Vascular Endothelial Cell Angiogenesis Research Articles

Inhibitory effect of insulin-like growth factor-binding protein-7 (IGFBP7) on in vitro angiogenesis of vascular endothelial cells in the rat corpus luteum.

Angiogenesis in the developing corpus luteum (CL) is a prerequisite for establishment and maintenance of an early pregnancy. To explore the physiological significance of insulin-like growth factor-binding protein-7 (IGFBP7) in the developing CL, the effects of IGFBP7 on vascular endothelial growth factor (VEGFA)- and luteinizing hormone (LH)-induced in vitro tube formation were tested using isolated luteal microvascular endothelial cells (LECs). Capillary-like tube formation of LECs and their proliferation were stimulated by both VEGFA and LH. IGFBP7 treatment suppressed VEGFA- or LH-induced tube formation. The proliferation and migration of LECs, and phosphorylation of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase 1/2 were inhibited by IGFBP7. Furthermore, IGFBP7 attenuated VEGFA-enhanced cyclooxygenase (COX)-2 mRNA expression and prostaglandin E2 secretion. These findings suggest the possibility that luteal IGFBP7 secretion may suppress the stimulatory effect of VEGFA on angiogenesis in the early CL.

Human lung telocytes could promote the proliferation and angiogenesis of human pulmonary microvascular endothelial cells in vitro.

BackgroundIn the previous studies, telocytes were found near the capillaries in many tissues, especially on the extracellular matrix of blood vessels and positive to CD34 and c-kit. Therefore, the present study aimed to explore if telocytes could produce angiogenesis associated cytokines, promote the proliferation and the angiogenesis of vascular endothelial cells in vitro.MethodsHuman lung telocytes were isolated and cultured, and were identified by immunofluorescence cytochemistry with CD34, c-kit and vimentin. Telocytes conditional media (TCM) was prepared, and the expressions of angiogenesis associated cytokines in TCM were detected by ELISA. Human pulmonary microvascular endothelial cells (HPMECs) were cultured with DMEM media or TCM for 72 hours. The proliferation of HPMECs was continuously detected with CCK-8 kit at an interval of 12 hours. HPMECs were also injured by lipopolysaccharide, and cultured with TCM and DMEM respectively, and the tube formation capacity was detected.ResultsTelocytes were positive for CD34, c-kit and vimentin. The expressions of VEGF and EGF in TCM were significantly higher, the proliferation of HPMECs cultured with TCM significantly increased, and the tube formation of HPMECs injured by endotoxin was improved with the culture of TCM, as compared with the culture of DMEM.ConclusionThe present study provides the evidence that human lung telocytes could produce the growth factors, such as VEGF and EGF. Telocytes conditional media induced the proliferation of pulmonary endothelial cells and prevented from endotoxin-induced compromise of pulmonary endothelial angiogenesis.

Granulocyte colony-stimulating factor induces in vitro lymphangiogenesis

Granulocyte-colony stimulating factor (G-CSF) is reported to induce differentiation in cells of the monocyte lineage and angiogenesis in vascular endothelial cells, but its effects on lymphangiogenesis is uncertain. Here we examined the effects and the mechanisms of G-CSF-induced lymphangiogenesis using human lymphatic endothelial cells (hLECs). Our results showed that G-CSF induced capillary-like tube formation, migration and proliferation of hLECs in a dose- and time-dependent manner and enhanced sprouting of thoracic duct. G-CSF increased phosphorylation of Akt and ERK1/2 in hLECs. Supporting the observations, specific inhibitors of phosphatidylinositol 3′-kinase and MAPK suppressed the G-CSF-induced in vitro lymphangiogenesis and sprouting. Intraperitoneal administration of G-CSF to mice also stimulated peritoneal lymphangiogenesis. These findings suggest that G-CSF is a lymphangiogenic factor.

