Endothelial Cell Capillary Formation Research Articles

Treatment with an Angiopoietin-1 mimetic peptide promotes neurological recovery after stroke in diabetic rats.

AimVasculotide (VT), an angiopoietin‐1 mimetic peptide, exerts neuroprotective effects in type one diabetic (T1DM) rats subjected to ischemic stroke. In this study, we investigated whether delayed VT treatment improves long‐term neurological outcome after stroke in T1DM rats.MethodsMale Wistar rats were induced with T1DM, subjected to middle cerebral artery occlusion (MCAo) model of stroke, and treated with PBS (control), 2 µg/kg VT, 3 µg/kg VT, or 5.5 µg/kg VT. VT treatment was initiated at 24 h after stroke and administered daily (i.p) for 14 days. We evaluated neurological function, lesion volume, vascular and white matter remodeling, and inflammation in the ischemic brain. In vitro, we evaluated the effects of VT on endothelial cell capillary tube formation and inflammatory responses of primary cortical neurons (PCN) and macrophages.ResultsTreatment of T1DM‐stroke with 3 µg/kg VT but not 2 µg/kg or 5.5 µg/kg significantly improves neurological function and decreases infarct volume and cell death compared to control T1DM‐stroke rats. Thus, 3 µg/kg VT dose was employed in all subsequent in vivo analysis. VT treatment significantly increases axon and myelin density, decreases demyelination, decreases white matter injury, increases number of oligodendrocytes, and increases vascular density in the ischemic border zone of T1DM stroke rats. VT treatment significantly decreases MMP9 expression and decreases the number of M1 macrophages in the ischemic brain of T1DM‐stroke rats. In vitro, VT treatment significantly decreases endothelial cell death and decreases MCP‐1, endothelin‐1, and VEGF expression under high glucose (HG) and ischemic conditions and significantly increases capillary tube formation under HG conditions when compared to non‐treated control group. VT treatment significantly decreases inflammatory factor expression such as MMP9 and MCP‐1 in macrophages subjected to LPS activation and significantly decreases IL‐1β and MMP9 expression in PCN subjected to ischemia under HG conditions.ConclusionDelayed VT treatment (24 h after stroke) significantly improves neurological function, promotes vascular and white matter remodeling, and decreases inflammation in the ischemic brain after stroke in T1DM rats.

CD133+Exosome Treatment Improves Cardiac Function after Stroke in Type 2 Diabetic Mice.

Cardiac complications post-stroke are common, and diabetes exacerbates post-stroke cardiac injury. In this study, we tested whether treatment with exosomes harvested from human umbilical cord blood derived CD133+ cells (CD133+Exo) improves cardiac function in type 2 diabetes mellitus (T2DM) stroke mice. Adult (3-4m), male, BKS.Cg-m+/+Leprdb/J (db/db, T2DM) and non-DM (db+) mice were randomized to sham or photothrombotic stroke groups. T2DM-stroke mice were treated with phosphate-buffered saline (PBS) or CD133+Exo (20μg, i.v.) at 3days after stroke. T2DM sham and T2DM+CD133+Exo treatment groups were included as controls. Echocardiography was performed, and mice were sacrificed at 28days after stroke. Cardiomyocyte hypertrophy, myocardial capillary density, interstitial fibrosis, and inflammatory factor expression were measured in the heart. MicroRNA-126 expression and its target gene expression were measured in the heart. T2DM mice exhibit significant cardiac deficits such as decreased left ventricular ejection fraction (LVEF) and shortening fraction (LVSF), increased left ventricular diastolic dimension (LVDD), and reduced heart rate compared to non-DM mice. Stroke in non-DM and T2DM mice significantly decreases LVEF compared to non-DM and T2DM-sham, respectively. Cardiac dysfunction is worse in T2DM-stroke mice compared to non-DM-stroke mice. CD133+Exo treatment of T2DM-stroke mice significantly improves cardiac function identified by increased LVEF and decreased LVDD compared to PBS treated T2DM-stroke mice. In addition, CD133+Exo treatment significantly decreases body weight and blood glucose but does not decrease lesion volume in T2DM-stroke mice. CD133+Exo treatment of T2DM mice significantly decreases body weight and blood glucose but does not improve cardiac function. CD133+Exo treatment in T2DM-stroke mice significantly decreases myocardial cross-sectional area, interstitial fibrosis, transforming growth factor beta (TGF-β), numbers of M1 macrophages, and oxidative stress markers 4-HNE (4-hydroxynonenal) and NADPH oxidase 2 (NOX2) in heart tissue. CD133+Exo treatment increases myocardial capillary density in T2DM-stroke mice as well as upregulates endothelial cell capillary tube formation in vitro. MiR-126 is highly expressed in CD133+Exo compared to exosomes derived from endothelial cells. Compared to PBS treatment, CD133+Exo treatment significantly increases miR-126 expression in the heart and decreases its target gene expression such as Sprouty-related, EVH1 domain-containing protein 1 (Spred-1), vascular cell adhesion protein (VCAM), and monocyte chemoattractant protein 1 (MCP1) in the heart of T2DM-stroke mice. CD133+Exo treatment significantly improves cardiac function in T2DM-stroke mice. The cardio-protective effects of CD133+Exo in T2DM-stroke mice may be attributed at least in part to increasing miR-126 expression and decreasing its target protein expression in the heart, increased myocardial capillary density and decreased cardiac inflammatory factor expression.

