Inhibition of angiogenic factor- and tumour-induced angiogenesis by gamma linolenic acid.

Angiogenesis, the formation of new blood vessels, is an essential feature of malignant tumour development. Gamma linolenic acid (GLA), a n-6 polyunsaturated fatty acid (PUFA), inhibits the growth and metastasis of a variety of tumour cells, including breast, prostate, pancreatic cancer and hepatoma cells and also has anti-metastatic effects on endothelial cells. In the current study, we tested whether GLA inhibited angiogenesis induced by tumour cells. A rat aortic ring assay and in vitro tube formation of human vascular endothelial cells were used to determine angiogenesis (spontaneous, angiogenic factor- and tumour cells-induced). Inclusion of GLA in this 3-D matrix culture system significantly inhibited angiogenesis from aortic rings in a concentration-dependent manner. The results from tube formation of vascular endothelial cell further confirmed that GLA suppressed angiogenesis. Furthermore, in the cell motility assay (phagokinetic assay and endothelial wounding assay), a significant reduction of the motility of vascular endothelial cells by GLA was seen. It is concluded that gamma linolenic acid inhibits angiogenic factor and tumour-induced angiogenesis in vitro at least in part via its inhibitory effect on the motility of vascular endothelial cells.     

Alkaline phosphatase dual‐binding sites for collagen dictate cell migration and microvessel assembly in vitro

Interactions between cell types, growth factors, and extracellular matrix components involved in angiogenesis are crucial for new vessel formation leading to tissue regeneration. This study investigated whether cocultures of fibroblasts and endothelial cells (ECs; from macro‐ or microvasculature) play a role in the formation of microvessel‐like structures by ECs, as well as modulate fibroblast differentiation and growth factors production (vascular endothelial cell growth factor, basic fibroblast growth factor, active transforming growth factor‐β1, and interleukin‐8), which are important for vessel sprouting and maturation. Data obtained revealed that in vitro coculture systems of fibroblasts and human ECs stimulate collagen synthesis and growth factors production by fibroblasts that ultimately affect the formation and distribution of microvessel‐like structures in cell cultures. In this study, areas with activated fibroblasts and high alkaline phosphatase (ALP) activity were also observed in cocultures. Molecular docking assays revealed that ALP has two binding positions for collagen, suggesting its impact in collagen proteins’ aggregation, cell migration, and microvessel assembly. These findings indicate that bioinformatics and coculture systems are complementary tools for investigating the participation of proteins, like collagen and ALP in angiogenesis.     

Nuclear exosome HMGB3 secreted by nasopharyngeal carcinoma cells promotes tumour metastasis by inducing angiogenesis

Distant metastasis accompanied by angiogenesis is the main cause of nasopharyngeal carcinoma (NPC)-related death. Nuclear exosomes (nEXOs) are potential tumour biomarkers. High mobility group box 3 (HMGB3), a nuclear protein, is known to be overexpressed in cancers. However, its role in NPC has not been elucidated. Here, we explore for the first time the function of nEXO HMGB3 in tumour angiogenesis involved in NPC metastasis using a series of in vitro experiments with NPC cell lines and clinical specimens and in vivo experiments with tumour xenograft zebrafish angiogenesis model. We found a high expression of HMGB3 in NPC, accompanied by the formation of micronuclei, to be associated with metastasis. Furthermore, the NPC-secreted HMGB3 expression was associated with tumour angiogenesis. Moreover, HMGB3-containing nEXOs, derived from the micronuclei of NPC cells, were ingested by the human umbilical vein endothelial cells (HUVECs), and accelerated angiogenesis in vitro and in vivo. Importantly, western blotting and flow cytometry analysis showed that circulating nEXO HMGB3 positively correlated with NPC metastasis. In summary, nEXO HMGB3 can be a significant biomarker of NPC metastasis and provide a novel basis for anti-angiogenesis therapy in clinical metastasis.Subject terms: Metastasis, Tumour angiogenesis     

Contact-dependent inhibition of angiogenesis by cardiac fibroblasts in three-dimensional fibrin gels in vitro: implications for microvascular network remodeling and coronary collateral formation