Dkk‐1 promotes angiogenic responses and cartilage matrix proteinase secretion in synovial fibroblasts from osteoarthritic joints

Synovial hypervascularity is a prominent pathologic feature in osteoarthritic (OA) joints. Wnt inhibitor Dkk-1 contributes to joint remodeling. We undertook this study to investigate whether Dkk-1 regulates cartilage destruction activities in OA synovial fibroblasts. Synovial tissues were harvested from knees of patients with OA and from injured knees of non-OA patients who underwent arthroscopy. Expression of Dkk-1, angiogenic factors (stromal cell-derived factor 1 and colony-stimulating factor 1), and cartilage proteinases (ADAMTS-5 and matrix metalloproteinase 3 [MMP-3]) as well as vascularity in synovium and synovial fluid were quantified using enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction, and histomorphometry. Synovial fibroblasts were treated with interleukin-1β (IL-1β), anti-Dkk-1 antibody, and RNA interference to characterize their angiogenic activity. Rats with OA knees were administered Dkk-1 antisense oligonucleotide to verify synovial angiogenesis and cartilage integrity. OA synovium exhibited increased vascularity and expression of angiogenic factors and proteinases in association with up-regulated Dkk-1 levels. Neutralizing Dkk-1 reduced the inhibitory effects of OA synovial fluid on aggrecan expression in chondrocyte cultures. IL-1β induction of Dkk-1 increased expression of hypoxia-inducible factor 1α (HIF-1α), angiogenic factors, ADAMTS-5, and MMP-3 in synovial fibroblasts and promoted angiogenesis in vascular endothelial cells. Knockdown of HIF-1α decreased Dkk-1 enhancement of angiogenic factor expression. Stabilization of glycogen synthase kinase 3β phosphorylated at Ser(9) , β-catenin, T cell factor 4, and ERK signaling attenuated Dkk-1 up-regulation of angiogenic factor and proteinase expression in synovial fibroblasts. In vivo, Dkk-1 interference reduced the expression of angiogenic factors and proteinases and ameliorated synovial vascularity and cartilage deterioration in knees of rats with OA. Dkk-1 promoted angiogenic and cartilage degradation activities in synovial fibroblasts, which accelerated synovial angiogenesis and cartilage destruction. Dkk-1 blockade has therapeutic potential for reducing OA-induced synovitis and joint deterioration.

An easily accessible sulfated saccharide mimetic inhibits in vitro human tumor cell adhesion and angiogenesis of vascular endothelial cells

Oligosaccharides aberrantly expressed on tumor cells influence processes such as cell adhesion and modulation of the cell’s microenvironment resulting in an increased malignancy. Schmidt’s imidate strategy offers an effective method to synthesize libraries of various oligosaccharide mimetics. With the aim to perturb interactions of tumor cells with extracellular matrix proteins and host cells, molecules with 3,4-bis(hydroxymethyl)furan as core structure were synthesized and screened in biological assays for their abilities to interfere in cell adhesion and other steps of the metastatic cascade, such as tumor-induced angiogenesis.The most active compound, (4-{[(β-D-galactopyranosyl)oxy]methyl}furan-3-yl)methyl hydrogen sulfate (GSF), inhibited the activation of matrix-metalloproteinase-2 (MMP-2) as well as migration of the human melanoma cells of the lines WM-115 and WM-266-4 in a two-dimensional migration assay. GSF inhibited completely the adhesion of WM-115 cells to the extracellular matrix (ECM) proteins, fibrinogen and fibronectin.In an in vitro angiogenesis assay with human endothelial cells, GSF very effectively inhibited endothelial tubule formation and sprouting of blood vessels, as well as the adhesion of endothelial cells to ECM proteins. GSF was not cytotoxic at biologically active concentrations; neither were 3,4-bis{[(β-D-galactopyranosyl)oxy]methyl}furan (BGF) nor methyl β-D-galactopyranoside nor 3,4-bis(hydroxymethyl)furan, which were used as controls, eliciting comparable biological activity. In silico modeling experiments, in which binding of GSF to the extracellular domain of the integrin αvβ3 was determined, revealed specific docking of GSF to the same binding site as the natural peptidic ligands of this integrin. The sulfate in the molecule coordinated with one manganese ion in the binding site.These studies show that this chemically easily accessible molecule GSF, synthesized in three steps from 3,4-bis(hydroxymethyl)furan and benzoylated galactose imidate, is nontoxic and antagonizes cell physiological processes in vitro that are important for the dissemination and growth of tumor cells in vivo.