Inhibitor of growth 4 affects hypoxia-induced migration and angiogenesis regulation in retinal pigment epithelial cells.

Inhibitor of growth 4 (ING4), a potential tumor suppressor, is implicated in cell migration and angiogenesis. However, its effects on diabetic retinopathy (DR) have not been elucidated. In this study, we aimed to evaluate ING4 expression in normal and diabetic rats and clarify its effects on hypoxia-induced dysfunction in human retinal pigment epithelial (ARPE-19) cells. A Type 1 diabetic model was generated by injecting rats intraperitoneally with streptozotocin and then killed them 4, 8, or 12 weeks later. ING4 expression in retinal tissue was detected using western blot analysis, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and immunohistochemistry assays. After transfection with an ING4 overexpression lentiviral vector or small interfering RNA (siRNA), ARPE-19 migration under hypoxia was tested using wound healing and transwell assays. The angiogenic effect of conditioned medium (CM) from ARPE-19 cells was examined by assessing human retinal endothelial cell (HREC) capillary tube formation. Additionally, western blot analysis and RT-qPCR were performed to investigate the signaling pathways in which ING4, specificity protein 1 (Sp1), matrix metalloproteinase 2 (MMP-2), MMP-9, and vascular endothelial growth factor A (VEGF-A) were involved. Here, we found that ING4 expression was significantly reduced in the diabetic rats' retinal tissue. Silencing ING4 aggravated hypoxia-induced ARPE-19 cell migration. CM collected from ING4 siRNA-transfected ARPE-19 cells under hypoxia promoted HREC angiogenesis. These effects were reversed by ING4 overexpression. Furthermore, ING4 suppressed MMP-2, MMP-9, and VEGF-A expression in an Sp1-dependent manner in hypoxia-conditioned ARPE-19 cells. Overall, our results provide valuable mechanistic insights into the protective effects of ING4 on hypoxia-induced migration and angiogenesis regulation in ARPE-19 cells. Restoring ING4 may be a novel strategy for treating DR.

Abstract 1953: Expression and activity of MT1-MMP in endothelial cells is regulated by Semaphorin 4D