Angiogenesis and coronary artery collateral formation can improve blood flow and thereby prevent myocardial ischemia. The role of perivascular fibroblasts in neovascularization remains incompletely understood. Here we investigated the effects of epicardial and myocardial fibroblasts on angiogenesis in vitro by using a serum-free microcarrier-based fibrin gel angiogenesis system. To clearly distinguish between different cell types, we either stained endothelial cells or fibroblasts in the living with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine-perchlorate (DiI). In cocultures, low numbers of heart fibroblasts stimulated endothelial sprouting, and capillary growth was also induced by fibroblast-conditioned media, indicating a paracrine mechanism. Capillary formation was decreased by increasing the density of fibroblasts in the cocultures, indicating contact-dependent inhibition. Using time-lapse studies, it turned out that close contacts between fibroblasts and endothelial cells resulted in rapid retraction of endothelial cells or, rarely, in cell death. Depending on the local ratio of fibroblasts to endothelial cell numbers, fibroblasts determined the location of capillary growth and the size of developing capillaries and thereby contributed to capillary network remodeling. In contrast to primary heart fibroblasts, NIH 3T3 fibroblasts did not display contact-dependent inhibition of endothelial sprouts. NIH fibroblasts were frequently seen in close association with endothelial capillaries, resembling pericytes. Contact-dependent inhibition of angiogenesis by epicardial fibroblasts could not be reversed by addition of neutralizing anti-TGF-β1 antibodies, by addition of serum, of medium conditioned by hypoxic tumor cells or myocardium, by various cytokines or by growing cocultures under hypoxic conditions. Our results implicate a pivotal role of periendothelial mesenchymal cells for the regulation of microvascular network remodeling and collateral formation. Received: 15 September 1997 / Accepted: 6 April 1998     

Thymidine phosphorylase promotes angiogenesis and tumour growth in intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer, and thymidine phosphorylase (TP) is a regulator of angiogenesis. To investigate the biological activities of TP in ICC, we established human cholangiocarcinoma RBE cell lines overexpressing TP or silencing TP. Overexpression of TP enhanced viability, suppressed apoptosis and increased tube formation in human umbilical vein endothelial cells, while downregulation of TP reversed these effects. Moreover, an orthotopic xenograft mouse model of ICC was built to further explore TP's function in ICC in vivo. Histological analysis using H&E, TUNEL and Ki67 staining showed that TP promoted tumour growth and inhibited cell apoptosis. Immunostaining for CD31 revealed an elevation in microvessel density in the presence of TP. Besides, upregulation of TP increased the expression of vascular endothelial growth factor, basic fibroblast growth factor, interleukin-8 and tumour necrosis factor alpha. In contrast, TP knockdown inhibited tumour growth, suppressed microvessel formation and decreased the expression of angiogenesis-related proteins. Therefore, we suggest that TP promotes angiogenesis and tumour growth in ICC, which can be a potent therapeutic target for ICC treatment.     

Phenotypic modulations of human umbilical vein endothelial cells and human dermal fibroblasts using two angiogenic assays.

Different angiogenic assays in vitro have helped to define various events underlying angiogenesis. In this report we have compared the phenotypic modifications of human umbilical vein endothelial cells (HUVE cells) and human dermal fibroblasts using Matrigel and collagen gels. Both HUVE cells and human dermal fibroblasts form a network of anastomosing cords that apparently resemble blood capillaries when grown on Matrigel. The whole network was formed by several cellular aggregates joined to each other by cellular cords. Lumen formation was not observed in this angiogenic system. In opposite, considerable differences between HUVE cells and human dermal fibroblasts were observed in the three-dimensional angiogenic assay on collagen gels described by Montesano et al [14]. These results indicate that data obtained with angiogenic systems using Matrigel must be interpreted with caution and that the assay described by Montesano et al [14], is more reliable to describe angiogenesis.     

Maspin is an angiogenesis inhibitor

Maspin, a unique member of the serpin family, is a secreted protein encoded by a class II tumor suppressor gene whose downregulation is associated with the development of breast and prostate cancers. Overexpression of maspin in breast tumor cells limits their growth and metastases in vivo. In this report we demonstrate that maspin is an effective inhibitor of angiogenesis. In vitro, it acted directly on cultured endothelial cells to stop their migration towards basic fibroblast growth factor and vascular endothelial growth factor and to limit mitogenesis and tube formation. In vivo, it blocked neovascularization in the rat cornea pocket model. Maspin derivatives mutated in the serpin reactive site lost their ability to inhibit the migration of fibroblasts, keratinocytes, and breast cancer cells but were still able to block angiogenesis in vitro and in vivo. When maspin was delivered locally to human prostate tumor cells in a xenograft mouse model, it blocked tumor growth and dramatically reduced the density of tumor-associated microvessels. These data suggest that the tumor suppressor activity of maspin may depend in large part on its ability to inhibit angiogenesis and raise the possibility that maspin and similar serpins may be excellent leads for the development of drugs that modulate angiogenesis.     

