Aminopeptidase N/CD13 induces angiogenesis through interaction with a pro-angiogenic protein, galectin-3

Aminopeptidase N/CD13 is an endothelial cell surface ectoenzyme involved in one of the initial steps of angiogenesis. However, little is known how APN induces angiogenesis in endothelial cells. Using human cDNA library-encoding phage display biopanning method, we here identified a galactoside-specific lectin family protein, galectin-3, as an interacting protein of APN. Galectin-3 specifically binds to APN both in vitro and in human umbilical vein endothelial cells (HUVECs) in a carbohydrate recognition-dependent manner. Immunohistochemical analysis demonstrates that both APN and galectin-3 are exclusively coexpressed during the angiogenic stage of mouse forebrain development. Finally, exogenous addition of galectin-3 into HUVECs induced angiogenesis in an APN-dependent manner, implying that APN is a crucial mediator of galectin-3-induced angiogenesis in endothelial cells.     

Endothelial connexin32 enhances angiogenesis by positively regulating tube formation and cell migration

The gap junction proteins connexin32 (Cx32), Cx37, Cx40, and Cx43 are expressed in endothelial cells, and regulate vascular functions involving inflammation, vasculogenesis and vascular remodeling. Aberrant Cxs expression promotes the development of atherosclerosis which is modulated by angiogenesis; however the role played by endothelial Cxs in angiogenesis remains unclear. In this study, we determined the effects of endothelial Cxs, particularly Cx32, on angiogenesis. EA.hy926 cells that had been transfected to overexpress Cx32 significantly increased capillary length and the number on branches compared to Cx-transfectant cells over-expressing Cx37, Cx40, and Cx43 or mock-treated cells. Treatment via intracellular transfer of anti-Cx32 antibody suppressed tube formation of human umbilical vein endothelial cells (HUVECs) compared to controls. In vitro wound healing assays revealed that Cx32-transfectant cells significantly increased the repaired area while anti-Cx32 antibody-treated HUVECs reduced it. Ex vivo aorta ring assays and in vivo matrigel plaque assays showed that Cx32-deficient mice impaired both vascular sprouting from the aorta and cell migration into the implanted matrigel. Therefore endothelial Cx32 facilitates tube formation, wound healing, vascular sprouting, and cell migration. Our results suggest that endothelial Cx32 positively regulates angiogenesis by enhancing endothelial cell tube formation and cell migration.     

Covalently grafted VEGF(165) in hydrogel models upregulates the cellular pathways associated with angiogenesis

Angiogenesis is an important biological response known to be involved in many physiological and pathophysiological situations. Cellular responses involved in the formation of new blood vessels, such as increases in endothelial cell proliferation, cell migration, and the survival of apoptosis-inducing events, have been associated with vascular endothelial growth factor isoform 165 (VEGF(165)). Current research in the areas of bioengineering and biomedical science has focused on developing polyethylene glycol (PEG)-based systems capable of initiating and sustaining angiogenesis in vitro. However, a thorough understanding of how endothelial cells respond at the molecular level to VEGF(165) incorporated into these systems has not yet been established in the literature. The goal of the current study was to compare the upregulation of key intracellular proteins involved in angiogenesis in human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC) seeded on PEG hydrogels containing grafted VEGF(165) and adhesion peptides Arg-Gly-Asp-Ser (RGDS). Our data suggest that the covalent incorporation of VEGF(165) into PEG hydrogels encourages the upregulation of signaling proteins responsible for increases in endothelial cell proliferation, cell migration, and the survival after apoptosis-inducing events.     

Human placental multipotent mesenchymal stromal cells modulate placenta angiogenesis through Slit2-Robo signaling

abstract

The objective of this study was to investigate whether human placental multipotent mesenchymal stromal cell (hPMSC)-derived Slit2 and endothelial cell Roundabout (Robo) receptors are involved in placental angiogenesis. The hPMSC-conditioned medium and human umbilical vein endothelial cells were studied for Slit2 and Robo receptor expression by immunoassay and RT-PCR. The effect of the conditioned medium of hPMSCs with or without Slit2 depletion on endothelial cells was investigated by in vitro angiogenesis using growth factor-reduced Matrigel. hPMSCs express Slit2 and both Robo1 and Robo4 are present in human umbilical vein endothelial cells. Human umbilical vein endothelial cells do not express Robo2 and Robo3. The hPMSC-conditioned medium and Slit2 recombinant protein significantly inhibit the endothelial cell migration, but not by the hPMSC-conditioned medium with Slit2 depletion. The hPMSC-conditioned medium and Slit2 significantly enhance endothelial tube formation with increased cumulated tube length, polygonal network number and vessel branching point number compared to endothelial cells alone. The tube formation is inhibited by the depletion of Slit2 from the conditioned medium, or following the expression of Robo1, Robo4, and both receptor knockdown using small interfering RNA. Furthermore, co-immunoprecipitation reveals Slit2 binds to Robo1 and Robo4. Robo1 interacts and forms a heterodimeric complex with Robo4. These results suggest the implication of both Robo receptors with Slit2 signaling, which is involved in endothelial cell angiogenesis. Slit2 in the conditioned medium of hPMSCs has functional effect on endothelial cells and may play a role in placental angiogenesis.     