Abstract The semaphorins and plexins, originally shown to control axon growth cone guidance, are now known to influence many aspects of cell adhesion and migration. Our group and others have demonstrated that carcinomas over express Semaphorin 4D (S4D), particularly under conditions of hypoxia, for the purpose of supporting tumor-induced angiogenesis. However, S4D is a transmembrane protein that must be proteolytically cleaved to attract endothelial cells into a malignancy, a process that in head and neck squamous cell carcinoma (HNSCC) is controlled by membrane type 1-matrix metalloproteinase (MT1-MMP). We wanted to test the possibility that S4D acts through its receptor, Plexin-B1 (PB1), to enhance activity of endothelial cell MT1-MMP, facilitating extracellular matrix degradation and further promoting angiogenesis. We observed upregulated transcription, expression and cell surface localization of MT1-MMP in endothelial cells treated with S4D, occurring in a PB1, RhoA and NF-κB-dependent manner. We also demonstrated that S4D-induced endothelial cell collagen degradation, migration and capillary tube formation are suppressed when PB1 is silenced or its downstream effectors RhoA and NF-κB are inhibited. Endothelial MT1-MMP contributes to the ability of these cells to digest their way through the basement membrane and extracellular matrix, a requirement for the creation of the vascular networks that supply a developing tumor with oxygen and nutrients and allow it access to the systemic circulation for metastasis. These results demonstrate that S4D promotes angiogenesis at multiple levels, and suggest that it would be a valid target for anti-angiogenic intervention in the treatment of solid malignancies. Citation Format: Amr Bugshan, Zaid Khoury, Asma Buhamrah, John R. Basile. Expression and activity of MT1-MMP in endothelial cells is regulated by Semaphorin 4D [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1953.

Novel leucine ureido derivatives as aminopeptidase N inhibitors using click chemistry

The over-expression of aminopeptidase N on diverse malignant cells is associated with the tumor angiogenesis and metastasis. In this report, one new series of leucine ureido derivatives containing the triazole moiety was designed, synthesized and evaluated as APN inhibitors. Among them, compound 13v showed the best APN inhibition with an IC50 value of 0.089 ± 0.007 μM, which was two orders of magnitude lower than that of bestatin (IC50 = 9.4 ± 0.5 μM). Compound 13v also showed dose-dependent anti-angiogenesis activities. Even at the lower concentration (10 μM), compound 13v presented similar anti-angiogenesis activity compared with bestatin at 100 μM in both the human umbilical vein endothelial cells (HUVECs) capillary tube formation assay and the rat thoracic aorta rings test. Moreover, compared with bestatin, 13v exhibited comparable, if not better in vivo anti-metastasis activity in a mouse H22 pulmonary metastasis model.

Design, Synthesis, and Biological Evaluation of Pyrazoline-Based Hydroxamic Acid Derivatives as Aminopeptidase N (APN) Inhibitors.

Aminopeptidase N (APN) has been recognized as a target for anticancer treatment due to its overexpression on diverse malignant tumor cells and association with cancer invasion, metastasis and angiogenesis. Herein we describe the synthesis, biological evaluation, and structure-activity relationship study of two new series of pyrazoline analogues as APN inhibitors. Among these compounds, 5-(2-(2-(hydroxyamino)-2-oxoethoxy)phenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide (compound 13 e) showed the best APN inhibition with an IC50 value of 0.16±0.02 μm, which is more than one order of magnitude lower than that of bestatin (IC50 =9.4±0.5 μm). Moreover, compound 13 e was found to inhibit the proliferation of diverse carcinoma cells and to show potent anti-angiogenesis activity. At the same concentration, compound 13 e presents significantly higher anti-angiogenesis activity than bestatin in human umbilical vein endothelial cells (HUVECs) capillary tube formation assays. The putative binding mode of 13 e in the active site of APN is also discussed.

Poly(ethylmethacrylate-co-diethylaminoethyl acrylate) coating improves endothelial re-population, bio-mechanical and anti-thrombogenic properties of decellularized carotid arteries for blood vessel replacement