Primary breast cancer tryptase expression as novel molecular drug target.

e21113 Background: Tryptase, a serine protease stored and released from mast cells granules has been identified as a new non-classical angiogenetic factor. Mast cells can release tryptase following c-Kit receptor activation. We have evaluated the correlations among the number of MCs positive to tryptase (MCDPT), the number of c-Kit receptor expressing cells (C-KREC) and microvascular density (MVD) in a series of 88 primary T1-3, N0-2 M0 female breast cancer by means of immunohistochemistry and image analysis methods. Methods: Six-micrometers thick serial sections of formalin-fixed and paraffin-embedded bioptic tumor samples were microwaved at 500 W for 10 min. and treated with a 3% hydrogen peroxide solution. Sections were incubated with primary antibodies: anti-tryptase (AA1; Dako, Glostrup, Denmark), anti-c-Kit receptor (A4502;Dako, Glostrup, Denmark) and anti-CD34 (QB-END 10; Bio-Optica Milan, Italy). In serial sections MVD, MCDPT and C-KREC were counted by means of image analysis at x400. Results: Data demonstrated a significantly (r= ranging from 0.71 to 0.91; p: ranging from 0.001 to 0.003 by Pearson’s analysis respectively) correlation between MVD, MCDPT and C-KREC to each other. Conclusions: Published in vitro data suggest that tryptase induce angiogenesis in vascular endothelial cells and breast cancer cells lines. According to these data we shown that MVD, MCDPT and C-KREC paralleled to each other suggesting a role in in vivo breast cancer angiogenesis. In this context several tryptase inhibitors such as gabexate mesilate or nafamostat mesilate might be evaluated in clinical trials as a new antiangiogenetic approach.

Inhibition of neuroblastoma cell growth by TREX1-mutated human lymphocytes

T lymphocytes play a major role in counteracting cancer occurrence and development. Immune therapies against cancer are focused on eliciting a cytotoxic Tcell response. This anticancer activity is related to a variety of mechanisms including the activation of cytokines and proapoptotic mediators. Interferonα is an established inhibitor of cancer cell growth. A clinical situation involving the coexistence of high interferonα levels and lymphocyte activation is the Aicardi-Goutières syndrome, a progressive encephalopathy arising usually during the first year of life characterized by intracranial basal ganglia calcifications, leukodystrophy and microcephaly. Aicardi-Goutières syndrome1 mutation silences the TREX1 gene, a major endogenous nuclease. The invitro study presented herein evaluates the efficacy of the TREX1 mutation in potentiating the anticancer properties of Tcells. A TREX1-mutated lymphocyte cell line was derived from an Aicardi-Goutières syndrome patient and co-cultured with neuroblastoma cells and vascular endothelial cells in the presence of interferonα. TREX1-mutated lymphocytes exerted marked inhibitory action on neuroblastoma cell growth. CathepsinD was recognized by qPCR as the main mediator produced by TREX1-mutated lymphocytes involved in the inhibition of neuroblastoma cell growth. These effects were enhanced in the presence of interferonα. Similar inhibitory effects in cell growth were exerted by TREX1-mutated lymphocytes towards vascular endothelial cell angiogenesis as evaluated on Matrigel. The results obtained provide evidence that mutations of the TREX1 gene increase the capability of T-lymphocytes to inhibit growth of neoplastic neuronal cells and related angiogenesis.