Cyclooxygenase Regulates Angiogenesis Induced by Colon Cancer Cells

To explore the role of cyclooxygenase (COX) in endothelial cell migration and angiogenesis, we have used two in vitro model systems involving coculture of endothelial cells with colon carcinoma cells. COX-2-overexpressing cells produce prostaglandins, proangiogenic factors, and stimulate both endothelial migration and tube formation, while control cells have little activity. The effect is inhibited by antibodies to combinations of angiogenic factors, by NS-398 (a selective COX-2 inhibitor), and by aspirin. NS-398 does not inhibit production of angiogenic factors or angiogenesis induced by COX-2-negative cells. Treatment of endothelial cells with aspirin or a COX-1 antisense oligonucleotide inhibits COX-1 activity/expression and suppresses tube formation. Cyclooxygenase regulates colon carcinoma-induced angiogenesis by two mechanisms: COX-2 can modulate production of angiogenic factors by colon cancer cells, while COX-1 regulates angiogenesis in endothelial cells.     

Antiangiogenic potential of 10-hydroxycamptothecin

To investigate the antiangiogenic potential of 10-hydroxycamptothecin (HCPT), the proliferation of human microvascular endothelial cells (HMEC) and seven human tumor cell lines were detected by SRB assay, and the endothelial cell migration and tube formation were assessed using two in vitro model systems. Also, inhibition of angiogenesis was determined with a modification of the chick embryo chorioallantoic membrane (CAM) assay in vivo. Morphological assessment of apoptosis was performed by fluorescence microscope. HCPT 0.313-5 micromol x L(-1) treatment resulted in a dose-dependent inhibition of proliferation, migration and tube formation in HMEC cells, and HCPT 6.25-25 nmol x egg(-1) inhibited angiogenesis in CAM assay. HCPT 1.25-5 micromol x L(-1) elicited typical morphological changes of apoptosis including condensed chromatin, nuclear fragmentation, and reduction in volume in HMEC cells. HCPT significantly inhibited angiogenesis both in vitro and in vivo at relatively low concentrations, and this effect was related with induction of apoptosis in HMEC cells. These results taken collectively suggest that HCPT may be a potent antiangiogenetic and cytotoxic drug and further investigation is warranted.     

Spontaneous fibroblast‐derived pericyte recruitment in a human tissue‐engineered angiogenesis model in vitro

Cooperation between endothelial cells and pericytes is essential to the stabilization and maturation of blood microvessels. We developed a unique in vitro tissue‐engineered model to study angiogenesis. The human endothelialized reconstructed connective tissue model promotes the formation of a three‐dimensional branching network of capillary‐like tubes (CLT) with closed lumens. The purpose of this work was to investigate whether pericytes were spontaneously recruited around CLT in the model. We demonstrated that smooth muscle α‐actin (SMA)‐positive cells were found closely associated with PECAM‐1‐positive capillaries in the model. Twelve percent (±2.6) of SMA‐positive cells were detected along with 15% (±1.64) von Willebrand factor‐positive endothelial cells in the culture system after 31 days of in vitro maturation. Conversely, no SMA‐positive cells were detected in reconstructed connective tissues made solely of fibroblasts. Knowing that PDGF is a major factor in the recruitment of pericytes, we showed that blockade of the PDGFB receptor using the inhibitor AG1296 induced an overall 5, 2.6, and 2.4‐fold decrease in the SMA‐positive cells, von Willebrand factor‐positive cells, and number of capillaries, respectively. Using combinations of human GFP‐positive fibroblasts and endothelial cells, we demonstrated that pericytes were recruited from the fibroblast population in the model. In conclusion, our tissue‐engineered culture system promotes the spontaneous formation of a network of capillaries and the recruitment of pericytes derived from fibroblasts. Since pericytes are essential components of the blood microvasculature, this culture system is a powerful model to study angiogenesis and endothelial cell/pericyte interactions in vitro. J. Cell. Physiol. 227: 2130–2137, 2012. © 2011 Wiley Periodicals, Inc.     

