQ4 by tumor cells and reactive astroglia were major factors of cerebral edema.     

Tumoral micro-blood vessels and vascular microenvironment in human astrocytic tumors. A transmission electron microscopy study

The development of peritumoral edema is thought to be due to extravasation of plasma water and macromolecules through a defective blood–brain barrier (BBB), but the exact mechanism by which occurs is poorly understood. The aim of this study was analyze at submicroscopic level the morphological changes in both micro-blood vessels and vascular microenvironment of astrocytic tumors in an attempt of understanding the pathological aspects that may help in the future researches for the design of future therapeutic strategies. Biopsies of 25 patients with pathological diagnosis of astrocytic tumors were examined with the transmission electron microscope. Both open and close tight junctions were observed in the micro-blood vessels, inclusive in a same tumor. Cytoskeletal disorganization associated with disintegrated perijunctional actin filaments were seen. The paracellular space showed enlargement and commonly occupied by fluid proteinaceous, endothelial cells display oncotic and ischemic changes, basal lamina reveals enlargement, edema, vacuolization and collagen fibers disposed in irregular array. Pericytes exhibited edema and phagocytoced material, astrocytic perivascular-feet showed signs of oncosis and necrosis, cooption vessels totally surrounding by neoplastic cells also were seen. The ultrastructural abnormalities observed in both junctional complexes and vascular microenvironment suggest a multi-factorial pathobiology process, probably hypoxia intratumoral, calcium overload in endothelial cells, and degradative effects of metalloproteinases over the basal membrane appear as determinant factors that leading to structural modifications of junctional complexes, therefore, treatment with both HIF-1α and metalloproteinases inhibitors possibly can contribute with the pharmacological handling of the peritumoral edema associated with astrocytic tumors.     

Angiogenesis in primary central nervous system lymphoma (PCNSL)

Angiogenesis and angiogenic growth factors have a major role in the pathogenesis of malignancies. However, very little is known about the clinical and histopathological relevance of angiogenesis in primary central nervous system lymphoma (PCNSL). We investigated that expression of vascular endothelial growth factor (VEGF) of the lymphoma cells and microvessel density (MVD) were examined in 19 patients with PCNSL. Additionally, the presence of the blood–brain barrier (BBB) was examined using immunohistochemistry and the electron microscopy. MVD was significantly higher in nine cases with VEGF immunoreactivity (VEGF+) than in ten cases with negative immunoreactivity for VEGF (VEGF−) (P < 0.001). VEGF expression was significantly associated with a longer survival (P < 0.005). BBB markers were negative in angiogenic vessels of VEGF+. BBB markers were identified in vessels surrounding tumor cells and tight junctions were also preserved in the capillary endothelium surrounding tumor cells in VEGF−. Angiogenesis is associated with VEGF expression and an absent BBB in the vessels of PCNSL. The BBB may be preserved in lesions with lymphoma cell infiltration, especially in VEGF− PCNSL. VEGF may have a prognostic effect in PCNSL.     

Stromal cell-derived factor-1 stimulates vasculogenesis and enhances Ewing's sarcoma tumor growth in the absence of vascular endothelial growth factor

Stromal cell-derived Factor-1alpha (SDF-1alpha) stimulates the migration of bone marrow (BM) cells, similar to vascular endothelial growth factor (VEGF). We previously demonstrated that inhibition of VEGF(165) by small interfering RNA inhibited Ewing's sarcoma tumor growth, tumor vessel formation and recruitment of BM cells to the tumor. To determine the importance of BM cells in tumor vessel development, we investigated the effects of SDF-1alpha on VEGF-inhibited TC/siVEGF(7-1) Ewing's tumor neovasculature formation and growth. The effect of SDF-1alpha on CD34(+) progenitor cell chemotaxis was determined in vivo. Using a BM transplantation model with GFP(+) transgenic mice as BM donors and nude mice as recipients, we evaluated the effect of SDF-1alpha on the recruitment of BM-derived cells to VEGF(165)-inhibited TC/siVEGF(7-1) tumors, as well as its effect on neovasculature development, vessel morphology and tumor growth. SDF-1alpha stimulated the migration of CD34(+) progenitor cells to Matrigel plugs in vivo and promoted the retainment of BM-derived pericytes in close association with perfused, functional tumor vessels. Intratumor inoculation of Ad-SDF-1alpha into TC/siVEGF(7-1) tumors resulted in increased SDF-1 and PDGF-BB expression, augmented tumor growth, an increase in the number of large, lumen-bearing vascular structures, and enhanced vessel pericyte coverage, with no change in VEGF(165). SDF-1alpha stimulates BM cell chemotaxis and the association of these cells with functional tumor vessels. Furthermore, SDF-1alpha enhances tumor neovascularization and growth with no alteration in VEGF(165). Our work suggests that SDF-1-mediated vasculogenesis may represent an alternate pathway that could potentially be utilized by tumors to sustain growth and neovasculature expansion after anti-VEGF therapy.     

