Correlation between PSMA and VEGF expression as markers for LNCaP tumor angiogenesis

Our aim is the identification and correlation of changes in tumor-associated protein expression which results from therapy. LNCaP tumors, excised from nude mice treated either by orchiectomy or with the chemotherapeutic agent paclitaxel, were evaluated for the expression of proteins and receptors associated with growth, differentiation, and angiogenesis using immunohistologic procedures. Compared to untreated control tumors, both treatments reduced the expression of vascular endothelial growth factor (VEGF), prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), androgen receptor (AR), and epidermal growth factor receptor (EGFR). The effect of paclitaxel treatment on AR expression was the most significant (P = .005). Of particular interest was identifying a significant correlation (P < .000801) between PSMA and VEGF expression regardless of treatment modality. These altered expressions suggest that PSMA may also be a marker for angiogenesis and could represent a target for deliverable agents recognizing either prostatic tumors or endothelial development. Cell surface PSMA would then present a unique target for treatment of patients early in their development of prostatic metastases.     

Endogenous angiogenesis inhibitor blocks tumor growth via direct and indirect effects on tumor microenvironment

Tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) belongs to a small family of endogenous proteins that inhibits a group of enzymes, the matrix metalloproteinases (MMPs). TIMP-2 inhibits endothelial cell proliferation and migration in vitro and angiogenesis in vivo, through MMP-dependent and -independent mechanisms. However, little is known regarding the contribution of these mechanisms to the antitumor effects of TIMP-2. Using a retroviral delivery system, we stably overexpressed TIMP-2 and its mutant Ala+TIMP-2 (devoid of MMP inhibitory activity) in human adenocarcinoma A549 cells. Using real time PCR, and enzyme-linked immunosorbent assay (ELISA), we confirmed enhanced TIMP-2 expression and its MMP inhibitory activity by reverse zymography. In vitro, growth assays suggested that TIMP-2 and Ala+TIMP-2 did not alter basal cell proliferation rates, however, tumor cell migration and invasion were inhibited. In vivo, both TIMP-2 and Ala+TIMP-2 A549 xenografts exhibited reduced growth rate, CD31 immunostaining indicated decreased intratumoral microvascular density, and TUNEL demonstrated enhanced tumor cell apoptosis. Immunoblotting and immunohistochemical analyses of A549 xenograft tissues with either phospho-FAK (Tyr397) or phospho-AKT (Ser473) showed decreased activation in both TIMP-2 and Ala+TIMP-2 tumor cells. We conclude that TIMP-2-mediated inhibition of tumor growth occurs, at least in part, independently of MMP inhibition, and is a consequence of both direct effects of TIMP-2 on tumor cells and modulation of the tumor microenvironment.     

VEGF通过上调CCN2促HUVEC迁移和血管生成

目的 探讨血管内皮生长因子(VEGF)诱导的成骨细胞中结缔组织生长因子(CTGF/CCN2)对人脐静脉血管内皮细胞(HUVECs)的影响.方法 用Real time PCR法及ELISA法检测VEGF诱导成骨细胞(OSE)中CCN2含量;制备成骨细胞(OSE)上清液;将细胞分为control组、OSE组和VEGF-OSE组(n=3).用小干扰RNA (siRNA)转染法抑制成骨细胞中CCN2的表达;Transwell法检测内皮细胞迁移;Matrigel实验检测管样结构形成能力.结果 VEGF呈时间和剂量依赖性上调成骨细胞中CCN2 mRNA和蛋白的表达;CCN2可促进内皮细胞的迁移和管样结构形成(P＜0.05),当CCN2被siRNA基因沉默或者加入CCN2抗体后,CCN2对内皮细胞迁移和管样结构形成的促进作用均受到明显抑制(P＜0.05).结论 VEGF通过上调成骨细胞中CCN2的表达,促内皮细胞(HUVECs)的迁移和血管生成.     

Control of angiogenesis by VEGF and endostatin-encapsulated protein microcrystals and inhibition of tumor angiogenesis

Encapsulation of cytokines within protein microcrystals (polyhedra) is a promising approach for the stabilization and delivery of therapeutic proteins. Here, we investigate the influence of vascular endothelial growth factor (VEGF) microcrystals and endostatin microcrystals on angiogenesis. VEGF was successfully encapsulated into microcrystals derived from insect cypovirus with overexpression of protein disulfide bond isomerase. VEGF microcrystals were observed to increase the phosphorylation of p42/p44 MAP kinase and to stimulate the proliferation, migration, and network and tube formation of human umbilical vein endothelial cells (HUVECs). Endostatin was also successfully encapsulated into microcrystals. Endostatin microcrystals showed antiangiogenesis activities and inhibited the migration, and network and tube formation of HUVECs. Local administration of endostatin microcrystals in mice inhibited both angiogenesis and tumor growth with clear significant differences between treatment and control groups. Endostatin microcrystals only affected angiogenesis, but had no significant effect on lymphangiogenesis compared to controls. Local therapy using endostatin microcrystals offers a potential approach to achieve sustained therapeutic release of antiangiogenic molecules for cancer treatment.     