Collagen vitrigel membrane useful for paracrine assays in vitro and drug delivery systems in vivo

We previously succeeded in converting a soft and turbid disk of type-I collagen gel into a strong and transparent vitrigel membrane utilizing a concept for the vitrification of heat-denatured proteins and have demonstrated its protein-permeability and advantage as a scaffold for reconstructing crosstalk models between two different cell types. In this study, we observed the nano-structure of the type-I collagen vitrigel membrane and verified its utility for paracrine assays in vitro and drug delivery systems in vivo. Scanning electron microscopic observation revealed that the vitrigel membrane was a dense network architecture of typical type-I collagen fibrils. In the crosstalk model between PC-12 pheochromocytoma cells and L929 fibroblasts, nerve growth factor (NGF) secreted from L929 cells passed through the collagen vitrigel membrane and induced the neurite outgrowth of PC-12 cells by its paracrine effect. Also, the collagen vitrigel membrane containing vascular endothelial growth factor (VEGF) showed sustained-release of VEGF in vitro and its subcutaneous transplantation into a rat resulted in remarkable angiogenesis. These data suggest that the collagen vitrigel membrane is useful for paracrine assays in vitro and drug delivery systems in vivo.     

Fibroblast Growth Factor-2 (FGF-2) Induces Vascular Endothelial Growth Factor (VEGF) Expression in the Endothelial Cells of Forming Capillaries: An Autocrine Mechanism Contributing to Angiogenesis

FGF-2 and VEGF are potent angiogenesis inducers in vivo and in vitro. Here we show that FGF-2 induces VEGF expression in vascular endothelial cells through autocrine and paracrine mechanisms. Addition of recombinant FGF-2 to cultured endothelial cells or upregulation of endogenous FGF-2 results in increased VEGF expression. Neutralizing monoclonal antibody to VEGF inhibits FGF-2–induced endothelial cell proliferation. Endogenous 18-kD FGF-2 production upregulates VEGF expression through extracellular interaction with cell membrane receptors; high-M_r FGF-2 (22–24-kD) acts via intracellular mechanism(s). During angiogenesis induced by FGF-2 in the mouse cornea, the endothelial cells of forming capillaries express VEGF mRNA and protein. Systemic administration of neutralizing VEGF antibody dramatically reduces FGF-2-induced angiogenesis. Because occasional fibroblasts or other cell types present in the corneal stroma show no significant expression of VEGF mRNA, these findings demonstrate that endothelial cell-derived VEGF is an important autocrine mediator of FGF-2-induced angiogenesis. Thus, angiogenesis in vivo can be modulated by a novel mechanism that involves the autocrine action of vascular endothelial cell-derived FGF-2 and VEGF.     

Vasculogenic and angiogenic potential of adipose stromal vascular fraction cell populations in vitro

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous cell source that contains endothelial cells, pericytes, smooth muscle cells, stem cells, and other accessory immune and stromal cells. The SVF cell population has been shown to support vasculogenesis in vitro as well vascular maturation in vivo. Matrigel, an extracellular matrix (ECM) mixture has been utilized in vitro to evaluate tube formation of purified endothelial cell systems. We have developed an in vitro system that utilizes freshly isolated SVF and ECM molecules both in pure form (fibrin, laminin, collagen) as well as premixed form (Matrigel) to evaluate endothelial tip cell formation, endothelial stalk elongation, and early stages of branching and inosculation. Freshly isolated SVF rat demonstrate cell aggregation and clustering (presumptive vasculogenesis) on Matrigel ECM within the first 36 h of seeding followed by tip cell formation, stalk cell formation, branching, and inosculation (presumptive angiogenesis) during the subsequent 4 days of culture. Purified ECM molecules (laminin, fibrin, and collagen) promote cell proliferation but do not recapitulate events seen on Matrigel. We have created an in vitro system that provides a functional assay to study the mechanisms of vasculogenesis and angiogenesis in freshly isolated SVF to characterize SVF’s blood vessel forming potential prior to clinical implantation.     