In vivo prediction of vascular susceptibility to vascular susceptibility endothelial growth factor withdrawal: magnetic resonance imaging of C6 rat glioma in nude mice.

One of the hallmarks of tumor neovasculature is the prevalence of immature vessels manifested by the low degree of recruitment of vascular mural cells such as pericytes and smooth muscle cells. This difference in the architecture of the vascular bed provides an important therapeutic window for inflicting tumor-selective vascular damage. Here we demonstrate the application of gradient echo magnetic resonance imaging (MRI) for noninvasive in vivo mapping of vascular maturation, manifested by the ability of mature vessels to dilate in response to elevated levels of CO2. Histological alpha-actin staining showed a match between dilating vessels detected by MRI and vessels coated with smooth muscle cells. Switchable, vascular endothelial growth factor (VEGF)-overexpressing tumors (C6-pTET-VEGF rat glioma s.c. tumors in nude mice) displayed high vascular function and significant vascular damage upon VEGF withdrawal. However, damage was restricted to nondilating vessels, whereas mature dilating tumor vessels were resistant to VEGF withdrawal. Thus, MRI provides in vivo visualization of vascular maturity and prognosis of vascular obliteration induced by VEGF withdrawal.     

骨髓来源内皮前体细胞促进肿瘤新生血管生成

Objective:Neovascularization of tumor is a complex process.In this study,we aimed to reveal whether the bone marrow-originated endothelial progenitor calls (EPCs) contributed to neovasculature in tumor and the angiogenesis-associated factors,VEGF and B-FGF,enhanced this process.Methods:We had established a mouse model,which were deprived of bone marrow by radiation and transplanted with bone marrow of syngenetic GFP (Green Fluorescence Protein)-transgened mice,then implanted Lewis calls.Immunohistochemical and immunoflourensenca proved the EPCs location in tumors by indentifying colocalization of GFP expression in cells staining with endothelial progenitor cell markers,CD 133,ICAM-1,CD31.The growth statue and MVD of tumor was observed after injection of VEGF or B-FGF.ICAM-1 and VE-cadherin in tumor were detected by Western blot.Results:By immunohistochemical and immunoflourensence,we proved part of bone marrow precursors located in area of tumor angiogenesis and VEGF or B-FGF increased the MVD of tumor.In Western blot,it was found and VEGF or B-FGF up-regulate the expression of ICAM-1,VE-Cadherin.Conclusion:Bone marrow-derived endothelial progenitor cell seem to be recruited in neovasculature induced by tumor.VEGF and B-FGF are key regulators of this process.     