T-cadherin attenuates insulin-dependent signalling, eNOS activation, and angiogenesis in vascular endothelial cells

T-cadherin (T-cad) is a glycosylphosphatidylinositol-anchored cadherin family member. Experimental, clinical, and genomic studies suggest a role for T-cad in vascular disorders such as atherosclerosis and hypertension, which are associated with endothelial dysfunction and insulin resistance (InsRes). In endothelial cells (EC), T-cad and insulin activate similar signalling pathways [e.g. PI3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR)] and processes (e.g. angiogenesis). We hypothesize that T-cad is a regulatory component of insulin signalling in EC and therefore a determinant of the development of endothelial InsRes. We investigated T-cad-dependent effects on insulin sensitivity using human EC stably transduced with respect to T-cad overexpression or T-cad silencing. Responsiveness to insulin was examined at the level of effectors of the insulin signalling cascade, EC nitric oxide synthase (eNOS) activation, and angiogenic behaviour. Overexpression and ligation of T-cad on EC attenuates insulin-dependent activation of the PI3K/Akt/mTOR signalling axis, eNOS, EC migration, and angiogenesis. Conversely, T-cad silencing enhances these actions of insulin. Attenuation of EC responsiveness to insulin results from T-cad-mediated chronic activation of the Akt/mTOR-dependent negative feedback loop of the insulin cascade and enhanced degradation of the insulin receptor (IR) substrate. Co-immunoprecipitation experiments revealed an association between T-cad and IR. Filipin abrogated inhibitory effects of T-cad on insulin signalling, demonstrating localization of T-cad-insulin cross-talk to lipid raft plasma membrane domains. Hyperinsulinaemia up-regulates T-cad mRNA and protein levels in EC. T-cad expression modulates signalling and functional responses of EC to insulin. We have identified a novel signalling mechanism regulating insulin function in the endothelium and attribute a role for T-cad up-regulation in the pathogenesis of endothelial InsRes.

P2-05-03: Is Breast Cancer Tryptase a Novel Anti-Angiogenetic Molecular Target?

Abstract Tryptase, a serine protease stored and released from mast cells granules has been identified as a new non-classical angiogenetic factor. Tryptase is an agonist of proteinase-activated receptor-2 a G protein involved in cellular proliferation and angiogenesis. On the other hand mast cells can release tryptase following c-Kit receptor activation. We have evaluated the correlations among the number of MCs positive to tryptase (MCDPT), the number of c-Kit receptor expressing cells (C-KREC) and microvascular density (MVD) in a series of 91 primary T1-3, N0-2 M0 female breast cancer by means of immunohistochemistry and image analysis methods. Six-micrometers thick serial sections of formalin-fixed and paraffin-embedded bioptic tumor samples were microwaved at 500 W for 10 min. and treated with a 3% hydrogen peroxide solution. Sections were incubated with primary antibodies: anti-tryptase (AA1; Dako, Glostrup, Denmark), anti-c-Kit receptor (A4502; Dako, Glostrup, Denmark) and anti-CD34 (QB-END 10; Bio-Optica Milan, Italy). In serial sections MVD, MCDPT and C-KREC were counted by means of image analysis (Quantimet 500 Leica) at x400 microscopic fields. Data demonstrated a significantly (r= ranging from 0.72 to 0.91; p: ranging from 0.001 to 0.003 by Pearson's analysis respectively) correlation between MVD, MCDPT and C-KREC to each other. Published in vitro data suggest that tryptase induce angiogenesis in vascular endothelial cells and breast cancer cells lines. According to these data we shown that MVD, MCDPT and C-KREC paralleled to each other suggesting a role in in vivo breast cancer angiogenesis. In this context the inhibition of c-Kit mast cells tryptase degranulation by several tyrosin kinase inhibitors might be evaluated in clinical trials. Finally available tryptase inhibitors such as gabexate mesilate or nafamostat mesilate might be evaluated in clinical trials as a new antiangiogenetic approach. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-03.