Decellularized vascular scaffolds are promising materials for vessel replacements. However, despite the natural origin of decellularized vessels, issues such as biomechanical incompatibility, immunogenicity risks and the hazards of thrombus formation, still need to be addressed. In this study, we coated decellularized vessels obtained from porcine carotid arteries with poly (ethylmethacrylate-co-diethylaminoethylacrylate) (8g7) with the purpose of improving endothelial coverage and minimizing platelet attachment while enhancing the mechanical properties of the decellularized vascular scaffolds. The polymer facilitated binding of endothelial cells (ECs) with high affinity and also induced endothelial cell capillary tube formation. In addition, platelets showed reduced adhesion on the polymer under flow conditions. Moreover, the coating of the decellularized arteries improved biomechanical properties by increasing its tensile strength and load. In addition, after 5 days in culture, ECs seeded on the luminal surface of 8g7-coated decellularized arteries showed good regeneration of the endothelium. Overall, this study shows that polymer coating of decellularized vessels provides a new strategy to improve re-endothelialization of vascular grafts, maintaining or enhancing mechanical properties while reducing the risk of thrombogenesis. These results could have potential applications in improving tissue-engineered vascular grafts for cardiovascular therapies with small caliber vessels.

Effects of interleukin-17 on human retinal vascular endothelial cell capillary tube formation invitro.

The present study aimed to investigate the effect of and mechanism underlying interleukin (IL)‑17 on human retinal vascular endothelial cell (HREC) capillary tube formation invitro. The expression of IL‑17 receptor (IL‑17R) in human HRECs was quantified using reverse transcriptase‑polymerase chain reaction (RT‑PCR) and western blot analyses. The roles of IL‑17 in HREC migration and capillary tube formation were detected using a wound scratching assay and three‑dimensional Matrigel assay, respectively, invitro. HREC proliferation was examined using a cell counting kit‑8 assay with administration of serial doses of IL‑17. The effects of IL‑17 on the expression of vascular endothelial growth factor (VEGF), intercellular cell adhesion molecule (ICAM)‑1, IL‑6 and IL‑8 in HRECs were evaluated using RT‑PCR and western blot analyses. The results revealed that the HRECs expressed IL‑17R, and the number of intact capillary tubes formed by HRECs in the presence of IL‑17 was markedly higher, compared with that in the blank control group. The wound scratching assay showed that the numbers of migrated HRECs stimulated with IL‑17 at concentrations of 100 or 500ng/ml were significantly higher, compared with the number in the control group. The RT‑PCR and western blot analyses showed that IL‑17 significantly promoted the expression of VEGF, ICAM‑1, IL‑6 and IL‑8 by the HRECs. The proliferation of HRECs in the presence of IL‑17 was also significantly increased. Therefore, IL‑17 increased HREC capillary tube formation through promoting HREC migration, proliferation, and expression levels of VEGF, ICAM‑1, IL‑6 and IL-8.

Abstract LB-A23: Galectin-8 favors growth of endothelial cells and induces production of extracellular matrix by cancer associated fibroblasts

Abstract Galectins (Gal) belong to a family of endogenous lectins specifically recognizing sugar motives and playing an important role in the biology of tissue repair and cancer. It has been shown that the tandem-repeat lectin Gal-8 plays an essential role in tumor angiogenesis by improving endothelial cell migration and capillary tube formation. Interestingly by a more prominent way than the proangiogenic growth factor VEGF. Thus, new question arises, i.e. does Gal-8 improves endothelial cell proliferation? To provide answer to that question the MTT and BrDu proliferation assays using HUVEC (human umbilical vein endothelial cells) as a model were used. We found that Gal-8 induces cell proliferation in combination with VEGF, but Gal-8 alone does not exhibit any significant effect. Furthermore, we studied whether human Gal-8 induces production of a bioactive extracellular matrix (ECM) scaffold by cancer associated fibroblasts (CAF). In parallel, the effect of Gal-8 on ECM production by human dermal fibroblasts (HDF) was studied as well. Adding Gal-8 to the culture medium led the CAF to generate a very fine structured 3D meshwork especially rich in fibronectin. Contrary, the ECM production by HDF was not that apparent. Since ECM-cell interactions play an important role in the ability of tumors to spread and form metastasis further research focused on roles of the tandem-repeat-type Gal-8 in other steps of tumorigenesis is warranted by present in vitro study. This study was supported in part by the Grant Agency of Ministry of Education, Science, Research, and Sport of the Slovak Republic (VEGA-1/0404/15 and VEGA-1/0048/15) and the Agency for Science and Research under the contract no. APVV-0408-12 and APVV-14-0731. Citation Format: Peter Gal, Lenka Varinska, Martin Kello, Jan Mojzis, Barbora Dvorankova, Karel Smetana Jr.. Galectin-8 favors growth of endothelial cells and induces production of extracellular matrix by cancer associated fibroblasts. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A23.