Over expression of vascular endothelial growth factor and its receptor during the development of estrogen-induced rat pituitary tumors may mediate estrogen-initiated tumor angiogenesis

Estrogens, which have been associated with several types of human and animal cancers, can induce tumor angiogenesis in the pituitary of Fischer 344 rats. The mechanistic details of tumor angiogenesis induction, during estrogen carcinogenesis, are still unknown. To elucidate the role of estrogen in the regulation of tumor angiogenesis in the pituitary of female rats, the density of blood vessels was analysed using factor VIII related antigen (FVIIIRAg) immunohistochemistry and the expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) was examined by Western blot and immunohistochemical analysis. The expression of VEGF receptor (VEGFR-2/Flk-1/KDR) was also examined by immunohistochemistry. The results demonstrated that 17beta-estradiol (E2) induces neovascularization, as well as the growth and enlargement of blood vessels after 7 days of exposure. The high tumor angiogenic potential was associated with an elevated VEGF/VPF protein expression in the E2 exposed pituitary of ovariectomized (OVEX) rats. VEGF/VPF and FVIIIRAg immunohistochemistry and endothelial specific lectin (UEA1) binding studies, indicate that the elevation of VEGF protein expression initially occurred in both blood vessels and non-endothelial cells. After 15 days of E2 exposure, VEGF/VPF protein expression, in the non- endothelial cell population, sharply declined and was restricted to the blood vessels. The function of non-endothelial-derived VEGF is not clear. Furthermore, immunohistochemical studies demonstrated that VEGFR- 2 (flk-1/KDR), expression was elevated significantly in the endothelial cells of microblood vessels after 7 days of E2 exposure. These findings suggest that over expression of VEGF and its receptor (VEGFR-2) may play an important role in the initial step of the regulation of estrogen induced tumor angiogenesis in the rat pituitary.      

Vascular endothelial growth factor C promotes human gastric carcinoma lymph node metastasis in mice.

Vascular endothelial growth factor (VEGF)-C, one of several members of the VEGF family, is a relatively specific lymphangiogenic growth factor. VEGF receptor (VEGFR)-3 (or Flt4) is a VEGF-C receptor with expression restricted to lymphatic endothelial cells. Since the mechanisms by which carcinoma cells metastasize to lymph nodes remain unclear, we constructed a VEGF-C transfectant (AZ-VEGF-C) from the AZ521 human gastric carcinoma cell line, which ordinarily shows little nodal metastatic potential and little VEGF-C expression. We orthotopically implanted transfected tumor cells into the stomachs of nude mice. The number of mice developing lymph node metastases and the number of lymph node metastases per mouse with nodal metastases were higher than with implants of mock-transfected control cells. Specifically, percentages of mice with lymph node metastases were 95.5% (21/22) for AZ-VEGF-C and 29.4% (5/17) for controls (P<0.01), while mean numbers of involved lymph nodes were 3.76 for AZ-VEGF-C and 1.00 for controls (P<0.01). No difference was found between AZ-VEGF-C and controls regarding cell growth and chemotactic responses in vitro, or in volumes of tumors arising from implanted cells. When we performed immunohistochemical staining for VEGFR-3 in these tumors to investigate lymphangiogenesis by VEGF-C, the number of vessels stained for VEGFR-3 in tumors and surrounding tissues was higher for AZ-VEGF-C than for controls. VEGFR-3-positive vessels occupied 14.9/1000 of microscopically examined areas for AZ-VEGF-C, but only 1.30/1000 for controls (P<0.001). Our results suggest that VEGF-C is a specific lymphangiogenic growth factor with an important role in lymph node metastasis.     

Highly aggressive behavior and poor prognosis of small cell carcinoma in the stomach: flow cytometric and immunohistochemical analysis.

Small cell carcinoma of the stomach has an aggressive feature, and the survival rate of the patients is poor. The purpose of this study was to determine the clinical course, and effects of histopathologic characteristics of specific tumors including DNA contents and immunohistochemical aspects in patients with small cell carcinoma of the stomach. Medical records of 8 patients who presented with small cell carcinoma of the stomach were retrospectively reviewed. Primary tumors were studied by flow cytometric analysis and immunohistochemical staining for the p53 protein, PCNA (proliferating cell nuclear antigen), factor VIII related antigen (specific for endothelial cells), VEGF (vascular endothelial growth factor) and PD-ECGF (platelet-derived endothelial cell growth factor). DNA aneuploid was observed in 4 cases. Staining for the p53 product was positive in 50% of all the cases. The average PCNA labeling rate (LR) was 71.3+/-9.9%. Positive VEGF expression was found in 7 tumors and positive PD-ECGF expression was found in all tumors. The estimated median survival was 252 days for all the patients. Liver metastases were observed in 4 of the 8 patients, however, surgery and chemotherapy have given us one long-term survivor (43 months). Higher PCNA LR of small cell carcinoma may be an unfavorable characteristic of biological behavior. Moreover, both VEGF and PD-ECGF positivity are well-characterized inducers of hepatic metastasis.     