Pleiotropic and Age-dependent Effects of Decreased Protein Modification by O-Linked N-Acetylglucosamine on Pancreatic β-Cell Function and Vascularization

The hexosamine biosynthesis pathway (HBP) regulates the post-translational modification of nuclear and cytoplasmic protein by O-linked N-acetylglucosamine (O-GlcNAc). Numerous studies have demonstrated increased flux through this pathway contributes to the development of β-cell dysfunction. The effect of decreased O-GlcNAc on the maintenance of normal β-cell function, however, is not well understood. We studied transgenic mice that over express β-N-acetylglucosaminidase (O-GlcNAcase), an enzyme that catalyzes the removal of O-GlcNAc from proteins, in the pancreatic β-cell under control of the rat insulin promoter. 3-4-Month-old O-GlcNAcase transgenic mice have higher glucose excursions with a concomitant decrease in circulating insulin levels, insulin mRNA levels, and total islet insulin content. In older (8-9-month-old) O-GlcNAcase transgenic mice glucose tolerance is no longer impaired. This is associated with increased serum insulin, islet insulin content, and insulin mRNA in the O-GlcNAcase transgenic mice. These improvements in β-cell function with aging are associated with increased angiogenesis and increased VEGF expression, with parallel increases in activation of Akt and expression of PGC1α. The biphasic effects as a function of age are consistent with published observations of mice with increased O-GlcNAc in islets and demonstrate that O-GlcNAc signaling exerts multiple effects on both insulin secretion and islet survival.

N-benzyl-5-phenyl-1H-pyrazole-3-carboxamide promotes vascular endothelial cell angiogenesis and migration in the absence of serum and FGF-2

To investigate the effect of N-benzyl-5-phenyl-1H-pyrazole-3-carboxamide (BPC) on angiogenesis in human umbilical vein endothelial cells (HUVECs). Capillary-like tube formation on matrigel and cell migration analyses were performed in the absence of serum and fibroblast growth factor (FGF-2). Reactive oxygen species (ROS) were measured using a fluorescent probe, 2', 7'- dichlorodihydrofluorescein (DCHF). The nitric oxide (NO) production of HUVECs was examined using a NO detection kit. Morphological observation under a phase contrast microscope, a viability assay using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium (MTT) and a lactate dehydrogenase (LDH) activity analysis by a detection kit were performed to evaluate the toxicity of BPC on HUVECs in the presence of serum and FGF-2. The level of hypoxia-inducible factor 1α (HIF-1α) and the release of vascular endothelial growth factor (VEGF) were measured by Western blot and ELISA, respectively. In the absence of serum and FGF-2, cells treated with BPC (5-20 μmol/L) rapidly aligned with one another and formed tube-like structures within 12 h. In the presence of serum and FGF-2, cells treated with BPC for 24, 48 and 72 h had no changes in morphology, viability or LDH release compared with the control group. Cell migration in the BPC-treated group was significantly increased compared with the control group. During this process, NO production and ROS level were elevated dramatically, and the levels of HIF-1α and VEGF were increased dependent on the generation of ROS. BPC most effectively promoted angiogenesis and migration in HUVECs in the absence of FGF-2 and serum.

Angiogenic activities of YKL-40 in cancer development: A potential biomarker and target for cancer diagnosis and therapy

Pioneer Valley Life Sciences Institute, University of Massachusetts, USA A evidence has shown that elevated serum levels of a secreted glycoprotein YKL-40 are associated with a worse prognosis from a variety of advanced human cancers, including breast cancer, colorectal cancer, ovarian cancer, and brain tumor. Furthermore, these increased levels correlate with poorer survival of cancer patients, suggesting that serum levels of YKL-40 might be a prognostic biomarker. Yet the role of YKL-40 activity in these cancers is poorly understood. To explore a functional role of YKL-40 in tumor development, we engineered cancer cells with YKL-40 cDNA to express ectopic YKL-40. Over-expression of YKL-40 in these cancer cells led to larger tumor formation with an extensive angiogenic phenotype than did control cancer cells in mice. Affi nity purifi ed recombinant YKL-40 protein promoted vascular endothelial cell angiogenesis in vitro, the eff ects of which are similar to the activities observed using cancer cell conditioned medium aft er transfection with YKL-40. Immunohistochemical analysis of human breast cancer revealed a correlation between YKL-40 expression and blood vessel density. YKL-40 levels were positively correlated with tumor grade and Her2/neu, but negatively correlated with estrogen and progesterone receptor. In complementary approaches, we found that blockade of YKL-40 by YKL40 siRNA gene knockdown and a YKL-40 neutralizing antibody restrained tumor growth, angiogenesis, and progression. Taken together, these fi ndings have shed light on the mechanisms by which YKL-40 promotes tumor angiogenesis and progression; thus pointing to a valuable cancer diagnostic and prognostic biomarker as well as a target for cancer therapy.

