Formation Of Capillary-like Structures Research Articles

A novel monoclonal antibody targeting a novel epitope of VE‑cadherin inhibits vasculogenic mimicry of lung cancer cells.

Vascular endothelial cadherin (VE‑cadherin) was first found in vascular endothelial cells to maintain normal vascular structures and regulate endothelial cell permeability by homology adhesion. New evidence indicates that certain invasive tumor cells also express VE‑cadherin, which is involved in vasculogenic mimicry to provide a blood supply required for tumor growth and metastasis. EC1 and EC3 domains of VE‑cadherin were reported to be important for intercellular homology adhesion. In the present study, a monoclonal antibody specific to the outer-membrane immunoglobulin-like domains of VE‑cadherin was generated and the binding epitope was identified as peptide LDREVVPWYNLTVEA in the EC4 domain. This antibody inhibited proliferation and capillary-like structure formation of lung cancer Glc‑82 cells in 3D Matrigel culture invitro. This effect was mediated by the inhibition of AKT phosphorylation. Our results suggested that the EC4 domain participates in VE‑cadherin clustering and the antibody targeting the EC4 domain of VE‑cadherin may be a promising anti‑vasculogenic mimicry agent for cancer treatment.

Protective effects of human induced pluripotent stem cell-derived exosomes on high glucose-induced injury in human endothelial cells.

Exosomes are a family of extracellular vesicles that are secreted from almost all types of cells and are associated with cell-to-cell communication. The present study was performed to investigate the effects of human induced pluripotent stem cell-derived exosomes (hiPSC-exo) on cell viability, capillary-like structure formation and senescence in endothelial cells exposed to high glucose. Exosomes were isolated from the conditional medium of hiPSCs and confirmed by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis using Alix and cluster of differentiation-63 as markers. hiPSC-exo were labeled with PKH26 for tracking, and it was determined that spherical exosomes, with a typical cup-shape, were absorbed by human umbilical vascular endothelial cells (HUVECs). Cultured HUVECs were treated with high glucose (33 mM) with or without hiPSC-exo (20 µg/ml) for 48 h, and cell viability, capillary tube formation and senescence were assessed. When exposed to high glucose, viability and tube formation in HUVECs was significantly reduced (P<0.0001), whereas the proportion of senescent cells was higher compared with that in control HUVECs (P<0.0001). Furthermore, hiPSC-exo restored cell viability and capillary-like structure formation, and reduced senescence in HUVECs exposed to high glucose (P<0.0001). However, hiPSC-exo had minimal effects on normal HUVECs. These findings suggest that stem cell-derived exosomes are able to promote cell proliferation, enhance capillary-like structure formation and reduce senescence in endothelial cells exposed to high glucose.

Functionalized thermosensitive hydrogel combined with tendon stem/progenitor cells as injectable cell delivery carrier for tendon tissue engineering

Thermosensitive hydrogels have been studied for potential application as promising alternative cell carriers in cell-based regenerative therapies. In this study, a thermosensitive butane diisocyanate (BDI)-collagen hydrogel (BC hydrogel) was designed as an injectable cell delivery carrier of tendon stem/progenitor cells (TSPCs) for tendon tissue engineering. We functionalized the BDI hydrogel with the addition of 20% (v/v) collagen I gel to obtain the thermosensitive BC hydrogel, which was then seeded with TSPCs derived from human Achilles tendons. The BC hydrogel compatibility and TSPC behavior and molecular response to the 3D hydrogel were investigated. Collagen (COL) I gel served as a control group. Our findings demonstrated that the BC hydrogel was thermosensitive, and hardened above 25 °C. It supported TSPC survival, proliferation, and metabolic activity with satisfactory dimension stability and biocompatibility, as revealed by gel contraction assay, live/dead staining, DNA quantification, and resazurin metabolic assay. Phalloidin-based visualization of F-actin demonstrated that the TSPCs were stretched within COL I gel with classical spindle cell shapes; similar cell morphologies were also found in the BC hydrogel. The gene expression profile of TSPCs in the BC hydrogel was comparable with that in COL I gel. Moreover, the BC hydrogel supported capillary-like structure formation by human umbilical vein endothelial cells (HUVECs) in the hydrogel matrix. Taken together, these results suggest that the thermosensitive BC hydrogel holds great potential as an injectable cell delivery carrier of TSPCs for tendon tissue engineering.