A novel compound T7 (N-{4′-[(1E)-N-hydroxyethanimidoyl]-3′,5,6-trimethoxybiphenyl-3-yl}-N′-[4-(3-morpholin-4-ylpropoxy)phenyl]urea) screened by tissue angiogenesis model and its activity evaluation on anti-angiogenesis

A tissue model for angiogenesis that imitated new blood vessels formation in vivo had been established in the previous study. Here, it was used to screen and evaluate a series of synthesized compounds and the results indicated that compound T7 (N-{4′-[(1E)-N-hydroxyethanimidoyl]-3′,5,6-trimethoxybiphenyl-3-yl}-N′-[4-(3-morpholin-4-ylpropoxy)phenyl]urea) could effectively inhibit the blood vessels formation. Then the anti-angiogenic potential of T7 and its related molecular mechanisms against lung carcinoma in vitro and in vivo were investigated. Treatment with T7 significantly inhibited human umbilical vein endothelial cells and A549 cells proliferation and migration. T7 reduced human umbilical vein endothelial cells tube formation as well. Western blotting analysis of cell signaling molecules indicated that T7 reduced phosphorylation of KDR and its downstream signaling players AKT and ERK1/2 activation in endothelial cells and A549 cells. Moreover, T7 inhibited tumor growth in A549 xenografted model of athymic mice and reduced CD34 expression levels in tumor-bearing mice by immunohistochemistry. In sum, our findings showed that T7 was a candidate of tumor angiogenesis inhibitors, and it functioned by interrupting the autophosphorylation of KDR, AKT and ERK1/2.     

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.     

Albendazole inhibits endothelial cell migration, tube formation, vasopermeability, VEGF receptor-2 expression and suppresses retinal neovascularization in ROP model of angiogenesis

The angiogenic process begins with the cell proliferation and migration into the primary vascular network, and leads to vascularization of previously avascular tissues and organs as well to growth and remodeling of the initially homogeneous capillary plexus to form a new microcirculation. Additionally, an increase in microvascular permeability is a crucial step in angiogenesis. Vascular endothelial growth factor (VEGF) plays a central role in angiogenesis. We have previously reported that albendazole suppresses VEGF levels and inhibits malignant ascites formation, suggesting a possible effect on angiogenesis. This study was therefore designed to investigate the antiangiogenic effect of albendazole in non-cancerous models of angiogenesis. In vitro, treatment of human umbilical vein endothelial cells (HUVECs) with albendazole led to inhibition of tube formation, migration, permeability and down-regulation of the VEGF type 2 receptor (VEGFR-2). In vivo albendazole profoundly inhibited hyperoxia-induced retinal angiogenesis in mice. These results provide new insights into the antiangiogenic effects of albendazole.     

Autophagy triggered by magnolol derivative negatively regulates angiogenesis

Angiogenesis has a key role in the tumor progression and metastasis; targeting endothelial cell proliferation has emerged as a promising therapeutic strategy for the prevention of cancer. Previous studies have revealed a complex association between the process of angiogenesis and autophagy and its outcome on tumorigenesis. Autophagy, also known as type-II cell death, has been identified as an alternative way of cell killing in apoptotic-resistant cancer cells. However, its involvement in chemoresistance and tumor promotion is also well known. In this study, we used a derivate of natural product magnolol (Ery5), a potent autophagy inducer, to study the association between the autophagy and angiogenesis in both in vitro and in vivo model system. We found that the robust autophagy triggered by Ery5, inhibited angiogenesis and caused cell death independent of the apoptosis in human umbilical cord vein endothelial cells and PC-3 cells. Ery5 induced autophagy effectively inhibited cell proliferation, migration, invasion and tube formation. We further demonstrated that Ery5-mediated autophagy and subsequent inhibition of angiogenesis was reversed when autophagy was inhibited through 3-methyl adenine and knocking down of key autophagy proteins ATG7 and microtubule-associated protein light chain 3. While evaluating the negative regulation of autophagy on angiogenesis, it was interesting to find that angiogenic environment produced by the treatment of VEGF and CoCl2 remarkably downregulated the autophagy and autophagic cell death induced by Ery5. These studies, while disclosing the vital role of autophagy in the regulation of angiogenesis, also suggest that the potent modulators of autophagy can lead to the development of effective therapeutics in apoptosis-resistant cancer.     