Toona sinensis (leaf extracts) inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in vascular endothelial cells

Toona sinensis is well known as a traditional Chinese medicine; also, it has been shown to exhibit anticancer and anti-inflammatory effects. This study was aimed at evaluating the anti-angiogenesis effect of the aqueous extracts of Toona sinensis (TS extracts) or gallic acid, a major component of TS extracts, against both VEGF-induced EA.hy 926 and human umbilical vein endothelial cells (HUVECs). Anti-proliferative activity of TS extracts or gallic acid, was determined against EA.hy 926 and HUVECs by trypan blue exclusion method. Invasion, tube formation and chick chorioallantoic membrane assay were carried out to determine the in vitro and in vivo anti-angiogenic effects. Non-cytotoxic concentration of TS extracts (50-100μg/mL) and gallic acid (5μg/mL) inhibited the proliferation of VEGF-stimulated EA.hy 926 and HUVECs. Inhibitory effects of TS extracts and gallic acid on angiogenesis were assessed by VEGF-induced migration/invasion and capillary-like tube formation by EA.hy 926 and HUVECs. Additionally, gelatin zymography assays showed that TS extracts and gallic acid suppressed the activity of metalloproteinase (MMP)-9 and MMP-2 activated by VEGF. In vivo, TS extracts and gallic acid strongly suppressed neovessel formation in the chorioallantoic membrane of chick embryos. Flow cytometry analyses and Western blot demonstrated that treatment with TS extracts and gallic acid induced G(0)/G(1) arrest in VEGF-stimulated EA.hy 926 cells via a reduction in the amounts of cyclin D1, cyclin E, CDK4, hyperphosphorylated retinoblastoma protein (pRb), VEGFR-2, and eNOS. These results support an anti-angiogenic activity of Toona sinensis that may contribute critically to its cancer and inflammation chemopreventive potentials.

Abstract 1732: Elevated expression of YKL-40, a cancer biomarker, is associated with tumor angiogenesis

Abstract Growing evidence from clinical investigations has shown that increased serum levels of YKL-40 are correlated with short survival and metastasis in a broad type of human cancers. These data suggest that YKL-40 in the blood may present a powerful tool for cancer diagnosis and prognosis. However, pathological activity of YKL-40 in cancer development is poorly understood. Here, we explored a functional role for YKL-40 in vasculature development of human cancer. In a survey of twenty cases of breast ductal carcinoma in situ (DCIS) and thirty-eight cases of infiltrating ductal carcinoma (IDC), we found that expression levels of YKL-40 in specimens from DCIS were barely detectable, but different levels of YKL-40 were present in IDC. Nine out of thirty-eight IDC cases showed high levels of YKL-40 and nine cases displayed medium levels of YKL-40, whereas the rest twenty cases were low or negative. Interestingly, in an analysis of vasculature formation in these IDC samples, we found that different blood vessel densities significantly correlated with different degrees of YKL-40 expression. To test this possible angiogenic activity of YKL-40 in tumor development, we utilized angiogenic xenograft models by subcutaneous injection of SCID/Beige mice with a brain tumor line U87 cells which express strong endogenous YKL-40 and U87 cells with siRNA knockdown of YKL-40. siRNA knockdown of YKL-40 notably suppressed tumor angiogenesis and tumor development relative to high levels of YKL-40 expressed in control U87 cells. Further, to dissect cellular and molecular mechanisms underlying YKL-40-induced tumor angiogenesis, we treated human microvascular endothelial cells with YKL-40 and found that YKL-40 was capable of binding to heparan sulfate on ectodomain of membrane receptor syndecan-1 (S1). The activated S1 prompted its interaction with adjacent receptor integrin αvβ3, leading to activation of intracellular cascades including phosphorylation of focal adhesion kinase and MAP kinase. A series of individual signaling inhibitors blocked YKL-40-induced angiogenesis in vitro. Collectively, these data strongly suggest that YKL-40 expressed in cancer activates vascular endothelial cell angiogenesis to promote tumor development. These findings may hold promise for developing novel therapeutic agents targeting YKL-40, a cancer biomarker that is overexpressed in a wide range of human cancers. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1732.