Efficacy of combined use of plasmid constructs containing HGF and angiopoietin-1 genes to restore blood flow in ischemic tissues

New methods to stimulate blood supply of the ischemic organs and tissues are being intensively developed worldwide. These approaches are based on revascularization and remodeling of the newly formed blood vessels. This strategy was called therapeutic angiogenesis. Using in vitro, ex vivo and in vivo models we investigated the specific biological activity and angiogenic potential of Vascopoietin, which contained the plasmids for HGF and angiopoietin-1 expression. Vascopoietin stimulated vascular cell migration, proliferation and the formation of capillary-like structures in vitro and ex vivo. Using in vivo model of posterior limb ischemia in mice we demonstrated that Vascopoietin administration mediated stable HGF and angiopoietin-1 production resulting in new blood vessel formation and their stabilization in the ischemic muscles. In addition, Vascopoietin injection led to the restoration of the blood flow, decrease in the size of necrosis in ischemic limb and the reduction in the amputation frequency. The current data suggest Vascopoietin a promising drug for therapeutic angiogenesis.

Isomangiferin, a Novel Potent Vascular Endothelial Growth Factor Receptor 2 Kinase Inhibitor, Suppresses Breast Cancer Growth, Metastasis and Angiogenesis.

PurposeVascular endothelial growth factor (VEGF) signal transduction mainly depends on its binding to VEGF receptor 2 (VEGFR-2). VEGF downstream signaling proteins mediate several of its effects in cancer progression, including those on tumor growth, metastasis, and blood vessel formation. The activation of VEGFR-2 signaling is a hallmark of and is considered a therapeutic target for breast cancer. Here, we report a study of the regulation of the VEGFR-2 signaling pathway by a small molecule, isomangiferin.MethodsA human breast cancer xenograft mouse model was used to investigate the efficacy of isomangiferin in vivo. The inhibitory effect of isomangiferin on breast cancer cells and the underlying mechanism were examined in vitro.ResultsIsomangiferin suppressed tumor growth in xenografts. In vitro, isomangiferin treatment inhibited cancer cell proliferation, migration, invasion, and adhesion. The effect of isomangiferin on breast cancer growth was well coordinated with its suppression of angiogenesis. A rat aortic ring assay revealed that isomangiferin significantly inhibited blood vessel formation during VEGF-induced microvessel sprouting. Furthermore, isomangiferin treatment inhibited VEGF-induced proliferation of human umbilical vein endothelial cells and the formation of capillary-like structures. Mechanistically, isomangiferin induced caspase-dependent apoptosis of breast cancer cells. Furthermore, VEGF-induced activation of the VEGFR-2 kinase pathway was down-regulated by isomangiferin.ConclusionOur findings demonstrate that isomangiferin exerts anti-breast cancer effects via the functional inhibition of VEGFR-2. Pharmaceutically targeting VEGFR-2 by isomangiferin could be an effective therapeutic strategy for breast cancer.

PHD-2 Suppression in Mesenchymal Stromal Cells Enhances Wound Healing.

Cell therapy with mesenchymal stromal cells is a promising strategy for tissue repair. Restoration of blood flow to ischemic tissues is a key step in wound repair, and mesenchymal stromal cells have been shown to be proangiogenic. Angiogenesis is critically regulated by the hypoxia-inducible factor (HIF) superfamily, consisting of transcription factors targeted for degradation by prolyl hydroxylase domain (PHD)-2. The aim of this study was to enhance the proangiogenic capability of mesenchymal stromal cells and to use these modified cells to promote wound healing. Mesenchymal stromal cells harvested from mouse bone marrow were transduced with short hairpin RNA (shRNA) against PHD-2; control cells were transduced with scrambled shRNA (shScramble) construct. Gene expression quantification, human umbilical vein endothelial cell tube formation assays, and wound healing assays were used to assess the effect of PHD knockdown mesenchymal stromal cells on wound healing dynamics. PHD-2 knockdown mesenchymal stromal cells overexpressed HIF-1α and multiple angiogenic factors compared to control (p < 0.05). Human umbilical vein endothelial cells treated with conditioned medium from PHD-2 knockdown mesenchymal stromal cells exhibited increased formation of capillary-like structures and enhanced migration compared with human umbilical vein endothelial cells treated with conditioned medium from shScramble-transduced mesenchymal stromal cells (p < 0.05). Wounds treated with PHD-2 knockdown mesenchymal stromal cells healed at a significantly accelerated rate compared with wounds treated with shScramble mesenchymal stromal cells (p < 0.05). Histologic studies revealed increased blood vessel density and increased cellularity in the wounds treated with PHD-2 knockdown mesenchymal stromal cells (p < 0.05). Silencing PHD-2 in mesenchymal stromal cells augments their proangiogenic potential in wound healing therapy. This effect appears to be mediated by overexpression of HIF family transcription factors and up-regulation of multiple downstream angiogenic factors.