Characterization of a novel angiogenic model based on stable, fluorescently labelled endothelial cell lines amenable to scale-up for high content screening

Background. Blood vessel formation is important for many physiological and pathological processes and is therefore a critical target for drug development. Inhibiting angiogenesis to starve a tumour or promoting ‘normalization’ of tumour vasculature in order to facilitate delivery of anticancer drugs are both areas of active research. Recapitulation of vessel formation by human cells in vitro allows the investigation of cell—cell and cell—matrix interactions in a controlled environment and is therefore a crucial step in developing HCS (high content screening) and HTS (high throughput screening) assays to search for modulators of blood vessel formation. HUVECs (human umbilical‐vein endothelial cells) exemplify primary cells used in angiogenesis assays. However, primary cells have significant limitations that include phenotypic decay and/or senescence by six to eight passages in culture, making stable integration of fluorescent markers and large‐scale expansion for HTS problematic. To overcome these limitations for HTS, we developed a novel angiogenic model system that employs stable fluorescent endothelial cell lines based on immortalized HMECs (human microvascular endothelial cell). We then evaluated HMEC cultures, both alone and co‐cultured with an EMC (epicardial mesothelial cell) line that contributes vascular smooth muscle cells, to determine the suitability for HTS or HCS. Results. The endothelial and epicardial lines were engineered to express a panel of nuclear‐ and cytoplasm‐localized fluorescent proteins to be mixed and matched to suit particular experimental goals. HMECs retained their angiogenic potential and stably expressed fluorescent proteins for at least 13 passages after transduction. Within 8 h after plating on Matrigel, the cells migrated and coalesced into networks of vessel‐like structures. If co‐cultured with EMCs, the branches formed cylindrical‐shaped structures of HMECs surrounded by EMC derivatives reminiscent of vessels. Network formation measurements revealed responsiveness to media composition and control compounds. Conclusions. HMEC‐based lines retain most of the angiogenic features of primary endothelial cells and yet possess long‐term stability and ease of culture, making them intriguing candidates for large‐scale primary HCS and HTS (of ～10000–1000000 molecules). Furthermore, inclusion of EMCs demonstrates the feasibility of using epicardial‐derived cells, which normally contribute to smooth muscle, to model large vessel formation. In summary, the immortalized fluorescent HMEC and EMC lines and straightforward culture conditions will enable assay development for HCS of angiogenesis.     

Scutellarin promotes in vitro angiogenesis in human umbilical vein endothelial cells

Angiogenesis is critical to a wide range of physiological and pathological processes. Scutellarin, a major flavonoid of a Chinese herbal medicine Erigeron breviscapus (Vant.) Hand. Mazz. has been shown to offer beneficial effects on cardiovascular and cerebrovascular functions. However, scutellarin’s effects on angiogenesis and underlying mechanisms are not fully elucidated. Here, we studied angiogenic effects of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. Scutellarin was found by MTT assay to induce proliferation of HUVECs. In scutellarin-treated HUVECs, a dramatic increase in migration was measured by wound healing assay; Transwell chamber assay found significantly more invading cells in scutellarin-treated groups. Scutellarin also promoted capillary-like tube formation in HUVECs on Matrigel, and significantly upregulated platelet endothelial cell adhesion molecule-1 at both mRNA and protein levels. Scutellarin’s angiogenic mechanism was investigated in vitro by measuring expression of angiogenic factors associated with cell migration and invasion. Scutellarin strongly induced MMP-2 activation and mRNA expression in cultured HUVECs in a concentration-dependent manner. Taken together, these results suggest that scutellarin promotes angiogenesis and may form a basis for angiogenic therapy.     