Abstract P041: Inhibition of MicroRNA-221 by Estradiol Contributes to its Differential Effects on Smooth Muscle Cell Growth and Endothelial Cell Capillary Formation

MicroRNAs play a key role in vascular remodeling associated with cardiovascular disease. MiR-221 actively contributes to injury-induced neointima formation by inhibiting endothelial cell (EC) growth and promoting smooth muscle cell (SMC) growth. Since estradiol (E2) prevents neointimal thickening, we hypothesize that E2 mediates its vasoprotective actions by downregulating miR-221 expression and abrogating its effects on SMC and EC growth. RT-qPCR confirmed that both Human Umbilical Vein ECs (HUVECs) and Human Coronary Artery SMCs (HCASMCs) produce miR-221. Treatment of HCASMCs with PDGF-BB (20ng/ml) induced miR-221 levels from 100±8% to 189±9% (p<.05) and treatment with E2 (100nM) inhibited this to 126±4% (p<.05 vs PDGF). PDGF-BB stimulated DNA synthesis (BrdU incorporation), CyclinD1 expression (Western Blot) and migration (Scratch assay) in HCASMCs and these effects were mimicked by miR-221 (25nM) overexpression; and abrogated in HCASMCs transfected with miR-221 antimiR (25nM). E2 inhibited PDGF-induced HCASMC numbers by 30±4% and these effects were reversed by miR-221 (p<0.05). Inhibitory effects of E2 on PDGF-induced miR-221 production in HCASMCs were mimicked by estrogen receptor (ER) α agonist PPT, but not by ERβ and GPER agonists; and blocked by ERα antagonist MPP, suggesting these effects are ERα mediated. In contrast to SMCs, transfection of HUVECs with miR-221 mimic inhibited capillary formation and wound healing by 39±8% and 27±6%, respectively (p<.05). Neutralization of miR-221 with antimiR induced capillary formation and wound closure by 26±3 % and 51±15%, respectively (p<.05). E2 (10nM) inhibited miR-221 levels in HUVECs from 100 to 73±6% (p<.05). Moreover, transfection of HUVECs with miR-221 mimic inhibited E2-induced capillary formation (from137±9% to 85±13%; p<.05 vs E2) and wound closure (from 125±5% to 82±12%; p<.05 vs E2). Our findings provide the first evidence that E2 inhibits miR-221 production in HCASMCs and HUVECs and these effects contribute to its antimitogenic effects on HCASMCs and capillary promoting effects in HUVECs. Modulation of miR-221 by E2 represents a novel mechanism by which E2 may mediate its differential effects on SMC and EC growth, and confer vascular protection.

DNA Aptamer Assembly as a Vascular Endothelial Growth Factor Receptor Agonist

Controlling receptor-mediated processes in cells is paramount in many research areas. The activity of small molecules and growth factors is difficult to control and can lead to off-target effects through the activation of nonspecific receptors as well as binding affinity to nonspecific cell types. In this study, we report the development of a molecular trigger in the form of a divalent nucleic acid aptamer assembly toward vascular endothelial growth factor receptor-2 (VEGFR2). The assembly binds to VEGFR2 and functions as a receptor agonist with targeted receptor binding, promoting receptor phosphorylation, activation of the downstream Akt pathway, upregulation of endothelial nitric oxide synthase, and endothelial cell capillary tube formation. The agonist action we report makes this aptamer construct a promising strategy to control VEGFR2-mediated cell signaling.

Paxillin regulates vascular endothelial growth factor A-induced in vitro angiogenesis of human umbilical vein endothelial cells.