Natural Killer Cells from Malignant Pleural Effusion Are Endowed with a Decidual-Like Proangiogenic Polarization.

Natural killer (NK) cells are crucial in tumor recognition and eradication, but their activity is impaired in cancer patients, becoming poorly cytotoxic. A particular type of NK cells, from the decidua, has low cytotoxicity and shows proangiogenic functions. We investigated whether NK cells from peripheral blood (PB) and pleural effusions of patients develop decidual-like NK phenotype and whether exposure to IL-2 can restore their killing ability in the presence of pleural fluids. NK cells from pleural effusion of patients with inflammatory conditions (iPE, n = 18), primary tumor (ptPE, n = 18), and metastatic tumor (tmPE, n = 27) acquired the CD56brightCD16− phenotype. NK cells from both ptPE and tmPE showed increased expression for the CD49a and CD69 decidual-like (dNK) markers and decreased levels of the CD57 maturation marker. NK from all the PE analyzed showed impaired degranulation capability and reduced perforin release. PE-NK cells efficiently responded to IL-2 stimulation in vitro. Addition of TGFβ or cell-free pleural fluid to IL-2 in the culture medium abrogated NK cell CD107a and IFNγ expression even in healthy donors (n = 14) NK. We found that tmPE-NK cells produce VEGF and support the formation of capillary-like structures in endothelial cells. Our results suggest that the PE tumor microenvironment can shape NK cell polarization towards a low cytotoxic, decidual-like, highly proangiogenic phenotype and that IL-2 treatment is not sufficient to limit this process.

Constitutive expression of the active fragment of human vasostatin Vs30 in Pichia pastoris SMD1168H

Vasostatin 30 (Vs30) is an active fragment derived from the N-terminal region (135–164 aa) of human calreticulin and has the ability to inhibit angiogenesis. In this work, the expression of Vs30 was performed using a protease-deficient strain of the methylotrophic yeast Pichia pastoris. The vs30 gene was optimized for P. pastoris preferential codon usage and inserted into constitutive expression vector pGAPZαA. In addition, a plasmid with four copies of the expression cassette was obtained and transformed into P. pastoris. The flask fermentation conditions were: culture volume of 25 mL in 250 mL baffled flasks at 28 °C, pH 6 and harvest time of 48 h. Up to 21.07 mg/L Vs30 were attained and purified by ultrafiltration with a 30-kDa cut-off membrane and the recovery was 49.7%. Bioactivity of Vs30 was confirmed by the inhibition of cell proliferation, as well as the inhibition of the capillary-like structures formation of EA.hy926 cells in vitro. This work constitutes the first report on the expression of Vs30 in Pichia pastoris using a constitutive promoter and multi-copy approach such as strategies to improve the recombinant Vs30 expression.

Triptonide potently suppresses pancreatic cancer cell-mediated vasculogenic mimicry by inhibiting expression of VE-cadherin and chemokine ligand 2 genes