脑星形细胞肿瘤中FAK、Pyk2表达及其与血管生成的关系

背景与目的：非受体酪氨酸蛋白激酶家族成员FAK和Pyk2具有高度同源性,对肿瘤细胞增殖和侵袭具有重要的调控作用,但其是否参与肿瘤血管生成过程尚不明确。本研究旨在探讨FAK和Pyk2在脑星形细胞肿瘤中表达与血管生成的关系。方法：应用免疫组化法检测58例脑星形细胞肿瘤中FAK、Pyk2和血管内皮生长因子（VEGF）表达,并用CD31标记计数瘤内微血管密度。结果：FAK、Pyk2蛋白主要表达于肿瘤细胞胞浆,Pyk2阳性表达也见于肿瘤血管内皮细胞。在Ⅰ、Ⅱ、Ⅲ、Ⅳ级星形细胞肿瘤中,FAK阳性率分别为20.0%（1/5）、26.7%（4/15）、44.4%（8/18）、50.0%（10/20）,Pyk2阳性率分别为40.0%（2/5）、60.0%（9/15）、77.8%（14/18）、85.0%（17/20）;FAK和Pyk2阳性表达强度评分在Ⅱ级星形细胞肿瘤与Ⅲ、Ⅳ级星形细胞肿瘤中具有显著差异性（P〈0.05）。FAK、Pyk2表达与VEGF和微血管密度呈正相关,相关系数分别为rs=0.423（P=0.001）和rs=0.729（P〈0.005）。结论：FAK、Pyk2可能通过与VEGF的相互作用而参与脑星形细胞肿瘤的血管生成过程。     

Immunohistochemically Detection of Angiogenesis in Oral Pre-Cancerous Lesions Compared with Oral Invasive Carcinomas

Background: Angiogenic activity is an important event in oral carcinogenesis. During transition of normaloral mucosa to different grades of dysplasia and to invasive carcinoma, significant increase of vascularity occurs.Angiogenesis can be determined by immunohistochemical assessment of several endothelial cell markers likeEndogelin (CD 105), expressed in activated endothelial cells and associated with neovasculature, and the vascularendothelial growth factor (VEGF). This study was conducted to evaluate angiogenic activity in oral precancerous lesionscompared with oral invasive carcinomas by immunohistochemical staining of VEGF and CD 105 proteins. Methods:In the present cross-sectional study, 20 normal, 20 pre-cancerous mucosa and 20 oral invasive carcinoma sampleswere immunohistochemically stained. Positive cells were counted in each section and micro vessel density (MVD)was determined. The data were statistically analyzed by Mann-Whitney and Kruskal-Wallis tests, with a P-value ≤0.05considered significant. Results: The mean expression value for VEGF was 24.6 in oral invasive carcinoma, 16.4 inprecancerous mucosa and 15.5 in normal mucosa, with no significant differences between the latter two. Endoglinwas negative in all normal mucosa samples, but had scores of 7.58 for precancerous mucosa and 19.4 in oral invasivecarcinoma specimens. MVD was significantly higher in SCC than in dysplastic mucosa. Conclusion: Oral invasivecarcinoma has more angiogenic activity in comparison with pre-cancerous lesions and normal mucosa. Given the highexpression of CD105 positive vessels in malignant lesions, we can argue that determination of mean vessel density (MVD)by application of the CD105 marker could be a useful parameter to differentiate cancerous from pre-cancerous lesions.     