Inhibition of prostate tumor angiogenesis by the tumor suppressor CEACAM1

We have previously shown that CEACAM1, a cell-adhesion molecule, acts as a tumor suppressor in prostate carcinoma. Expression of CEACAM1 in prostate cancer cells suppresses their growth in vivo. However, CEACAM1 has no effect on the growth of prostate cancer cells in vitro. This difference suggests that the antitumor effect of CEACAM1 may be due to inhibition of tumor angiogenesis, perhaps by increased secretion of antiangiogenic molecules from the cells. In this study, we have demonstrated that expression of CEACAM1 in DU145 prostate cancer cells induced the production of a factor or factors that specifically blocked the growth of endothelial but not epithelial cells. Conditioned medium from the CEACAM1-expressing cells but not control luciferase-expressing cells inhibited endothelial cell migration up a gradient of stimulatory vascular endothelial growth factor in vitro and inhibited corneal neovascularization induced by basic fibroblast growth factor in vivo. Moreover, conditioned medium from CEACAM1-expressing cells induced endothelial cell apoptosis in vitro. Only medium conditioned by CEACAM1 mutants that were able to suppress tumor growth in vivo could cause endothelial cell apoptosis. These observations suggest that CEACAM1-mediated tumor suppression in vivo is, at least in part, due to the ability of CEACAM1 to inhibit tumor angiogenesis.     

Mathematical Modeling of Cellular Cross-Talk Between Endothelial and Tumor Cells Highlights Counterintuitive Effects of VEGF-Targeted Therapies

Tumor growth and progression are critically dependent on the establishment of a vascular support system. This is often accomplished via the expression of pro-angiogenic growth factors, including members of the vascular endothelial growth factor (VEGF) family of ligands. VEGF ligands are overexpressed in a wide variety of solid tumors and therefore have inspired optimism that inhibition of the different axes of the VEGF pathway—alone or in combination—would represent powerful anti-angiogenic therapies for most cancer types. When considering treatments that target VEGF and its receptors, it is difficult to tease out the differential anti-angiogenic and anti-tumor effects of all combinations experimentally because tumor cells and vascular endothelial cells are engaged in a dynamic cross-talk that impacts key aspects of tumorigenesis, independent of angiogenesis. Here we develop a mathematical model that connects intracellular signaling responsible for both endothelial and tumor cell proliferation and death to population-level cancer growth and angiogenesis. We use this model to investigate the effect of bidirectional communication between endothelial cells and tumor cells on treatments targeting VEGF and its receptors both in vitro and in vivo. Our results underscore the fact that in vitro therapeutic outcomes do not always translate to the in vivo situation. For example, our model predicts that certain therapeutic combinations result in antagonism in vivo that is not observed in vitro. Mathematical modeling in this direction can shed light on the mechanisms behind experimental observations that manipulating VEGF and its receptors is successful in some cases but disappointing in others.     

Angiogenesis inhibitors identified by cell-based high-throughput screening: synthesis, structure-activity relationships and biological evaluation of 3-[(E)-styryl]benzamides that specifically inhibit endothelial cell proliferation

Proliferation of endothelial cells is critical for angiogenesis. We report orally available, in vivo active antiangiogenic agents which specifically inhibit endothelial cell proliferation. After identifying human umbilical vein endothelial cell (HUVEC) proliferation inhibitors from a cell-based high-throughput screening (HTS), we eliminated those compounds which showed cytotoxicity against HCT116 and vascular endothelial growth factor receptor 2 (VEGFR-2) inhibitory activity. Evaluations in human Calu-6 xenograft model delivered lead compound 1. Following extensive lead optimization and alteration of the scaffold we discovered 32f and 32g, which both inhibited the proliferation and tube formation of HUVEC without showing inhibitory activity against any of 25 kinases or cytotoxicity against either normal fibroblasts or 40 cancer cell lines. Upon oral administration, 32f and 32g had good pharmacokinetic profiles and potent antitumor activity and decreased microvessel density (MVD) in Calu-6 xenograft model. Combination therapy with a VEGFR inhibitor enhanced the in vivo efficacy. These results suggest that 32f and 32g may have potential for use in cancer treatment.     