CXCL8和VEGF在胰腺癌组织中的表达及其与血管生成的关系

目的探讨CXCL8和VEGF在胰腺癌组织中的表达及其与肿瘤血管生成的关系。方法应用免疫组化方法检测23例胰腺癌组织和27例正常胰腺组织微血管密度和CXCL8、VEGF的表达,分析MVD、CXCL8、VEGF与胰腺癌临床病理因素的关系,并分析三者之间的相关性。结果胰腺癌组织中MVD显著高于正常胰腺组织(t=11.187,P0.05),胰腺癌分化程度差、CA199分泌多、临床分期晚肿、瘤直径越大以及淋巴结转移患者肿瘤组织MVD值越高(P0.05);胰腺癌组织中VEGF的表达显著高于正常胰腺组织(χ~2=40.338,P0.05);胰腺癌组织中CXCL8的表达显著高于正常胰腺组织(χ~2=25.343,P0.05),胰腺癌患者中CA199水平越高,分化程度越差,CXCL8的阳性表达率越高(P0.05);胰腺癌组织中MVD与VEGF的表达无相关性;在所有统计病例中,VEGF表达阳性者MVD值比阴性者高(P0.05);胰腺癌组织中CXCL8阳性者MVD值比阴性者高(P0.05),所有统计病例中,CXCL8阳性者MVD值比阴性者高(P0.05);胰腺癌组中CXCL8与VEGF的表达无相关性,在所有统计病例中,CXCL8与VEGF的表达呈正相关(r=-0.079,P0.05)。结论胰腺癌中微血管新生高于正常胰腺组织,CXCL8和VEGF与肿瘤血管生成相关。     

VEGF分泌量及分泌来源对肿瘤血管生长影响的数值模拟

目的 数值模拟研究血管内皮生长因子（vascular endothelial growth factor, VEGF）分泌量及分泌来源对肿瘤血管生成的影响。方法 建立肿瘤内外血管生成的二维离散数学模型。围绕VEGF的分泌及其诱导新生血管形成肿瘤富血管区的过程,考虑细胞外基质的旁分泌作用以及对内皮细胞运动的趋触作用,以微血管密度作为定量指标,探讨VEGF的分泌量及不同的分泌来源对血管生成的影响。结果 肿瘤增殖细胞区、VEGF高浓度区、富血管区三者统一,微血管密度与VEGF的表达有关,随着增殖细胞区域的扩大,即VEGF的表达越来越多,微血管密度也越来越大,但在不同类型的肿瘤中,VEGF不同分泌来源的比重与微血管密度无明显相关性。结论 模型探讨了VEGF分泌量及分泌来源对肿瘤血管生成的影响,其中对VEGF的不同分泌来源的考虑可作为研究靶向VEGF治疗肿瘤的模型基础。     

Pancreas transplantation: an immunohistochemical analysis of pancreatic hormones and HLA-DR expression

The immunohistochemical features of pancreatic grafts in eight patients with pancreatic transplants were analyzed and compared with pancreases from five patients with chronic pancreatitis and with three pancreatic tissues without histological abnormalities. There was a significant increase in glucagon producing cells in patients with transplanted pancreases compared with those with chronic pancreatitis (P less than 0.05). A significant decline in insulin-producing cells was seen in the transplanted pancreases with rejection in comparison with normal pancreatic tissue. Immunohistochemical staining for HLA-DR (Ia) antigens revealed expression of HLA-DR by endothelial cells, mononuclear cells, and by some ductal epithelial cells, but not by the endocrine islet cells. These results suggest that significant changes in insulin and glucagon production occur in the transplanted pancreas with rejection and that HLA-DR is not expressed by islet cells during graft rejection or with chronic inflammation.     

血管内皮生长因子及相关促肿瘤血管生成分子及靶向治疗

1971年,Folkman首次提出了"肿瘤生长浸润依赖于肿瘤血管生成"的假说.此后,以肿瘤新生血管为作用靶点的抗血管生成治疗成为重要的抗肿瘤策略之一.     