Various aggressive cancers, including pancreatic cancer, produce functional blood vessels by neovascularization. Tumor vasculogenic mimicry (VM) promotes cancer progression and is closely associated with the poor prognosis of the cancer patients. Therefore, tumor VM is a sensible target for novel anti-cancer drug discovery. However, there is a lack of effective anti-tumor VM drugs in the clinical setting. In this study, we aim to explore novel agents to effectually inhibit pancreatic cancer cell-mediated tumor VM for anti-cancer therapy. Pancreatic cancer cell lines Patu8988 and Panc1 were utilized as a model. A mouse model was used for in vitro capillary-like structure formation and in vivo Matrigel plug assays to evaluate the anti-tumor VM efficacy of a small molecule triptonide from traditional Chinese herbs. Various methods, including RT-PCR, immunohistochemical staining, and the luciferase gene transcription reporter system, were applied to study the mechanisms of triptonide-exerted anti-tumor VM. Triptonide effectively inhibited pancreatic cancer cell-formed capillary-like structures in vitro and blood vessels in vivo through suppressing pancreatic cancer cell migration, invasion, and VM via inhibiting expression of tumor VM master gene VE-cadherin and pro-migratory gene chemokine C-X-C motif ligand 2 (CXCL2), mainly via reduction of gene promoter activity. Triptonide potently suppresses pancreatic cancer cell-mediated VM by reducing tumor cell migration and invasion and inhibiting expression of VE-cadherin and CXCL2 genes. Our results provide a novel and potent anti-tumor VM drug candidate for further development of effective anti-pancreatic cancer therapy.

Development of Endothelial Cell Networks in 3D Tissues by Combination of Melt Electrospinning Writing with Cell-Accumulation Technology.

A remaining challenge in tissue engineering approaches is the in vitro vascularization of engineered constructs or tissues. Current approaches in engineered vascularized constructs are often limited in the control of initial vascular network geometry, which is crucial to ensure full functionality of these constructs with regard to cell survival, metabolic activity, and potential differentiation ability. Herein, the combination of 3D-printed poly-ε-caprolactone scaffolds via melt electrospinning writing with the cell-accumulation technique to enable the formation and control of capillary-like network structures is reported. The cell-accumulation technique is already proven itself to be a powerful tool in obtaining thick (50 µm) tissues and its main advantage is the rapid production of tissues and its ease of performance. However, the applied combination yields tissue thicknesses that are doubled, which is of outstanding importance for an improved handling of the scaffolds and the generation of clinically relevant sample volumes. Moreover, a correlation of increasing vascular endothelial growth factor secretion to hypoxic conditions with increasing pore sizes and an assessment of the formation of neovascular like structures are included.

Mechanotransduction-modulated fibrotic microniches reveal the contribution of angiogenesis in liver fibrosis.

The role of pathological angiogenesis on liver fibrogenesis is still unknown. Here, we developed fibrotic microniches (FμNs) that recapitulate the interaction of liver sinusoid endothelial cells (LSECs) and hepatic stellate cells (HSCs). We investigated how the mechanical properties of their substrates affect the formation of capillary-like structures and how they relate to the progression of angiogenesis during liver fibrosis. Differences in cell response in the FμNs were synonymous of the early and late stages of liver fibrosis. The stiffness of the early-stage FμNs was significantly elevated due to condensation of collagen fibrils induced by angiogenesis, and led to activation of HSCs by LSECs. We utilized these FμNs to understand the response to anti-angiogenic drugs, and it was evident that these drugs were effective only for early-stage liver fibrosis invitro and in an invivo mouse model of liver fibrosis. Late-stage liver fibrosis was not reversed following treatment with anti-angiogenic drugs but rather with inhibitors of collagen condensation. Our work reveals stage-specific angiogenesis-induced liver fibrogenesis via a previously unrevealed mechanotransduction mechanism which may offer precise intervention strategies targeting stage-specific disease progression.

Three-Dimensional Printing and Angiogenesis: Tailored Agarose-Type I Collagen Blends Comprise Three-Dimensional Printability and Angiogenesis Potential for Tissue-Engineered Substitutes.