The purpose of the present study was to investigate the role of paxillin in the vascular endothelial growth factor A (VEGF-A)-induced adhesion, proliferation, migration and capillary formation of endothelial cells (ECs) in vitro. Human umbilical vein ECs (HUVECs) were used to evaluate these four processes in vitro. The HUVECs were either mock-transfected (control), transfected with scramble small interference RNA (siRNA) or transfected with siRNA specifically targeting paxillin. VEGF-A (20 ng/ml) was used to stimulate angiogenesis. The VEGF-A treatment significantly increased the adhesion, proliferation, migration and tube formation of the HUVECs in the control and scramble siRNA groups, whereas the siRNA-mediated knockdown of paxillin inhibited these VEGF-A-induced effects. Paxillin is essential for VEGF-A-mediated angiogenesis in ECs and its inhibition may be a potential target for antiangiogenic therapies.

Four Jointed Box 1 Promotes Angiogenesis and Is Associated with Poor Patient Survival in Colorectal Carcinoma

Angiogenesis, the recruitment and re-configuration of pre-existing vasculature, is essential for tumor growth and metastasis. Increased tumor vascularization often correlates with poor patient outcomes in a broad spectrum of carcinomas. We identified four jointed box 1 (FJX1) as a candidate regulator of tumor angiogenesis in colorectal cancer. FJX1 mRNA and protein are upregulated in human colorectal tumor epithelium as compared with normal epithelium and colorectal adenomas, and high expression of FJX1 is associated with poor patient prognosis. FJX1 mRNA expression in colorectal cancer tissues is significantly correlated with changes in known angiogenesis genes. Augmented expression of FJX1 in colon cancer cells promotes growth of xenografts in athymic mice and is associated with increased tumor cell proliferation and vascularization. Furthermore, FJX1 null mice develop significantly fewer colonic polyps than wild-type littermates after combined dextran sodium sulfate (DSS) and azoxymethane (AOM) treatment. In vitro, conditioned media from FJX1 expressing cells promoted endothelial cell capillary tube formation in a HIF1-α dependent manner. Taken together our results support the conclusion that FJX1 is a novel regulator of tumor progression, due in part, to its effect on tumor vascularization.

Automated Image Analysis of In Vitro Angiogenesis Assay

Angiogenesis is the biological process of generating new capillary blood vessels. It is a fundamental component of a number of normal (reproduction and wound healing) and pathological processes (diabetic retinopathy, rheumatoid arthritis, tumor growth, and metastasis). In vitro angiogenesis assays provide a platform for evaluating the effects of pro- or antiangiogenic compounds. One of the most informative assays is the endothelial cells capillary tube formation assay performed on a biological matrix. This assay is based on quantification of the stimulatory and inhibitory effects of various agents, which is estimated through the measurement of the pseudo-tubules network length. This standard measurement is usually carried out manually by trained operators but requires time, attention, and dedication to achieve a reasonable degree of accuracy. Moreover, the screening is operator dependent. In this article, we propose an automated procedure to evaluate the pseudo-tubule network lengths. We propose a series of image analysis procedures developed using a freely available image analysis software library. More than 800 images from 12 experiments were analyzed automatically and manually, and their results were compared to improve and validate the proposed image analysis procedure. The resulting image analysis software is currently running on a dedicated server, with comparable accuracy to manual measurements. Using this new automated procedure, we are able to treat 540 images, or three complete assays per hour.

Abstract 3038: Epithelial Membrane Protein-2 (EMP2) promotes glioblastoma growth via upregulating invasion and angiogenesis.