Helicobacter pylori-derived Heat shock protein 60 enhances angiogenesis via a CXCR2-mediated signaling pathway

Helicobacter pylori is a potent carcinogen associated with gastric cancer malignancy. Recently, H. pylori Heat shock protein 60 (HpHSP60) has been reported to promote cancer development by inducing chronic inflammation and promoting tumor cell migration. This study demonstrates a role for HpHSP60 in angiogenesis, a necessary precursor to tumor growth. We showed that HpHSP60 enhanced cell migration and tube formation, but not cell proliferation, in human umbilical vein endothelial cells (HUVECs). HpHSP60 also indirectly promoted HUVEC proliferation when HUVECs were co-cultured with supernatants collected from HpHSP60-treated AGS or THP-1 cells. The angiogenic array showed that HpHSP60 dramatically induced THP-1 cells and HUVECs to produce the chemotactic factors IL-8 and GRO. Inhibition of CXCR2, the receptor for IL-8 and GRO, or downstream PLCβ2/Ca2+-mediated signaling, significantly abolished HpHSP60-induced tube formation. In contrast, suppression of MAP K or PI3 K signaling did not affect HpHSP60-mediated tubulogenesis. These data suggest that HpHSP60 enhances angiogenesis via CXCR2/PLCβ2/Ca2+ signal transduction in endothelial cells.     

Different roles of galectin-9 isoforms in modulating E-selectin expression and adhesion function in LoVo colon carcinoma cells

Galectin-9, a member of galectin family, plays multiple roles in a variety of cellular functions, including cell adhesion, aggregation, and apoptosis. Galectin-9 also has three isoforms (named galectin-9L, galectin-9M, and galectin-9S), but whether these isoforms differ in their functions remains poorly understood. In this study, we showed that transient expression of galectin-9L decreased E-selectin levels, while transient expression of galectin-9M or galectin-9S increased E-selectin levels in LoVo cells, which do not express endogenous galectin-9. We also found that over-expression of three galectin-9 isoforms led to increased attachment of LoVo cells to extracellular matrix proteins respectively, while over-expression of galectin-9M or galectin-9S increased the adhesion of LoVo cells to human umbilical vein endothelial cells in vitro. In summary, these findings indicate that different isoforms of galectin-9 exhibit distinct biological functions.     

Annexin A2 Is a Molecular Target for TM601, a Peptide with Tumor-targeting and Anti-angiogenic Effects

TM601 is a synthetic form of chlorotoxin, a 36-amino acid peptide derived from the venom of the Israeli scorpion, Leirius quinquestriatus, initially found to specifically bind and inhibit the migration of glioma cells in culture. Subsequent studies demonstrated specific in vitro binding to additional tumor cell lines. Recently, we demonstrated that proliferating human vascular endothelial cells are the only normal cell line tested that exhibits specific binding to TM601. Here, we identify annexin A2 as a novel binding partner for TM601 in multiple human tumor cell lines and human umbilical vein endothelial cell (HUVEC). We demonstrate that the surface binding of TM601 to the pancreatic tumor cell line Panc-1 is dependent on the expression of annexin A2. Identification of annexin A2 as a binding partner for TM601 is also consistent with the anti-angiogenic effects of TM601. Annexin A2 functions in angiogenesis by binding to tissue plasminogen activator and regulating plasminogen activation on vascular endothelial cells. We demonstrate that in HUVECs, TM601 inhibits both vascular endothelial growth factor- and basic fibroblast growth factor-induced tissue plasminogen activator activation, which is required for activation of plasminogen to plasmin. Consistent with inhibition of cell surface protease activity, TM601 also inhibits platelet-derived growth factor-C induced trans-well migration of both HUVEC and U373-MG glioma cells.     

Sphingosine 1-phosphate and cell migration: resistance to angiogenesis inhibitors

Naturally occurring angiogenesis inhibitors can inhibit different steps of the angiogenic process, such as endothelial cell migration. However, the mechanisms underlying this inhibition have not been elucidated. We demonstrate that migration of human umbilical vein endothelial cells induced by the potent endothelial cell chemoattractant sphingosine 1-phosphate is refractory to inhibition by well-characterized angiogenesis inhibitors such as endostatin and plasminogen-related protein-B. Our data support the contention that for effective blockage of tumor-induced angiogenesis, antagonists of both G protein-coupled receptor signaling and receptor tyrosine kinase signaling must be combined.     