Effect of physical exercise on tumor growth regulating factors of tumor microenvironment: Implications in exercise-dependent tumor growth retardation

Recently, we reported that treadmill exercise renders survival benefits in a murine tumor model of a transplantable lymphoma of spontaneous origin, designated as Dalton’s lymphoma (DL), owing to an augmented apoptosis of tumor cells. However, the underlying the mechanisms of the same remained unclear with respect to the role alterations if any in the components of tumor microenvironment following physical exercise. Therefore, in the present we investigated the role of oxygen, pH, lactate and cytokines of tumor micro-environment associated with physical exercise-dependent alteration in the growth properties of tumor cells to explore their contribution in modulation of tumor. Physical exercise of tumor-bearing host resulted in a decreased angiogenesis in the vicinity of tumor. This was also found to be accompanied by a decrease in erythrocyte count and increase in the level of oxygen while the content of lactate showed a concomitant decrease in the tumor microenvironment along with normalization of pH. Moreover, physical exercise also resulted in an inhibition of VEGF expression which was correlated to an altered expression of cytokines: IL-1, IL-4, IL-10, TGF-β and IFN-γ. Ascitic fluid of tumor-bearing host subjected to physical exercise showed an increase in nitric oxide content along with an increase in the expression of inducible nitric oxide synthase (iNOS). The study discusses the possible role of the aforesaid alterations in constituents of tumor microenvironment of tumor-bearing host following physical exercise in retardation of tumor growth.     

Hypoxia-induced expression of vascular endothelial growth factor by retinal glial cells promotes in vitro angiogenesis

To determine whether retinal glial cells (RGCs) participate in the paracrine regulation of retinal neovascularization, we investigated whether cultured RGCs synthesize and release vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) under normoxic or hypoxic conditions. Northern blot analysis demonstrated that cultured RGCs transcribed both VEGF mRNA with two molecular bands approximately 3.9 and 4.3 kilobases (kb), and bFGF mRNA with approximately 3.7 and 6.0 kb. The expression of VEGF mRNA was greatly enhanced by hypoxic cultivation (2% oxygen) when compared with normoxic cultivation (20% oxygen), while the expression of bFGF mRNA by RGCs was not significantly affected by hypoxia. The effects of RGCs-conditioned media (CM) on tritiated-thymidine incorporation and in vitro angiogenesis by retinal capillary endothelial cells (RECs) in producing the formation of capillary-like tubes in type I collagen gels, were evident in the observation that RGCs-CM harvested after hypoxic cultivation significantly enhanced tritiated-thymidine incorporation (1.9 times, P<0.01) and in vitro angiogenesis (2.4 times, P<0.01) compared with the normoxic RGCs-CM. These enhancing effects of RGCs-CM at hypoxia were suppressed by anti-VEGF neutralizing antibody. Furthermore, RECs were shown to express mRNA encoding the VEGF receptor flt-1 by northern blot analysis. These results suggest that VEGF expressed by RGCs under hypoxic conditions plays an integral role in the initiation and progression of retinal neovascularization in a paracrine manner.     

Hepatocyte growth factor promotes tumor growth in a novel in vivo model of human lung cancer

Significant progress has been made toward identifying growth factors that display autocrine or paracrine effects on the growth of lung cancer cells. Determining the in vivo relevance of specific growth factors on lung tumor formation, however, has not often been demonstrated in laboratory models. Although hepatocyte growth factor (HGF) has been shown to have mitogenic and motogenic effects on human lung cancer cells in vitro, and to have prognostic importance in patients with lung cancer, the effects of HGF on tumor behavior in vivo remain unknown. We therefore developed an airway tumor xenograft model that allowed us to test the hypothesis that HGF promotes human non-small cell lung cancer (NSCLC) growth in vivo. Human airway tumor xenografts were created in Severe Combined Immunodeficient mice by injecting human lung adenocarcinoma cells into human bronchial segments. After determining the optimal times for tumor-cell injection and the time course of tumor growth, we evaluated the effects of HGF on tumor growth by injecting recombinant HGF, or saline as a control, into the lumen of tumor xenografts for 10 consecutive days. Histologic evaluation 2 to 3 wk later revealed that the HGF-injected xenografts had a significantly greater tumor volume and more tumor cells were located in the submucosal space than were found in the saline-injected xenografts. These data demonstrate the usefulness of this novel in vivo model to study NSCLC, and show that HGF promotes both the growth and invasion of human lung cancer in vivo.     

胶质瘤中hTFERT、VEGF表达及其与肿瘤血管形成的关系

目的 探讨胶质瘤中端粒酶逆转录酶(hTERT)、血管内皮生长因子(VEGF)的表达与肿瘤血管形成问的相互关系.方法 分别用原位杂交和免疫组化染色检测106例胶质瘤hTERT和VEGF的表达;用CD34标记瘤组织血管内皮细胞,测定微血管密度(MVD).结果 hTERT、VEGF总阳性表达率分别为53.8%(57/106)、68.0%(72/106);hTERT或VEGF阳性分别定位于肿瘤细胞核内和细胞质内.hTERT阳性或VEGF阳性的瘤组织MVD分别为71.2±18.0和74.4±20.0;而相应的阴性组分别为60.3±21.8和58.4±23.1,两组差异均有显著性(P＜0.01).hTERT、VEGF的表达和MVD均与胶质瘤组织病理分级呈正相关(P＜0.01).结论 hTERT、VEGF的表达及MVD均与胶质瘤的恶性程度有关,前两者阳性表达,其MVD高于两者阴性表达,说明hTERT、VEGF在肿瘤血管形成中可能起促进作用.     