Abstract Recent data suggests that EMP2 is a novel oncogene in a number of tumors including breast, ovarian, endometrial and primary central nervous system malignancies. Its expression correlates with poor survival and can serve as a prognostic indicator in many tumors. Glioblastoma multiforme (GBM) is an aggressive brain tumor with a medium survival of 18 months, and we have found that EMP2 is expressed in the majority of GBM tumors but not in healthy brains. In order to therapeutically exploit the expression of EMP2 on these tumors, we have developed recombinant antibodies to this oncogene. In experimental models, use of the anti-EMP2 antibodies showed profound efficacy in reducing GBM tumor load. We further dissected the underlying molecular mechanisms and found that EMP2 accelerated GBM tumor growth through increasing αvβ3 integrin surface expression in GBM cells, activating downstream FAK and Src kinases and promoting cell migration and invasion. Furthermore, EMP2 upregulated vascular endothelial growth factor (VEGF) in several GBM cell lines. This increase appeared to be functionally relevant as conditioned medium from GBM cells with forced overexpression of EMP2 increased human umbilical vein endothelial cell (HUVEC) migration and capillary tube formation, suggesting a role for EMP2 in the regulation of tumor angiogenesis. Our results suggest the importance of this oncogene in the control of the tumor microenvironment and that targeting EMP2 expression may be a novel therapy for GBM. Citation Format: Yu Qin. Epithelial Membrane Protein-2 (EMP2) promotes glioblastoma growth via upregulating invasion and angiogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3038. doi:10.1158/1538-7445.AM2013-3038

Exploitation of in silico potential in prediction, validation and elucidation of mechanism of anti-angiogenesis by novel compounds: comparative correlation between wet lab and in silico data

This study describes in silico validation of the wet lab data from aeroplysinin-1, curcumin and halofuginone using Autodock Tools 4.0. The inhibition patterns of the vascular endothelial cell differentiation and capillary tube formation mediated by anti-angiogenic growth factors from these test compounds were found quite comparable with the in silico results using angiogenic targets (EFGR, bFGF and VEGFR-1). Successful validation of the wet lab results of the selected angiogenic targets by in silico method has led to exploit the hidden potential of in silico tools in preliminary screening of the unknown compounds for anti-angiogenic potential in a cost-effective manner.

Reconstruction of hepatic stellate cell-incorporated liver capillary structures in small hepatocyte tri-culture using microporous membranes

In liver sinusoids, hepatic stellate cells (HSCs) locate the outer surface of microvessels to form a functional unit with endothelia and hepatocytes. To reconstruct functional liver tissue in vitro, formation of the HSC-incorporated sinusoidal structure is essential. We previously demonstrated capillary formation of endothelial cells (ECs) in tri-culture, where a polyethylene terephthalate (PET) microporous membrane was intercalated between the ECs and hepatic organoids composed of small hepatocytes (SHs), i.e. hepatic progenitor cells, and HSCs. However, the high thickness and low porosity of the membranes limited heterotypic cell-cell interactions, which are essential to form HSC-EC hybrid structures. Here, we focused on the effective use of the thin and highly porous poly( d, l-lactide-co-glycolide) (PLGA) microporous membranes in SH-HSC-EC tri-culture to reconstruct the HSC-incorporated liver capillary structures in vitro. First, the formation of EC capillary-like structures was induced on Matrigel-coated PLGA microporous membranes. Next, the membranes were stacked on hepatic organoids composed of small SHs and HSCs. When the pore size and porosity of the membranes were optimized, HSCs selectively migrated to the EC capillary-like structures. This process was mediated in part by platelet-derived growth factor (PDGF) signalling. In addition, the HSCs were located along the outer surface of the EC capillary-like structures with their long cytoplasmic processes. In the HSC-incorporated capillary tissues, SHs acquired high levels of differentiated functions, compared to those without ECs. This model will provide a basis for the construction of functional, thick, vascularized liver tissues in vitro.

Antitumor and Angiostatic Activities of the Antimicrobial Peptide Dermaseptin B2

Recently, we have found that the skin secretions of the Amazonian tree frog Phyllomedusa bicolor contains molecules with antitumor and angiostatic activities and identified one of them as the antimicrobial peptide dermaseptin (Drs) B2. In the present study we further explored the in vitro and in vivo antitumor activity of this molecule and investigated its mechanism of action. We showed that Drs B2 inhibits the proliferation and colony formation of various human tumor cell types, and the proliferation and capillary formation of endothelial cells in vitro. Furthermore, Drs B2 inhibited tumor growth of the human prostate adenocarcinoma cell line PC3 in a xenograft model in vivo. Research on the mechanism of action of Drs B2 on tumor PC3 cells demonstrated a rapid increasing amount of cytosolic lactate dehydrogenase, no activation of caspase-3, and no changes in mitochondrial membrane potential. Confocal microscopy analysis revealed that Drs B2 can interact with the tumor cell surface, aggregate and penetrate the cells. These data together indicate that Drs B2 does not act by apoptosis but possibly by necrosis. In conclusion, Drs B2 could be considered as an interesting and promising pharmacological and therapeutic leader molecule for the treatment of cancer.