Matrix Metalloproteinase-13 (MMP-13) Directly and Indirectly Promotes Tumor Angiogenesis

Matrix metalloproteinases (MMPs) are extracellular zinc-dependent endopeptidases involved in the degradation and remodeling of extracellular matrix in physiological and pathological processes. MMPs also have a role in cell proliferation, migration, differentiation, angiogenesis, and apoptosis. We previously identified cancer invasion-related factors by comparing the gene expression profiles between parent and the highly invasive clone of cancer cells. Matrix metalloproteinase-13 (MMP-13) was identified as a common up-regulated gene by cancer invasion-related factors. Although MMP-13 slightly promoted tumor invasion, we found that MMP-13 was involved in tumor angiogenesis. Conditioned medium from MMP-13-overexpressing cells promoted capillary formation of immortalized human umbilical vein endothelial cells. Furthermore, treatment with recombinant MMP-13 protein enhanced capillary tube formation both in vitro and in vivo. MMP-13-promoted capillary tube formation was mediated by activation of focal adhesion kinase and ERK. Interestingly, MMP-13 promoted the secretion of VEGF-A from fibroblasts and endothelial cells. By immunohistochemical analysis, we found a possible correlation between MMP-13 expression and the number of blood vessels in human cancer cases. In summary, these findings suggest that MMP-13 may directly and indirectly promote tumor angiogenesis.     

Phenyl and Diaryl Ureas with Thiazolo[5,4‐d]pyrimidine Scaffold as Angiogenesis Inhibitors: Design,Synthesis and Biological Evaluation

Angiogenesis is crucial for tumor growth and inhibition of angiogenesis has been regarded as a promising approach for cancer therapy. Vascular endothelial growth factor receptor‐2 (VEGFR‐2) is an important factor in angiogenesis. In this work, a novel series of thiazolo[5,4‐d]pyrimidine derivatives inhibiting angiogenesis were rationally designed and synthesized. Their inhibitory activities against human umbilical vein endothelial cells (HUVEC) were investigated in vitro. 1‐(4‐Fluorophenyl)‐3‐{4‐[(5‐methyl‐2‐phenyl[1,3]thiazolo[5,4‐d]pyrimidin‐7‐yl)amino]phenyl}urea ( 19b ) and 1‐(3‐Fluorophenyl)‐3‐{4‐[(5‐methyl‐2‐phenyl[1,3]thiazolo[5,4‐d]pyrimidin‐7‐yl)amino]phenyl}urea ( 19g ) exhibited the most potent inhibitory effect on HUVEC proliferation (IC₅₀=12.8 and 5.3 μm , respectively). Compound 19g could inhibit the migration of human umbilical vein endothelial cells. These results support the further investigation of these compounds as potent anticancer agents.     

NSK-01105, a Novel Sorafenib Derivative,Inhibits Human Prostate Tumor Growth via Suppression of VEGFR2/EGFR-Mediated Angiogenesis

The purpose of this study is to investigate the anti-angiogenic activities of NSK-01105, a novel sorafenib derivative, in in vitro, ex vivo and in vivo models, and explore the potential mechanisms. NSK-01105 significantly inhibited vascular endothelial growth factor (VEGF)-induced migration and tube formation of human umbilical vein endothelial cells at non-cytotoxic concentrations as shown by wound-healing, transwell migration and endothelial cell tube formation assays, respectively. Cell viability and invasion of LNCaP and PC-3 cells were significantly inhibited by cytotoxicity assay and matrigel invasion assay. Furthermore, NSK-01105 also inhibited ex vivo angiogenesis in matrigel plug assay. Western blot analysis showed that NSK-01105 down-regulated VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2) and the activation of epidermal growth factor receptor (EGFR). Tumor volumes were significantly reduced by NSK-01105 at 60 mg/kg/day in both xenograft models. Immunohistochemical staining demonstrated a close association between inhibition of tumor growth and neovascularization. Collectively, our results suggest a role of NSK-01105 in treatment for human prostate tumors, and one of the potential mechanisms may be attributed to anti-angiogenic activities.     

3-O-Acetyloleanolic acid exhibits anti-angiogenic effects and induces apoptosis in human umbilical vein endothelial cells

3-O-Acetyloleanolic acid, a pentacyclic triterpenoid isolated from cowpea seeds, inhibited proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. HUVECs. The induced apoptosis was characterized by detection of cell surface annexin V and sub-G1 populations. The number of cells immunostained with annexin V-fluorescein isothiocyanate increased after treatment with 3-O-acetyloleanolic acid. The sub-G1 cell populations were also increased in treated HUVECs. 3-O-Acetyloleanolic acid induced activation of caspase 3, a critical mediator of apoptosis signaling. It also significantly inhibited angiogenesis in an in vivo Matrigel plug assay. 3-O-Acetyloleanolic acid thus exhibits anti-angiogenic effects and induces apoptosis in HUVECs and the results suggest that it has a potential use for suppression of the tumor growth stimulated by angiogenesis.     

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.