Three-dimensional (3D) bioprinting is a promising technology for manufacturing cell-laden tissue-engineered constructs. Larger tissue substitutes, however, require a vascularized network to ensure nutrition supply. Therefore, tailored bioinks combining 3D printability and cell-induced vascularization are needed. We hypothesize that tailored hydrogel blends made of agarose-type I collagen and agarose-fibrinogen are 3D printable and will allow the formation of capillary-like structures by human umbilical vein endothelial cells and human dermal fibroblasts. Samples were casted, incubated for 14 days, and analyzed by immunohistology and two-photon laser scanning microscopy. The 3D printability of the hydrogel blends was examined using a drop-on-demand printing system. The rheological behavior was also investigated. Substantial capillary network formation was observed in agarose-type I collagen hydrogel blends with concentrations of 0.2% or 0.5% collagen and 0.5% agarose. Furthermore, storage moduli of agarose-collagen blends were significantly increased compared to those of the corresponding single components (448 Pa for 0.5% agarose, 148 Pa for 0.5% collagen, and 1551 Pa for 0.5% agarose-0.5% collagen). Neither the addition of collagen nor fibrinogen significantly impaired the printing resolution. In conclusion, we present a tailored hydrogel blend that can be printed in 3D and in parallel exhibits cell-induced vascularization capability.

CD106 is a novel mediator of bone marrow mesenchymal stem cells via NF-\u03baB in the bone marrow failure of acquired aplastic anemia

BackgroundAcquired aplastic anemia (AA) is characterized by deficiency or dysfunction of the bone marrow (BM) microenvironment. However, little is known about the impairment of BM-derived mesenchymal stem cells (MSCs) in AA patients.MethodsWe used Illumina HiSeqTM 2000 sequencing, quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry (FCM), and Western blotting to test the expression of CD106 gene (vascular cell adhesion molecule 1 (VCAM1)) and CD106 protein of BM-MSCs. Furthermore, we used hematoxylin and eosin (H&E) and histochemical staining analysis, immunofluorescence, and the formation of capillary-like structures to analyze capillary tube-like formation in vitro; we also used the Matrigel plug assay to test in vivo vasculogenesis, and an assay of colony forming units (CFUs) and colony-forming unit-megakaryocyte (CFU-MK) to detect the support function of MSCs in vitro. The in vivo engraftment of CD34+ cells and MSCs in NOD/SCID mice was tested by FACS and survival assay; the expression of NF-κB was tested by NanoPro analysis and immunofluorescence. NF-κB-regulated CD106 gene (VCAM1) was confirmed by tumor necrosis factor alpha (TNF-α)-stimulated and lipopolysaccharide (LPS)-stimulated MSCs, blockade assay, and immunofluorescence.ResultsHere, we report that BM-MSCs from AA patients exhibited downregulation of the CD06 gene (VCAM1) and low expression of CD106 in vitro. Further analysis revealed that CD106+ MSCs from both AA patients and healthy controls had increased potential for in vitro capillary tube-like formation and in vivo vasculogenesis compared with CD106– MSCs, and the results were similar when healthy MSCs were compared with AA MSCs. CD106+ MSCs from both AA patients and healthy controls more strongly supported in vitro growth and in vivo engraftment of CD34+ cells in NOD/SCID mice than CD106– MSCs, and similar results were obtained when healthy MSCs and AA MSCs were compared. The expression of NF-κB was decreased in AA MSCs, and NF-κB regulated the CD106 gene (VCAM1) which supported hematopoiesis.ConclusionsThese results revealed the effect of CD106 and NF-κB in BM failure of AA.

Lysophosphatidic acid-triggered pathways promote the acquisition of trophoblast endovascular phenotype in vitro.

Successful implantation and placentation requires that extravillous cytotrophoblast acquires an endovascular phenotype and remodels uterine spiral arteries. Defects in this mechanism correlate with severe obstetric complications as implantation failure and preeclampsia. Lysophosphatidic acid (LPA) participates in embryo implantation and contributes to vascular physiology in different biological systems. However, the role of LPA on trophoblast endovascular transformation has not been studied. Due to difficulties in studying human pregnancy in vivo, we adopted a pharmacological approach in vitro to investigate LPA action in various aspects of trophoblast endovascular response, such as the formation of endothelial capillary-like structures, migration, and proliferation. The HTR-8/SVneo cell line established from human first trimester cytotrophoblast was used to model the acquisition of the endovascular phenotype by the invading trophoblast. LPA increased HTR-8/SVneo tube formation, migration (wound healing assay and phalloidin staining) and proliferation (MTT assay). LPA G protein-coupled receptors, LPA1 and LPA3 , were expressed in HTR-8/SVneo. By using selective antagonists, we showed that enhanced tubulogenesis was mediated by LPA3 . In addition, cyclooxygenase-2 and inducible nitric oxide synthase pathways participated in LPA-stimulated tubulogenesis. Inducible nitric oxide synthase was activated downstream cyclooxygenase-2. Furthermore, prostaglandin E2 and a nitric oxide donor (SNAP) increased trophoblast tube formation in a concentration-dependent manner. Finally, we observed that cyclooxygenase-2 and inducible nitric oxide synthase were localized in the nucleus, and LPA did not modify their cellular distribution. Our results show that LPA-triggered regulatory pathways promote trophoblast endovascular response in vitro, suggesting a new role for LPA during spiral artery remodeling at the maternal-fetal interface.