星形细胞肿瘤VEGF、MVD和Ki-67表达的临床意义

 目的　探讨VEGF和Ki- 6 7表达与星形细胞肿瘤恶性程度、微血管密度 (MVD)、瘤细胞增殖以及病人预后等方面的关系 ,评估两者在病理诊断和临床治疗中潜在的应用价值。方法　采用免疫组化S -P法 ,分析 4 8例脑星形细胞肿瘤标本中VEGF蛋白和Ki- 6 7的表达 ,并用FⅧ -RA标记血管内皮细胞以计算MVD。结果　VEGF阳性率在低、高度恶性星形细胞肿瘤中分别为 2 6 .0 9%、6 4 .0 0 % ;在低度恶性星形细胞肿瘤中Ki- 6 7LI和MVD分别为 8.89± 5 .80和 6 .6 9± 4 .81,但在高度恶性肿瘤中分别为 17.4 9± 13.13和 2 3.6 2± 6 .78,其差异具有显著性 ;术后生存两年以上者 ,VEGF表达、MVD和Ki- 6 7LI明显低于术后生存两年以下者 ;相关分析表明VEGF蛋白表达、MVD与Ki- 6 7LI间存在相关性。结论　VEGF可促进肿瘤血管形成和恶性进展 ,对病人的预后产生影响 ;同时定量检测MVD和Ki- 6 7,对星形细胞肿瘤病理分级和预后判断有一定帮助。     

Impact of vascular endothelial growth factor receptor 1, 2, and 3 expression on the outcome of patients with gastric cancer

Tumor angiogenesis is a multistep interactive process in which vascular endothelial growth factor (VEGF) and its receptors have a major role. However, the clinical significance of these molecules in gastric cancer (GC) remains unclear. Our study group comprised 86 patients who underwent gastrectomy and subsequently received chemotherapy for recurrent or residual tumor. Using immunohistochemical techniques, we analyzed the expression of VEGF receptors (VEGF-R) 1, 2, and 3. VEGF-R1 expression (defined as >5% staining) was found in the tumor cells of 65 tumors (76%) and in the stromal vessels of 36 tumors (42%). VEGF-R2 expression was found in tumor cells and stromal vessels of 0 and 46 tumors (0 and 53%), respectively, and VEGF-R3 expression was found in tumor cells and stromal vessels of 0 and 75 tumors (0 and 87%), respectively. Univariate analysis revealed that VEGF-R expression correlated with shorter survival (VEGF-R1 in stromal vessels, P  = 0.001; VEGF-R2 in stromal vessels, P  = 0.009; VEGF-R3 in stromal vessels, P  = 0.005) and lower response to S-1 (VEGF-R1 in stromal vessels, P  = 0.039). Multivariate analysis of potential prognostic factors showed that VEGF-R1 and VEGF-R2 in stromal vessels were independent predictors of poor outcome. Our data suggest that VEGF-R expression can be a predictor of unfavorable clinical outcome in GC. VEGF-R are promising candidates as therapeutic targets. ( Cancer Sci 2009; 100: 310–315)     

Angiogenesis in metastatic verrucous carcinoma of the uterine cervix

A 65-year old woman operated on for verrucous carcinoma of the uterine cervix 13 years previously was found to have multiple small recurrent tumors in the retroperitoneal space. Tumor cell expression of vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) was investigated at the second operation. Expression of VEGF was strongly positive in the tumor cells and expression of PD-ECGF was strongly positive in both the tumor cells and the interstitial cells.     

Possible Pathophysiological Role of Vascular Endothelial Growth Factor (VEGF) and Matrix Metalloproteinases (MMPs) in Metastatic Brain Tumor-associated Intracerebral Hemorrhage

