Human serum-derived exosomes modulate macrophage inflammation to promote VCAM1-mediated angiogenesis and bone regeneration

During exogenous bone-graft-mediated bone defect repair, macrophage inflammation dictates angiogenesis and bone regeneration. Exosomes from different human cells have shown macrophage immunomodulation-mediated bone regeneration potential. However, the effect of human serum-derived exosomes (serum-Exo) on macrophage immunomodulation-mediated angiogenesis during bone defect repair has not been investigated yet. In this study, we explored the effects of serum-Exo on macrophage inflammation regulation-mediated angiogenesis during bone defect repair and preliminarily elucidated the mechanism. Healthy serum-Exo was isolated by ultracentrifugation. The effect of serum-Exo on LPS-induced M1 macrophage inflammation was analysed in vitro. The conditioned medium of serum-Exo-treated LPS-induced M1 macrophage (serum-Exo-treated M1 macrophage-CM) was used to culture human umbilical vein endothelial cells (HUVEC), and the effect on angiogenesis was analysed by western blot, qRT-PCR, etc. mRNA-sequencing of HUVECs was performed to identify deferentially expressed genes. Finally, the rat mandibular defect model was established and treated with Bio-Oss and Bio-Oss + Exo. The effect of the Bio-Oss + Exo combination on mandibular bone regeneration was observed by micro-computed tomography (micro-CT), haematoxylin and eosin (HE) staining, Masson staining, and immunohistochemical staining. Serum-Exo promoted the proliferation of RAW264.7 macrophages and reduced the expression of M1-related genes such as IL-6, IL-1β, iNOS, and CD86. Serum-Exo-treated M1 macrophage-CM induced the proliferation, migration, and angiogenic differentiation of HUVEC, as well as the expression of H-type blood vessel markers CD31 and endomucin (EMCN), compared with M1 macrophage-CM. Moreover, higher expression of vascular endothelial adhesion factor 1 (VCAM1) in HUVEC cultured with serum-Exo-treated M1 macrophage-CM compared with M1 macrophages-CM. Inhibition of VCAM1 signalling abrogated the pro-angiogenic effect of serum-Exo-treated M1 macrophage-CM on HUVEC. Local administration of serum-Exo during mandibular bone defect repair reduced the number of M1 macrophages and promoted angiogenesis and osteogenesis. Collectively, our results demonstrate the macrophage inflammation regulation-mediated pro-angiogenic potential of serum-Exo during bone defect repair possibly via upregulation of VCAM1 signalling in HUVEC.     

Isolated tumor endothelial cells maintain specific character during long-term culture

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1-2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells.In this study, we have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research.     

Nucleosomes bind fibroblast growth factor-2 for increased angiogenesis in vitro and in vivo

Solid tumors often display sites of necrosis near regions of angiogenesis in vivo. As tumor cell necrosis would result in the release of nucleosomes into the extracellular environment, we explored the potential role of nucleosomes in the promotion of angiogenesis. Data indicate that nucleosomes acted similar to heparin and bound to several heparin-binding, proangiogenic factors [i.e., fibroblast growth factor (FGF)-1, FGF-2, vascular endothelial growth factor, and transforming growth factor-beta1]. Nucleosomes modestly enhanced FGF-2 growth of human umbilical vein endothelial cells when grown in restricted media as well as increased human umbilical vein endothelial cell migration and primitive blood vessel tube formation in vitro. On s.c. injection in mice, nucleosomes aided FGF-2 in promoting angiogenesis. These results suggest that nucleosomes released from dying tumor cells aid in the formation of blood vessels and may provide a novel means by which tumor cells increase angiogenesis.     

Doxazosin inhibits human vascular endothelial cell adhesion, migration, and invasion

The quinazoline-derived alpha1-adrenoceptor antagonists, doxazosin and terazosin have been recently shown to induce an anoikis effect in human prostate cancer cells and to suppress prostate tumor vascularity in clinical specimens [Keledjian and Kyprianou, 2003]. This study sought to examine the ability of doxazosin to affect the growth of human vascular endothelial cells and to modulate vascular endothelial growth factor (VEGF)-mediated angiogenesis. Human umbilical vein endothelial cells (HUVECs) were used as an in vitro model to determine the effect of doxazosin on cell growth, apoptosis, adhesion, migration, and angiogenic response of endothelial cells. The effect of doxazosin on cell viability and apoptosis induction of human endothelial cells, was evaluated on the basis of trypan blue and Hoechst 33342 staining, respectively. Doxazosin antagonized the VEGF-mediated angiogenic response of HUVEC cells, by abrogating cell adhesion to fibronectin and collagen-coated surfaces and inhibiting cell migration, via a potential downregulation of VEGF expression. Furthermore there was a significant suppression of in vitro angiogenesis by doxazosin on the basis of VEGF-mediated endothelial tube formation (P < 0.01). Fibroblast growth factor-2 (FGF-2) significantly enhanced HUVEC cell tube formation (P < 0.01) and this effect was suppressed by doxazosin. These findings provide new insight into the ability of doxazosin to suppress the growth and angiogenic response of human endothelial cells by interfering with VEGF and FGF-2 action. This evidence may have potential therapeutic significance in using this quinazoline-based compound as an antiangiogenic agent for the treatment of advanced prostate cancer.     