Targeting STAT3 by HO3867 induces apoptosis in ovarian clear cell carcinoma.

Advanced ovarian clear cell carcinoma (OCCC) carries a very poor prognosis in large part secondary to the extremely high rate of resistance to standard platinum and taxane chemotherapy. Signal transducer and activator of transcription 3(STAT3) expression and activation has been shown to regulate tumor progression in various human cancers, though has not been well studied in OCCC. Preliminary work in our lab has demonstrated constitutive activation of STAT3 (pSTAT3Tyr705 or pSTAT3727) in OCCC cell lines as well as human OCCC tumor tissue samples. Significantly, pSTAT3 is expressed in the absence of other forms of activated STAT (pSTAT1, 2, 6). Therefore, this work was planned to investigate the role of STAT3 and examine the efficacy of a novel anti-cancer compound -HO-3867, which is an inhibitor of STAT3, using known OCCC cell lines. Results demonstrate that treatment with HO-3867 decreased expression of pSTAT3 Tyr705 as well pSTAT3 Ser727, while total STAT3 remained constant. STAT3 overexpression increased the migration capability in OVTOKO cells in vitro and led to an increased tumor size when injected in vivo. The inhibitory effect of HO-3867 on cell proliferation and cell survival was accompanied by increased apoptosis, within 24 h post treatment. Treatment with HO-3867 resulted in a decrease in Bcl-2 and increase of cleavage of caspase 3, caspase 7, and PARP, confirming induction of apoptosis after treatment with HO-3867. In addition, HO-3867 significantly inhibited formation of human umbilical vein endothelial cells capillary-like structures and invasion at both 5 and 10 µM concentrations. STAT3 expression plays an important role in the spread of OCCC in vitro as well as in vivo. Thus, we can exploit the STAT3 pathway for targeted drug therapy. Inhibition of pSTAT3 using HO-3867in OCCC cell lines appears to be a promising therapy. This is of utmost importance given the poor response of OCCC to standard chemotherapy regimens.

Abstract 790: Regulation of tumor angiogenesis by low dose Aspirin

Abstract Background and Objective: Tumor angiogenesis is a pathophysiological process in which new blood vessels are formed in the primary tumor site or distant organs for the nourishment of cancer cells and metastatic growth. Thereby, targeting tumor angiogenesis is an important area of research for cancer therapy. Abnormal structure of blood vessels (i.e. leakiness due to abnormal lining of pericytes on the microvessels) is one of the critical features of tumor angiogenesis that sensitizes vascular cells to cytokines and helps circulating tumor cells to metastasize to distant organs. Our long term goal is to repurpose the drugs that may prevent tumor angiogenesis or normalize the vessels by repairing leakiness via recruiting pericytes or both. Our recent studies found that Aspirin (ASA) has the potency to inhibit breast cancer growth and metastasis, as well as reprogram the mesenchymal to epithelial transition (MET). Given the importance of ASA, we tested whether ASA may be able to regulate tumor angiogenesis. Methods: To do so, we determined the effect of low dose ASA (1mM, which is equivalent to 80mg human dose), ASA-treated (2.5mM) conditioned media (231-CMASA) or vehicle-treated conditioned media (231-CMVT) of MDA-MB-231 cells on different endothelial cell physiology. These include endothelial cells’ migration towards serum using 3D modified Boyden chamber assay, in vitro capillary-like structure formation on Matrigel, cell permeability using in vitro endothelial permeability assay and interactions of pericytes-endothelial cells. We also determined the effect of ASA on various angiogenic factors associated with tumor angiogenesis. Finally, we determined the effect of ASA on in vivo tumor angiogenesis using in vivo Angiogenesis Assay (Trevigen) Results and Conclusions: We found that 231-CMASA significantly blocks in vitro migration, the formation of in vitro capillary-like structures parallel with leakiness via incomplete interaction of pericytes and endothelial cells as compared to 231-CMASA. The antiangiogenic effect of ASA was also documented in in vivo assays. Mechanistically, ASA treatment blocks several angiogenic factors including VEGF-A that are associated with these three events, implicating a low dose of Aspirin is potentially therapeutic for breast cancer via blocking and normalizing tumor angiogenesis. Citation Format: Jinia Chakraborty, Gargi Maity, Snigdha Banerjee, Sushanta K. Banerjee. Regulation of tumor angiogenesis by low dose Aspirin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 790. doi:10.1158/1538-7445.AM2017-790