Identification of the Gene Regulatory Region in Human rasgrp2 Gene in Vascular Endothelial Cells

Ras guanyl nucleotide-releasing protein 2 (RASGRP2) is a calcium- and diacylglycerol-responsive guanine nucleotide exchange factor. Previously, we reported that XRASGRP2, a homolog of human RASGRP2, is expressed in the vascular region of the Xenopus embryo during embryogenesis. Here, we report the regulatory mechanisms of human rasgrp2 gene expression in vascular endothelial cells. Although three alternative splicing variants, differing in the first exon of 5'-untranslated region (5'-UTR), have been found for the human rasgrp2 gene, reverse transcription-polymerase chain reaction (RT-PCR) showed that the cDNA containing the distal first exon (D1E) was expressed only in human umbilical artery endothelial cells. We analyzed the 5'-flanking region of the human rasgrp2 gene by a luciferase assay, which revealed that not only a promoter but also silencer regions were present upstream of D1E, suggesting that rasgrp2 expression is controlled by a combination of transcriptional promotion and repression. Gel super shift assay demonstrated that OCT1/POU2F1 bound to the silencer region. These findings may help furthering our understanding of vasculogenesis and/or angiogenesis in vascular endothelial cells.

YKL-40, a secreted glycoprotein, promotes tumor angiogenesis

Tumor angiogenesis is of paramount importance in solid tumor development. Elevated serum levels of YKL-40, a secreted heparin-binding glycoprotein have been associated with a worse prognosis from a variety of advanced human cancers. Yet the role of YKL-40 activity in these cancers is still missing. Here, we have shown that ectopic expression of YKL-40 in MDA-MB-231 breast cancer cells and HCT-116 colon cancer cells led to larger tumor formation with an extensive angiogenic phenotype than did control cancer cells in mice. Affinity purified recombinant YKL-40 protein promoted vascular endothelial cell angiogenesis in vitro, the effects similar to the activities observed using MDA-MB-231 and HCT-116 cell conditioned medium after transfection with YKL-40. Further, YKL-40 was found to induce the coordination of membrane-bound receptor syndecan-1 and integrin αvβ3 and activate an intracellular signaling cascade including focal adhesion kinase and MAP kinase Erk1/2 in endothelial cells. Also, blockade of YKL-40 using siRNA gene knockdown suppressed tumor angiogenesis in vitro and in vivo. Immunohistochemical analysis of human breast cancer revealed a correlation between YKL-40 expression and blood vessel density. These findings provide novel insights into angiogenic activities and molecular mechanisms of YKL-40 in cancer development.

Regulation of angiogenesis through a microRNA (miR-130a) that down-regulates antiangiogenic homeobox genes GAX and HOXA5

AbstractAngiogenesis is critical to tumor progression. The homeobox gene GAX inhibits angiogenesis in vascular endothelial cells (ECs). We have identified a microRNA (miR-130a) that regulates GAX expression and hypothesized that it plays a major role in modulating GAX activity in ECs. A 280-bp fragment from the GAX 3′-untranslated region (3′-UTR) containing 2 miR-130a targeting sites was observed to be required for the rapid down-regulation of GAX expression by serum and proangiogenic factors, whereas the activity of the GAX promoter did not vary with exposure to serum or proangiogenic factors. This same 280-bp sequence in the GAX 3′-UTR cloned into the psiCHECK2-Luciferase vector mediated serum-induced down-regulation of the reporter gene when placed 3′ of it. Finally, forced expression of miR-130a inhibits GAX expression through this specific GAX 3′-UTR sequence. A genome-wide search for other possible miR-130a binding sites revealed an miR-130a targeting site in the 3′-UTR of the antiangiogenic homeobox gene HOXA5, the expression and antiangiogenic activity of which are also inhibited by miR-130a. From these data, we conclude that miR-130a is a regulator of the angiogenic phenotype of vascular ECs largely through its ability to modulate the expression of GAX and HOXA5.

