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The synthetic compound fasentin has been described as a modulator of GLUT-1 and GLUT-4 transporters, thus inhibiting glucose uptake in some cancer cells. Endothelial glucose metabolism has been recently connected to angiogenesis and it is now an emerging topic in scientific research. Indeed, certain compounds with a known effect on glucose metabolism have also been shown to inhibit angiogenesis. In this work we tested the capability of fasentin to modulate angiogenesis in vitro and in vivo. We show that fasentin inhibited tube formation in endothelial cells by a mechanism that involves a negative effect on endothelial cell proliferation and invasion, without affecting other steps related to the angiogenic process. However, fasentin barely decreased glucose uptake in human dermal microvascular endothelial cells and the GLUT-1 inhibitor STF-31 failed to inhibit tube formation in these cells. Therefore, this modulatory capacity on endothelial cells function exerted by fasentin is most likely independent of a modulation of glucose metabolism. Taken together, our results show a novel biological activity of fasentin, which could be evaluated for its utility in cancer and other angiogenesis-dependent diseases.

Background and Aims: The endothelium is crucially involved in the pathogenesis of atherosclerosis according to accumulating evidence. Moreover, recent studies have showed that lncRNAs could serve as biomarkers of cardiovascular diseases, in particular atherosclerosis. However, the underlying mechanism of endothelial dysfunction involving lncRNAs in atherosclerosis remains unknown. This study investigated the mechanism of lncRNA XXYLT1-AS2 in endothelial dysfunction in atherosclerosis., Methods: The levels of lncRNA XXYLT1-AS2, FUS, VCAM-1, MCP-1, p-AKT, and p-P65 were measured in arteries and HUVEC cell lines via quantitative real-time PCR or Western blot. FISH assay demonstrated that XXYLT1-AS2 and FUS are localized in the nucleus. HUVECs were transfected with si-XXYLT1-AS2 or XXYLT1-AS2 to further assess cell proliferation, migration, and adhesion. Furthermore, bioinformatics analysis, RNA immunoprecipitation and immunofluorescence were performed to investigate the target genes of XXYLT1-AS2 and possible signal pathways., Results: Overexpression of XXYLT1-AS2 inhibited cell proliferation and migration, reduced the expression of adhesion molecules (VCAM-1) and chemoattractant proteins (MCP-1), and restrained monocyte adhesion to endothelial cells. Mechanistic investigations indicated that XXYLT1-AS2 directly interacts with the target gene FUS/cyclin D1 and modulates the proliferation and migration of endothelial cells (ECs). Moreover, XXYLT1-AS2 exerts a protective role against the inflammatory response in atherosclerosis by blocking NF-κB activity. Clinically, the involvement of XXYLT1-AS2/FUS was also observed in human arteries and the results were consistent with the in vitro analysis., Conclusions: Our study identified a novel long non-coding RNA (XXYLT1-AS2) and suggests that it might act as an underlying therapeutic target in atherosclerosis-related diseases by regulating ECs functions., Competing Interests: Declaration of competing interest The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Diabetic vascular complications caused by endothelial dysfunction play an important role in the pathogenesis of diabetic foot. A well understanding of the role of endothelial dysfunction in diabetic foot vasculopathy will help to further reveal the pathogenesis of diabetic foot. This study aimed to assess whether the RhoA/ROCK signaling pathway is controlled by Rho GTPase-activating proteins (RhoGAP, ARHGAP) and advanced glycosylation end products (AGEs), and to clarify the roles of ARHGAP and AGEs in the RhoA/ROCK signaling pathway or the mechanism by which AGEs regulated RhoA. Real-time PCR was applied to detect gene expression. Manipulation of endothelial biological functions by ARHGAP18 and AGEs were studied via cell counting kit-8 (CCK-8), Western blot, transwell, FITC-Dextran and TEER permeability experiments. RhoA-specific inhibitor Y-27632 was used to silence the activity of RhoA. Dual Luciferase Reporter Assay, Western blot and ELISA assays were used to detect molecular mechanism of endothelial biological functions. In this study, we found that ARHGAP18 was negatively correlated with RhoA, and the expression of ARHGAP18 in human umbilical vein endothelial cells (HUVECs) was decreased with gradient-increased AGEs. Furthermore, AGEs and ARHGAP18 could orchestrate RhoA activity, then activate NF-κB signaling pathway, affect the structural and morphological of VE-cadherin and tight junction protein, and cause endothelial cell contraction, thereby increasing permeability of endothelial cells. In conclusion, AGEs and ARHGAP18 orchestrate cell proliferation, invasion and permeability by controlling the RhoA/ROCK signaling pathway, affecting NF-κB signaling pathway as well as the structure and morphology of VE-cadherin and tight junction protein, and regulating endothelial cell contraction.