The involvement of autophagy in melanoma vasculogenic mimicry

Introduction. Autophagy, a catabolic process of protein and organelle recycling by transferring defective cytoplasm and organelles into double-membraned vesicles to degrade and regenerate materials, plays a critical role in maintaining energy homeostasis. Autophagy also protects against stress and infection, participates at the development of autoimmune disease. In recent years, the existence of alternative blood circulation system in tumors, vasculogenic mimicry (VM), which can partially compensate the lack of nutrients and oxygen under the hypoxic conditions, has been described. Objective. To elucidate the relationship between autophagy and VM. Materials and methods. In this study we used 2D- and 3D-culturing of melanoma cells derived from surgical species of patients with disseminated melanoma, electrophoresis and western blot, knockdown of the genes by using small interfering RNA (siRNA), flow cytometry, fluorescence microscopy. Results. We detected the basal level autophagy by examining the expression of autophagy-specific protein (LC-3B) by flow cytometry and cellular immunofluorescence staining by monodancylcadaverine. Both assays are the markers of autophagy late stage. Here we show that the level of autophagy in melanoma cells mel P, participated in capillary-like structures (CLS) formation in matrigel, was considerably higher than in mel Me cells which do not involve in VM. To explore the function of autophagy in the ability of melanoma cells to form CLS 3-methyladenine (3-MA) or chloroquine - inhibitors of initiation and terminal stage of autophagy - were used. Both inhibitors reduced the ability of melanoma cells to engage in VM. The data obtained were confirmed by siRNA-mediated gene silencing of BECN1 involved in the initiation of autophagy and ATG5 gene which is considered to be a marker of late stage of autophagy. Knockdown of BECN1 or ATG5 in mel P melanoma cells reduced the level of protein Beclin-1 and Atg5 about 70-75 %, and suppressed CLS formation in matrigel. Melanoma cells with the ATG5 gene knockdown changed the shape but maintained the ability to migrate and recognize each other, the formation of CLS was not observed. Low molecular weight VM inhibitor LCS-1269, significantly reduced the basic level of autophagy. Conclusion. Our data indicate that autophagy participates in CLS formation, and inhibition of autophagy suppresses CLS formation. We suggest that autophagy plays a dual role in the survival and development of tumors: autophagy helps cancer cells against environment stress and provides a temporary survival pathway by promoting energy regeneration, autophagy also promotes VM formation which supplies nutrients and oxygen to less vascularized area of tumor.

Exosomes secreted by cardiomyocytes subjected to ischaemia promote cardiac angiogenesis.

Myocardial infarction (MI) is the leading cause of morbidity and mortality worldwide and results from an obstruction in the blood supply to a region of the heart. In an attempt to replenish oxygen and nutrients to the deprived area, affected cells release signals to promote the development of new vessels and confer protection against MI. However, the mechanisms underlying the growth of new vessels in an ischaemic scenario remain poorly understood. Here, we show that cardiomyocytes subjected to ischaemia release exosomes that elicit an angiogenic response of endothelial cells (ECs). Exosomes secreted by H9c2 myocardial cells and primary cardiomyocytes, cultured either in control or ischaemic conditions were isolated and added to ECs. We show that ischaemic exosomes, in comparison with control exosomes, confer protection against oxidative-induced lesion, promote proliferation, and sprouting of ECs, stimulate the formation of capillary-like structures and strengthen adhesion complexes and barrier properties. Moreover, ischaemic exosomes display higher levels of metalloproteases (MMP) and promote the secretion of MMP by ECs. We demonstrate that miR-222 and miR-143, the relatively most abundant miRs in ischaemic exosomes, partially recapitulate the angiogenic effect of exosomes. Additionally, we show that ischaemic exosomes stimulate the formation of new functional vessels in vivo using in ovo and Matrigel plug assays. Finally, we demonstrate that intramyocardial delivery of ischaemic exosomes improves neovascularization following MI. This study establishes that exosomes secreted by cardiomyocytes under ischaemic conditions promote heart angiogenesis, which may pave the way towards the development of add-on therapies to enhance myocardial blood supply.