VEGF expression and angiogenesis in oral squamous cell carcinoma: an immunohistochemical and morphometric study

Angiogenesis is involved in tumor progression of oral squamous cell carcinoma (OSCC). In this study, we have investigated by immunohistochemistry vascular endothelial growth factor (VEGF) expression in tumor cells and we have correlated VEGF expression to microvessel area, evaluated by using CD105 as a marker of endothelial cells, in bioptic specimens of 54 human OSCC. Results demonstrated that VEGF is highly expressed in OSCC tumor specimens when compared to pre-neoplastic and normal tissues, without differences between the edge and inside the tumor. Moreover, VEGF expression is reduced in poor differentiated OSCC tumors when compared to moderate and good differentiated forms, and tumor microvessel area is higher in tumors when compared to pre-neoplastic lesions and normal tissues. Finally, VEGF and CD105 may be considered as reliable markers of tumor angiogenesis and progression in OSCC, even if we did not demonstrate any correlation between VEGF expression, tumor microvascular area, clinical stage, and lymph node status.     

Dual role of macrophages in tumor growth and angiogenesis

During the neoplastic progression, macrophages as well as dendritic and NK cells are attracted into the tumor site and initiate the immune response against transformed cells. They activate and present tumor antigens to T cells, which are then activated to kill tumor cells. However, tumor cells are often capable of escaping the immune machinery. As the immune surveillance is not sufficient anymore, tumor-associated macrophages contribute to tumor progression. It is notable that tumor-associated macrophages promote the proliferation of tumor cells directly by secreting growth factors. They also participate in tumor progression by acting on endothelial cells and thus promoting the neovascularization of the tumor. Tumor-associated macrophages are indeed key protagonists during angiogenesis and promote each step of the angiogenesis cascade.     

An intravital microscopic study of the hepatic microcirculation in cirrhotic mice models: relationship between fibrosis and angiogenesis

This intravital fluorescence microscopy (IVFM) study validates cirrhotic mice models and describes the different intrahepatic alterations and the role of angiogenesis in the liver during genesis of cirrhosis. Cirrhosis was induced by subcutaneous injection of carbon tetrachloride (CCl₄) and by common bile duct ligation (CBDL) in mice. Diameters of sinusoids, portal venules (PV), central venules (CV) and shunts were measured at different time points by IVFM. Thereafter, liver samples were taken for sirius red, CD31, Ki67, vascular endothelial growth factor (VEGF) and α‐smooth muscle actin (α‐SMA) evaluation by immunohistochemistry (IHC). In parallel with fibrogenesis, hepatic microcirculation was markedly disturbed in CCl₄ and CBDL mice with a significant decrease in sinusoidal diameter compared to control mice. In CCl₄ mice, CV were enlarged, with marked sinusoidal‐free spaces around CV. In contrast, PV were enlarged in CBDL mice and bile lakes were observed. In both mice models, intrahepatic shunts developed gradually after induction. During genesis of cirrhosis using CD31 IHC we observed a progressive increase in the number of blood vessels within the fibrotic septa area and a progressively increase in staining by Ki67, VEGF and α‐SMA of endothelial cells, hepatocytes and hepatic stellate cells respectively. In vivo study of the hepatic microcirculation demonstrated a totally disturbed intrahepatic architecture, with narrowing of sinusoids in both cirrhotic mice models. The diameters of CV and PV increased and large shunts, bypassing the sinusoids, were seen after both CCl₄ and CBDL induction. Thus present study shows that there is angiogenesis in the liver during cirrhogenesis, and this is probably due partially to an increased production of VEGF.     

Paracrine sonic hedgehog signaling derived from tumor epithelial cells: a key regulator in the pancreatic tumor microenvironment

Activation of the hedgehog (Hh) signaling pathway is involved in embryo development and tumorigenesis. While normal pancreatic tissue exhibits little Hh pathway activity, patients with pancreatic adenocarcinoma have high levels of Hh pathway signaling in both the tumor epithelia and the surrounding stromal tissue. Hh ligands expressed by pancreatic cancers promote tumor growth indirectly by activating Hh signaling in the surrounding stroma. This paracrine activation of Hh signaling in the tumor microenvironment provides a more favorable environment for tumor cellular proliferation, metastasis, and resistance to therapy. Taken together, these findings are of valuable implications for the use of Hh pathway inhibitors currently in development and inhibition of the Hh pathway paracrine loop in pancreatic cancer.