CEA-related cell adhesion molecule 1: a potent angiogenic factor and a major effector of vascular endothelial growth factor

CEA-related cell adhesion molecule 1 (CEACAM1) exhibits angiogenic properties in in vitro and in vivo angiogenesis assays. CEACAM1 purified from granulocytes and endothelial cell media as well as recombinant CEACAM1 expressed in HEK293 cells stimulate proliferation, chemotaxis, and capillary-like tube formation of human microvascular endothelial cells. They increase vascularization of chick chorioallantoic membrane and potentiate the effects of vascular endothelial growth factor (VEGF)165. VEGF165 increases CEACAM1 expression both on the mRNA and the protein level. VEGF165-induced endothelial tube formation is blocked by a monoclonal CEACAM1 antibody. These data suggest that CEACAM1 is a major effector of VEGF in the early microvessel formation. Since CEACAM1 is expressed in tumor microvessels but not in large blood vessels, CEACAM1 may be a target for the inhibition of tumor angiogenesis.     

Changes in tumorigenesis- and angiogenesis-related gene transcript abundance profiles in ovarian cancer detected by tailored high density cDNA arrays

BACKGROUND: Complementary DNA array analysis of gene expression has a potential application for clinical diagnosis of disease processes. However, accessibility, affordability, reproducibility of results, and management of the data generated remain issues of concern. Use of cDNA arrays tailored for studies of specific pathways, tissues, or disease states may render a cost- and time-effective method to define potential hallmark genotype alterations. MATERIALS AND METHODS: We produced a 332-membered human cDNA array on nylon membranes tailored for studies of angiogenesis and tumorigenesis in reproductive disease. We tested the system for reproducibility using a novel statistical approach for analysis of array data and employed the arrays to investigate gene expression alterations in ovarian cancer. RESULTS: Intra-assay analysis and removal of agreement outliers was shown to be a critical step prior to interpretation of cDNA array data. The system revealed highly reproducible results, with intermembrane coefficient of reproducibility of +/- 0.98. Comparison of placental and ovarian sample data confirmed expected differences in angiogenic profiles and tissue-specific markers, such as human placental lactogen (hPL). Analysis of expression profiles of five normal ovary and four poorly differentiated serous papillary ovarian adenocarcinoma samples revealed an overall increase in angiogenesis-related markers, including vascular endothelial growth factor (VEGF) and angiopoietin-1 in the diseased tissue. These were accompanied by increases in immune response mediators (e.g. HLA-DR, Ron), apoptotic and neoplastic markers (e.g. BAD protein, b-myb), and novel potential markers of ovarian cancer, such as cofilin, moesin, and neuron-restrictive silencer factor (REST) protein. CONCLUSIONS: In-house production of tailored cDNA arrays, coupled to comprehensive analysis of resulting hybridization profiles, provides an accessible, reliable, and highly effective method of applying array technology to study disease processes. In the ovary, abundance of specific tumor markers, increased macrophage recruitment mediators, a late-stage angiogenesis profile, and the presence of chemoresistance-related markers distinguished normal and advanced ovarian cancer tissue samples. Detection of such parallel changes in pathway- and tissue-specific markers may prove a hallmark ready for application in reproductive disease diagnostic and therapeutic developments.     

抑制消减杂交分离肿瘤血管生成相关基因

为获得肿瘤血管生成相关基因 ,以便为抗血管形成治疗肿瘤的新策略提供有价值的靶位 ,采用人新鲜的肝癌、肺癌组织匀浆活化人脐静脉内皮细胞 (HUVEC) ,并构建了cDNA表达文库 .利用抑制消减杂交 (SSH)获得活化HUVEC高表达的基因片段 ,放射标记后筛选文库 .获得的阳性克隆进一步进行差异杂交筛选 ,去除假阳性 .共获得了 177个阳性克隆 ,对其中 74个克隆进行序列分析 ,发现它们代表 32个基因 ,其中多个与肿瘤血管生成相关 ,1个为与细胞色素c氧化酶亚单位Ⅲ同源的新基因 ,2个为功能未知的假定蛋白基因 .研究结果表明 ,用肿瘤组织匀浆模拟肿瘤内环境活化HUVEC ,并通过比较活化前后基因表达谱的差异可以分离到肿瘤血管生成相关的基因 .     