Thymosin β4 promotes the survival and angiogenesis of transplanted endothelial progenitor cells in the infarcted myocardium.

The survival of transplanted stem cells in ischemic tissue is poor. In the present study, the effects of thymosin β4 (Tβ4) on the survival and angiogenesis of endothelial progenitor cells (EPCs) and improvement in cardiac functions after transplantation of Tβ4-treated EPCs in the infarcted myocardium were investigated. EPCs were isolated from bone marrow of adult male rats and incubated in Endothelial Basal Medium-2. Then the cells were treated with Tβ4 at different concentrations (0.05, 0.1 and 0.2 µM), and cells incubated with DMEM were set as controls. MTT assay, Transwell assay and tube formation in Matrigel were used to detect cell viability, migration and angiogenesis, respectively. For examining the protective effect of Tβ4 on EPCs, the cells were also incubated in the condition of hypoxia and serum deprivation. p-Akt expression was also examined using western blot analysis. Rat models of myocardial infarction (MI) were established by ligation of the anterior descending branch of the left coronary artery. At four weeks after intramyocardial injection of Tβ4-treated EPCs, the changes in cardiac functions, size of the scar tissue and density of microvessels were examined by echocardiography, Masson's trichrome staining, immunohistochemistry and fluorescence in situ hybridization (FISH) for the Y-chromosome. Tβ4 enhanced EPC viability, protected the cells from apoptosis in hypoxia and serum deprivation, and promoted the proliferation and migration of the cells and formation of capillary-like structures in the cells. Moreover, Tβ4 increased p-Akt expression in the cells. The cytoprotective and proangiogenic effects of Tβ4 were in a dose-dependent manner. Tβ4-treated EPCs improved cardiac function, enhanced the repair of the infarcted myocardium, and promoted angiogenesis after transplantation in the infarcted myocardium. In conclusion, pretreatment of EPCs with Tβ4 is a novel strategy for the repair of ischemic tissue after transplantation in MI.

MicroRNA-27b functions as a new inhibitor of ovarian cancer-mediated vasculogenic mimicry through suppression of VE-cadherin expression.

Aggressive cancer cells gain robust tumor vascular mimicry (VM) capability that promotes tumor growth and metastasis. VE-cadherin is aberrantly overexpressed in vasculogenic cancer cells and regarded as a master gene of tumor VM. Although microRNAs (miRNAs) play an important role in modulating tumor angiogenesis and cancer metastasis, the miRNA that targets VE-cadherin expression in cancer cells to inhibit tumor cell-mediated VM is enigmatic. In this study, we found that miR-27b levels are negatively co-related to VE-cadherin expression in ovarian cancer cells and tumor cell-mediated VM, and demonstrated that miR-27b could bind to the 3′-untranslated region (3′UTR) of VE-cadherin mRNA. Overexpression of miR-27b in aggressive ovarian cancer cell lines Hey1B and ES2 significantly diminished intracellular VE-cadherin expression; convincingly, the inhibitory effect of miR-27b could be reversed by miR-27b specific inhibitor. Intriguingly, miR-27b not only effectively suppressed ovarian cancer cell migration and invasion, but also markedly inhibited formation of ovarian cancer cell-mediated capillary-like structures in vitro and suppressed generation of functional tumor blood vessels in mice. Together, our study suggests that miR-27b functions as a new inhibitor of ovarian cancer cell-mediated VM through suppression of VE-cadherin expression, providing a new potential drug candidate for antitumor VM and anti-ovarian cancer therapy.