A novel approach for studying angiogenesis: A human skin equivalent with a capillary-like network

Angiogenesis results from an ordered set of events that can be modulated in vivo by a variety of angiogenesis-enhancing or inhibiting agents. We review in vitro angiogenesis models and the agents that enhance or inhibit angiogenesis. We also discuss a new in vitro angiogenesis model created within a skin equivalent. Briefly, endothelial cells were combined with the cutaneous cells of a standard skin equivalent and cultured in a chitosan cross-linked collagen-glycosaminoglycan scaffold of this endothelialized skin. This model enables the formation of capillary-like structures in a coculture environment containing newly synthesized extracellular matrix by fibroblasts and keratinocytes. Several morphological characteristics associated with the microvasculature in vivo were observed in the endothelialized skin equivalent such as histotypic organization of tubular structures, basement membrane deposition, and intercellular junction formation. This revised version was published online in July 2006 with corrections to the Cover Date.     

NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

Recent studies have shown that Notch signaling is involved in many types of cancers, including oral squamous cell carcinomas (OSCCs). However, the role of Notch signaling in the tumor microenvironment is not yet fully understood. In this study, we investigated the roles of NOTCH3 signaling in cancer associated fibroblasts (CAFs) in OSCCs. Immunohistochemical study of 93 human tongue OSCC cases indicated that about one third of OSCCs showed NOTCH3 expression in CAFs, and that this expression significantly correlated with tumor-size. In vitro study showed that OSCC cell lines, especially HO1-N-1 cells stimulated NOTCH3 expression in normal human dermal fibroblasts (NHDFs) through direct cell-to-cell contact. Immunohistochemical and morphometric analysis using human OSCC samples demonstrated that NOTCH3 expression in CAFs significantly correlated with micro-vessel density in cancer stroma. In vitro angiogenesis assays involving co-culture of NHDFs with HO1-N-1 and human umbilical endothelial cells (HUVECs), and NOTCH3 knockdown in NHDFs using siRNA, demonstrated that HO1-N-1 cells significantly promoted tube formation dependent on NOTCH3-expression in NHDFs. Moreover, NOTCH3 expression in CAFs was related to poor prognosis of the OSCC patients. This work provides a new insight into the role of Notch signaling in CAFs associated with tumor angiogenesis and the possibility of NOTCH3-targeted molecular therapy in OSCCs.     

Inactivation of Src family kinases inhibits angiogenesis in vivo: implications for a mechanism involving organization of the actin cytoskeleton

Inhibition of angiogenesis could be a treatment strategy for diseases such as cancer, rheumatoid arthritis, and diabetic retinopathy. PP2 is a pharmacological inhibitor of Src family kinases and was found to inhibit FGF-2 induced angiogenesis in vivo. Experiments in vitro showed that PP2 inhibited invasive growth and sprouting of both endothelial and vascular smooth muscle cells into a fibrin matrix. PP2 inhibited the formation of lamellopodia and expression of kinase inactive c-Src reduced phosphorylation of cortactin and paxillin, suggesting a model in which Src kinases are involved in organization of the actin cytoskeleton. Consequently, endothelial cells expressing kinase inactive c-Src failed to spread and form cord-like structures on a collagen matrix. These data suggest that pharmacological inactivation of Src family kinases inhibits FGF-2 stimulated angiogenesis by interference with organization of the actin cytoskeleton in both endothelial and vascular smooth muscle cells, which affects cell migration.     

Inhibition of osteopontin would suppress angiogenesis in gastric cancer.

Osteopontin (OPN) plays an important role in tumorigenesis, tumor invasion, and metastasis in many types of cancers, including gastric cancer. Recently, much interest has been focused on the role of OPN in tumor angiogenesis. Our previous studies have shown that OPN is overexpressed, and associated with mean microvessel density in, the tissue samples of patients with gastric cancer. In the present study, we aimed to further determine and provide evidence for the role of OPN in gastric-cancer-associated angiogenesis by diminishing OPN expression in gastric cancer cells using the small interference RNA method, and then evaluate the effects of OPN on gastric cancer-associated angiogenesis by in vivo and in vitro assays. Our results revealed that reduced OPN production by gastric cancer cells would reduce the proliferation, migration, and tube formation of human umbilical vein endothelial cells, and lead to a lower microvessel density, i.e., angiogenesis, in transplanted tumors of mice. These data confirm the positive role of OPN in gastric-cancer-associated angiogenesis.