The ratio of VEGF/PEDF expression in bone marrow mesenchymal stem cells regulates neovascularization

Angiogenesis, or neovascularization, is a finely balanced process controlled by pro- and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) is a major pro-angiogenic factor, whereas pigment epithelial-derived factor (PEDF) is the most potent natural angiogenesis inhibitor. In this study, the regulatory role of bone marrow stromal cells (BMSCs) during angiogenesis was assessed by the endothelial differentiation potential, VEGF/PEDF production and responses to pro-angiogenic and hypoxic conditions. The in vivo regulation of blood vessel formation by BMSCs was also explored in a SCID mouse model. Results showed that PEDF was expressed more prominently in BMSCs compared to VEGF. This contrasted with human umbilical vein endothelial cells (HUVECs) where the expression of VEGF was higher than that of PEDF. The ratio of VEGF/PEDF gene expression in BMSCs increased when VEGF concentration reached 40ng/ml in the culture medium, but decreased at 80ng/ml. Under CoCl(2)-induced hypoxic conditions, the VEGF/PEDF ratio of BMSCs increased significantly in both normal and angiogenic culture media. There was no expression of endothelial cell markers in BMSCs cultured in either pro-angiogenic or hypoxia culture conditions when compared with HUVECs. The in vivo study showed that VEGF/PEDF expression closely correlated with the degree of neovascularization, and that hypoxia significantly induced pro-angiogenic activity in BMSCs. These results indicate that, rather than being progenitors of endothelial cells, BMSCs play an important role in regulating the neovascularization process, and that the ratio of VEGF and PEDF may, in effect, be an indicator of the pro- or anti-angiogenic activities of BMSCs.     

Green tea extract inhibits angiogenesis of human umbilical vein endothelial cells through reduction of expression of VEGF receptors

Epidemiological and animal studies have indicated that consumption of green tea is associated with a reduced risk of developing certain forms of cancer. However, the inhibitory mechanism of green tea in angiogenesis, an important process in tumor growth, has not been well established. In the present study, green tea extract (GTE) was tested for its ability to inhibit cell viability, cell proliferation, cell cycle dynamics, vascular endothelial growth factor (VEGF) and expression of VEGF receptors fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/Kinase insert domain containing receptor (Flk-1/KDR) in vitro using human umbilical vein endothelial cells (HUVECs). GTE in culture media did not affect cell viability but significantly reduced cell proliferation dose-dependently and caused a dose-dependent accumulation of cells in the G1 phase. The decrease of the expression of Flt-1 and KDR/Flk-1 in HUVEC by GTE was detected with immunohistochemical and Western blotting methods. These results suggest that GTE may have preventive effects on tumor angiogenesis and metastasis through reduction of expression of VEGF receptors.     

Constitutive and inducible expression and regulation of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF), which was originally discovered as vascular permeability factor, is critical to human cancer angiogenesis through its potent functions as a stimulator of endothelial cell survival, mitogenesis, migration, differentiation and self-assembly, as well as vascular permeability, immunosuppression and mobilization of endothelial progenitor cells from the bone marrow into the peripheral circulation. Genetic alterations and a chaotic tumor microenvironment, such as hypoxia, acidosis, free radicals, and cytokines, are clearly attributed to numerous abnormalities in the expression and signaling of VEGF and its receptors. These perturbations confer a tremendous survival and growth advantage to vascular endothelial cells as manifested by exuberant tumor angiogenesis and a consequent malignant phenotype. Understanding the regulatory mechanisms of both inducible and constitutive VEGF expression will be crucial in designing effective therapeutic strategies targeting VEGF to control tumor growth and metastasis. In this review, molecular regulation of VEGF expression in tumor cells is discussed.     

Hypoxia stimulates pancreatic stellate cells to induce fibrosis and angiogenesis in pancreatic cancer

Pancreatic cancer is characterized by excessive desmoplastic reaction and by a hypoxic microenvironment within the solid tumor mass. Chronic pancreatitis is also characterized by fibrosis and hypoxia. Fibroblasts in the area of fibrosis in these pathological settings are now recognized as activated pancreatic stellate cells (PSCs). Recent studies have suggested that a hypoxic environment concomitantly exists not only in pancreatic cancer cells but also in surrounding PSCs. This study aimed to clarify whether hypoxia affected the cell functions in PSCs. Human PSCs were isolated and cultured under normoxia (21% O(2)) or hypoxia (1% O(2)). We examined the effects of hypoxia and conditioned media of hypoxia-treated PSCs on cell functions in PSCs and in human umbilical vein endothelial cells. Hypoxia induced migration, type I collagen expression, and vascular endothelial growth factor (VEGF) production in PSCs. Conditioned media of hypoxia-treated PSCs induced migration of PSCs, which was inhibited by anti-VEGF antibody but not by antibody against hepatocyte growth factor. Conditioned media of hypoxia-treated PSCs induced endothelial cell proliferation, migration, and angiogenesis in vitro and in vivo. PSCs expressed several angiogenesis-regulating molecules including VEGF receptors, angiopoietin-1, and Tie-2. In conclusion, hypoxia induced profibrogenic and proangiogenic responses in PSCs. In addition to their established profibrogenic roles, PSCs might play proangiogenic roles during the development of pancreatic fibrosis, where they are subjected to hypoxia.     