Immunomagnetic exclusion of E-cadherin-positive hepatoblasts in fetal mouse liver cell cultures impairs morphogenesis and gene expression of sinusoidal endothelial cells

Previous studies have shown that various cell-cell interactions between hepatoblasts and nonparenchymal cells, including sinusoidal endothelial cells and stellate cells, are indispensable for the development of fetal murine hepatic architecture. The present study was undertaken to determine the effects of hepatoblasts on the sinusoidal structural formation using a culture system of fetal mouse livers. Primitive sinusoidal structures extensively developed in fetal livers, and were composed of LYVE-1- and PECAM-1-positive endothelial cells, desmin-positive stellate cells and F4/80-positive macrophages. When fetal liver cells at 12.5 days of gestation were cultured in vitro, hepatoblasts spread on glass slides and gave rise to hepatocytes on day 5. Desmin-positive stellate cells also spread on the glass slides. PECAM-1-positive endothelial cells became slender and developed into anastomosing capillary networks. When fetal liver cells were cultured without hepatoblasts, which were excluded by an immunomagnetic method using anti-E-cadherin antibodies, endothelial cells had impaired growth and capillary formation. These results demonstrated that capillary formation of endothelial cells was induced by the presence of hepatoblasts. VEGF and the conditioned medium containing humoral factors produced by hepatoblasts/hepatocytes did not induce capillary formation of endothelial cells in cultures of nonparenchymal cells, although they significantly increased the number of endothelial cells on the glass slides. The presence of hepatoblasts also significantly stimulated expression of CD32b mRNA, which is a sinusoidal endothelial marker. Hepatoblasts may work as a positive stimulator of sinusoid morphogenesis and maturation in liver development, in which a signal other than VEGF may play a decisive role, together with VEGF.

Effect of therapeutic targeting of the oncogene EMP2 in triple-negative breast cancer on tumor load.

1052 Background: Understanding tumor induced angiogenesis is a challenging problem with important consequences for the diagnosis and treatment of cancer. In this study we define a novel function for the tetraspan epithelial membrane protein-2 (EMP2) in the control of neoangiogenesis. EMP2 is an oncogene whose expression has been shown to correlate with tumor progression and survival in a number of human cancers including triple negative breast, ovarian, and endometrial tumors. In breast cancer, EMP2 is highly expressed on the majority of invasive tumors examined, and in particular 70% of triple negative breast tumors showed surface expression of this marker. In this study, we examine the control of EMP2 on the tumor microenvironment and further characterize its therapeutic efficacy in breast cancer. Methods: A number of cancer cell lines were utilized both in vitro and in vivo to determine if EMP2 expression levels altered the tumor microenvironment. Cells were tested for their expression of pro-angiogenic proteins HIF-1a and VEGF-A as well as for their ability to induce endothelial cell migration and capillary like tube formation. Cells with modulated EMP2 levels were also tested in vivo for tumor formation and examined for histological changes in the tumor microenvironment. In addition, we recently constructed a fully human EMP2 IgG1 and determined its therapeutic efficacy in a number of triple negative breast cancer cells. Results: In vitro, upregulation of EMP2 promoted VEGF expression through a HIF-1a dependent pathway and resulted in successful capillary-like tube formation. In contrast, reduction of EMP2 correlated with reduced HIF-1a and VEGF expression with the net consequence of poorly vascularized tumors in vivo. Treatment of breast cancer cells with EMP2 IgG1 reduced tumor load with a significant improvement in survival. Conclusions: Clinically, EMP2 has been shown to correlate with poor survival, and these results suggest that this occurs in part through its control on tumor vasculature. In addition, we provide additional evidence on the potent cytotoxic effects of EMP2 treatment in vivo.