Transcription Factor Ets-1 Mediates Ischemia- and Vascular Endothelial Growth Factor-Dependent Retinal Neovascularization

Transcription factor Ets-1 has been reported to regulate angiogenesis in vascular endothelial cells. Here, we investigated a mechanism that may regulate the expression of Ets-1 in vascular endothelial growth factor (VEGF)- and hypoxia-induced retinal neovascularization and that may have potential to inhibit ocular neovascular diseases. VEGF and hypoxia increased Ets-1 expression in cultured bovine retinal endothelial cells. The VEGF-induced mRNA increase of Ets-1 was suppressed by a tyrosine kinase inhibitor (genistein), by inhibitors of MEK (mitogen-activated protein and extracellular signal-regulated kinase kinase) (PD98059 and UO126), and by inhibitors of protein kinase C (GF109203X, staurosporine, and Gö6976). Dominant-negative Ets-1 inhibited VEGF-induced cell proliferation, tube formation, and the expression of neuropilin-1 and angiopoietin-2. In a mouse model of proliferative retinopathy, Ets-1 mRNA was up-regulated. Intravitreal injection of dominant-negative Ets-1 suppressed retinal angiogenesis in a mouse model of proliferative retinopathy. In conclusion, VEGF induces Ets-1 expression in bovine retinal endothelial cells and its expression is protein kinase C/ERK pathway-dependent. Ets-1 up-regulation is involved in the development of retinal neovascularization, and inhibition of Ets-1 may be beneficial in the treatment of ischemic ocular diseases.

Stimulation of In Vitro Angiogenesis by Tetrahydrobiopterin in Bovine Aortic Endothelial Cells

This study examined whether tetrahydrobiopterin (BH4) stimulates angiogenesis by measuring the formation of tube-like structures in vascular endothelial cells. Bovine aortic endothelial cells that were cultured between two layers of collagen type I gel formed tube-like networks. Addition of BH4 or sepiapterin, a precursor of BH4 synthesis, stimulated the formation of tube-like structures. The sepiapterin-stimulated tube formation was completely inhibited by co-treatment with N-acetylserotonin, an inhibitor of sepiapterin reductase. These findings show that BH4 stimulates in vitro angiogenesis in vascular endothelial cells.

Tubular morphogenesis by genotoxic therapeutic agents that induce NF-kappaB activation in human vascular endothelial cells.

Angiogenic stimuli induce tubular morphogenesis and angiogenesis in vascular endothelial cells, but these cells are highly vulnerable to cytokines, oxidative stress, and genotoxic anticancer agents. A transcription factor, NF-kappaB, is involved in the protection against apoptosis and in angiogenesis in response to stimuli that could induce cell death. NF-kappaB was specifically activated by the genotoxic anticancer therapeutic agents etoposide and doxorubicin, but not by bleomycin, mitomycin C and cisplatin, in human vascular endothelial cells in three independent assay systems: nuclear translocation of NF-kappaB, binding of NF-kappaB to its consensus sequence, and NF-kappaB -dependent transcription. Exposure to etoposide and doxorubicin induced tubular morphogenesis by vascular endothelial cells in type I collagen gel at rates comparable to tumor necrosis factor-alpha. Co-administration of NF-kappaB antisense oligonucleotides inhibited the angiogenesis by doxorubicin and etoposide. In contrast, bleomycin, mitomycin C, and cisplatin did not induce angiogenesis. An angiogenic factor, interleukin 8, was dramatically induced in vascular endothelial cells treated with doxorubicin, but not in cells treated with cisplatin. Co-administration of anti-interleukin 8 antibody almost completely blocked the doxorubicin-induced angiogenesis in vitro, suggesting a paracrine/autocrine control through drug-induced angiogenic factor(s). The presence or absence of NF-kappaB activation may have an essential role in tubular morphogenesis by vascular endothelial cells during chemotherapeutic treatment, possibly through interleukin 8.