Paracrine and autocrine mechanisms of apelin signaling govern embryonic and tumor angiogenesis

Apelin and its G protein-coupled receptor APJ play important roles in blood pressure regulation, body fluid homeostasis, and possibly the modulation of immune responses. Here, we report that apelin-APJ signaling is essential for embryonic angiogenesis and upregulated during tumor angiogenesis. A detailed expression analysis demonstrates that both paracrine and autocrine mechanisms mark areas of embryonic and tumor angiogenesis. Knockdown studies in Xenopus reveal that apelin-APJ signaling is required for intersomitic vessel angiogenesis. Moreover, ectopic expression of apelin but not vascular endothelial growth factor A (VEGFA) is sufficient to trigger premature angiogenesis. In vitro, apelin is non-mitogenic for primary human endothelial cells but promotes chemotaxis. Epistasis studies in Xenopus embryos suggest that apelin-APJ signaling functions downstream of VEGFA. Finally, we show that apelin and APJ expression is highly upregulated in microvascular proliferations of brain tumors such as malignant gliomas. Thus, our results define apelin and APJ as genes of potential diagnostic value and promising targets for the development of a new generation of anti-tumor angiogenic drugs.     

Acquired expression of periostin by human breast cancers promotes tumor angiogenesis through up-regulation of vascular endothelial growth factor receptor 2 expression

The late stages of human breast cancer development are poorly understood complex processes associated with the expression of genes by cancers that promote specific tumorigenic activities, such as angiogenesis. Here, we describe the identification of periostin as a mesenchyme-specific gene whose acquired expression by human breast cancers leads to a significant enhancement in tumor progression and angiogenesis. Undetectable in normal human breast tissues, periostin was found to be overexpressed by the vast majority of human primary breast cancers examined. Tumor cell lines engineered to overexpress periostin showed a phenotype of accelerated growth and angiogenesis as xenografts in immunocompromised animals. The underlying mechanism of periostin-mediated induction of angiogenesis was found to derive in part from the up-regulation of the vascular endothelial growth factor receptor Flk-1/KDR by endothelial cells through an integrin alpha(v)beta(3)-focal adhesion kinase-mediated signaling pathway. These findings demonstrate the presence of a novel mechanism by which tumor angiogenesis is acquired with the expression of a mesenchyme-specific gene as a crucial step in late stages of tumorigenesis.     

Glioma cells under hypoxic conditions block the brain microvascular endothelial cell death induced by serum starvation

Angiogenesis is one of essential components for the growth of neoplasms, including malignant gliomas. However, tumor vascularization is often poorly organized and marginally functional due to tumor structural abnormalities, inducing regional or temporal hypoxic conditions and nutritional shortages in tumor tissues. We investigated how during angiogenesis migrating endothelial cells survive in these hypoxic and reduced nutritional conditions. Human brain microvascular endothelial cells (HBMECs) underwent apoptosis and necrosis after serum withdrawal. This endothelial cell death was blocked by recombinant VEGF protein or the culture medium of U251 glioma cells exposed to hypoxia (H-CM). Hypoxic treatment increased vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNF-alpha) expression in U251 glioma cells. H-CM activated nuclear factor-kappaB (NFkappaB) protein and increased the gene expression of antiapoptotic factors including Bcl-2, Bcl-X(L), survivin and X-chromosome-linked inhibitor of apoptosis protein (XIAP) in endothelial cells. The survival activity of H-CM for endothelial cells was abolished by two kinds of VEGF inhibitors {Cyclopeptidic VEGF inhibitor and a VEGF receptor tyrosine kinase inhibitor (4-[(4'-chloro-2'-fluoro) phenylamino]-6, 7-dimethoxyquinazoline)} or NFkappaB inhibitors (ALLN and BAY 11-7082). These VEGF inhibitors did not block the activation of NFkappaB induced by H-CM in endothelial cells. On the contrary, TNF-alpha antagonist WP9QY enhanced the survival activity of H-CM for endothelial cells and blocked NFkappaB activation induced by H-CM under serum-starved conditions. Taken together, our data suggest that both the secretion of VEGF from glioma cells and activation of NFkappaB in endothelial cells induced by TNF-alpha are necessary for endothelial cell survival as they increase the expression of antiapoptotic genes in endothelial cells under conditions of serum starvation. These pathways may be one of the mechanisms by which angiogenesis is maintained in glioma tissues.