Summary Background Intratumoral hemorrhage, as one of the cerebrovascular complications in various tumor-related conditions, occurs mainly in malignant brain tumors. Recent studies have shown that the overexpression of vascular endothelial growth factor (VEGF) and metalloproteinase (MMP) may play a role for the loss of vascular integrity and the subsequent hemorrhage in several instances, in addition to their well-known properties in tumor development and metastasis. Methods To investigate the potential role of VEGF and MMP in hemorrhagic complication of metastatic brain tumor, we estimated the expression of VEGF, MMP-2 & -9 by immunohistochemical studies in pathological specimens of metastatic brain tumors obtained from 16 patients, 7 in hemorrhagic and 9 in non-hemorrhagic group. We also examined the expression of collagen type IV, CD34, Factor VIII in order to evaluate the status of tumor vasculature. Results Patients in hemorrhagic group showed a higher VEGF expression with neovascularization than those in non-hemorrhagic group. The basement membranes of newly formed vessels were disrupted in cases with high expression in both MMP-2 and -9. These results indicate that rapid growing nascent blood vessels, responding vigorously to VEGF, are concentrated around the hemorrhagic tumors. Besides, these results suggest a possibility that the basement membranes of these nascent vessels could be disrupted proteolytically by MMP. Conclusion We conclude that overexpression of VEGF and MMP may play a role in metastatic brain tumor-associated hemorrhage. Presumably, the underlying pathophysiological mechanisms are through rapid growth and breakdown of vessels around the tumors caused by overexpression of VEGF and MMP of tumor cells.     

Signaling through Raf-1 in the Neovasculature and Target Validation by Nanoparticles

A recent study demonstrated that vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) activate Raf-1 kinase in an experimental neovasculature system. The study showed that bFGF and VEGF activate p21-activated protein kinase-1 (PAK-1) and Src kinase, respectively. PAK-1 and Src kinases phosphorylate specific serine and tyrosine residues within the activation loop of Raf-1 kinase. Their findings further suggest that phosphorylation at these sites protects endothelial cells from apoptosis induced by both intrinsic and extrinsic factors. The tumor neovasculature provides specific molecular markers or "zip codes". This group of investigators has previously shown that nanosphere-aided targeting of the neovasculature with mutant Raf-1 causes regression of the tumor vasculature. Thus, nanoparticles coated with "zip code"-specific homing biomolecules may be useful for delivering anti-angiogenic molecules that can induce tumor regression.     

Bevacizumab changes vascular structure and modulates the expression of angiogenic factors in recurrent malignant gliomas

Bevacizumab (BV), a monoclonal antibody against vascular endothelial growth factor (VEGF), is currently used in the treatment of malignant glioma. To understand mechanisms of resistance to BV, we investigated morphological changes in tumor vessels and expression of angiogenic factors, such as VEGF, Flt-1, basic fibroblast growth factor (bFGF), and platelet-derived growth factor-BB (PDGF-BB), in four autopsied tumors after BV treatment. Three patients had glioblastomas; the fourth had a secondary glioblastoma that developed from a diffuse astrocytoma. BV was administered because of recurrence following the use of the Stupp regimen in these four patients. We compared the initial surgical specimen with that obtained after death following BV treatment. Immunohistochemical staining of the autopsied tumors showed that Flt-1 expression increased while VEGF expression was significantly reduced. Additionally, other angiogenic factors, particularly bFGF, were enhanced. Interestingly, the proliferation of endothelial cells was reduced, but remarkable proliferation of pericytes was observed. These results suggest that following BV treatment, glioblastomas can grow tumor vessels by expressing various angiogenic factors. These mechanisms might be important for rapid regrowth and blood brain barrier repair after BV treatment. Inhibition of multiple angiogenic factors will be required to control tumor vessels in glioblastoma.     

血管内皮生长因子在膀胱移行细胞癌中表达的意义

目的:探讨膀胱移行细胞癌中血管内皮生长因子(VEGF)的表达及其与临床病理指标的关系.方法:应用免疫组织化学方法,对62例原发性膀胱移行细胞癌及8例正常膀胱组织中VEGF进行检测.结果:正常膀胱移行上皮均为阴性反应,膀胱癌组织中VEGF阳性表达率为56.5%.低分化和浸润性癌中VEGF阳性表达率明显高于高分化和表浅性癌组(P<0.05),复发者阳性表达率明显高于未复发者(P<0.05),WEGF阳性表达者3年生存率明显低于阴性表达者(P<0.05).结论:VEGF表达对膀胱癌生物学行为有重要影响,VEGF表达有可能成为预测膀胱癌复发、转移和预后的一种指标.