Calcineurin Inhibitors (CNIs) are routinely used for immunosuppression following solid organ transplantation. However, the prolonged use of these agents lead to organ fibrosis which limits their efficacy. CNIs induce TGFβ expression, which is reported to augment endothelial-to-mesenchymal transition (EndMT), but their role in this process is not known. In these studies, we find that the CNIs FK506 and cyclosporine (CsA) are potent to increase endothelial cell (EC) proliferation using established in vitro assays (P < 0.05). Furthermore, using phosphokinase arrays, we find that each CNI activates the MAPK and Akt/mTOR signaling pathways, and that pharmacological inhibition of each pathway targets CNI-induced proliferative responses (P < 0.001). EndMT was evaluated by FACS for N-cadherin and CD31 expression and by qPCR for the expression of α-smooth muscle actin, N-cadherin and Snail. We find that CNIs do not directly induce dedifferentiation, while TGFβ and hypoxia induce EndMT in small numbers of EC. In contrast, the treatment of EC with the inflammatory cytokine TNFα was potent to elicit an EndMT response, and its effects were most notably in EC following proliferation/doubling. Taken together, these observations suggest that CNIs elicit proliferative responses, which enhance EndMT in association with local inflammation. The clinical implications of these findings are that anti-proliferative therapeutics have high potential to target the initiation of this EndMT response., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Researches have been focusing on the role of Slit2 in angiogenesis, specifically in cell migration and vessel permeability. Nevertheless, the role of Slit2-N, the bioactive fragment of Slit2, in the proliferation of vascular endothelia in choroidal neovascularization and some related mechanisms have not been studied yet. Thus, our study aimed to explore the role of Slit2-N in proliferation of vascular endothelia and the related mechanisms in choroidal neovascularization. Fluorescein isothiocyanate perfusion and HE staining were performed to evaluate volumes of choroidal neovascularization lesions. The effect of Slit2-N on VEGF165-induced cell proliferation and some related mechanisms were detected by CCK8 assay, flow cytometry, siRNA transfection, and western blotting. We found that Slit2-N reduced volumes of laser-induced choroidal neovascularization networks in vivo. Results of the in vitro study showed Slit2-N reduced VEGF165-induced cell proliferation of both human umbilical vascular endothelial cells and human microvascular endothelial cells possibly via activation of AKT rather than that of ERK1/2. Additionally, Robo4, one of the receptors binding to Slit2-N, was involved in the inhibitory effect of Slit2-N. Generally, our findings revealed the inhibitory role of Slit2-N in proliferation of vascular endothelia and some related mechanisms, and presented some potential targets, molecules along Slit2-N-Robo4-AKT axis, to choroidal neovascularization therapy.

This work investigates the physicochemical properties and in vitro accuracy of a genetically engineered drug-delivery system based on elastin-like block recombinamers. The DNA recombinant techniques allowed us to create this smart complex polymer containing bioactive sequences for internalization, lysosome activation under acidic pH, and blockage of cellular growth by a small peptide inhibitor. The recombinant polymer reversibly self-assembled when the temperature was increased above 15 °C into nanoparticles with a diameter of 72 nm and negative surface charge. Furthermore, smart nanoparticles were shown to enter in the cells via clathrin-dependent endocytosis and properly blocked phosphorylation and consequent activation of Akt kinase. This system provoked apoptosis-mediated cell death in breast and colorectal cancer cells, which possess higher expression levels of Akt, whereas noncancerous cells, such as endothelial cells, fibroblasts, and mesenchymal stem cells, were not affected. Hence, we conclude that the conformational complexity of this smart elastin-like recombinamer leads to achieving successful drug delivery in targeted cells and could be a promising approach as nanocarriers with bioactive peptides to modulate multiple cellular processes involved in different diseases.

Angiogenesis plays a critical role in embryo development, tissue repair, tumor growth and wound healing. In the present study, we investigated the role of the serine/threonine kinase Akt in angiogenesis. Silencing of Akt1 in human umbilical vein endothelial cells significantly inhibited vascular endothelial growth factor (VEGF)-induced capillary-like tube formation. Mice lacking Akt1 exhibited impaired retinal angiogenesis with delayed endothelial cell (EC) proliferation. In addition, VEGF-induced corneal angiogenesis and tumor development were significantly inhibited in mice lacking Akt1. Loss of Akt1 resulted in reduced angiogenic sprouting, as well as the proliferation of ECs and mural cells. Addition of culture supernatant of vascular smooth muscle cells (VSMCs) in which Akt1 was silenced suppressed tube formation, the stability of preformed tubes and the proliferation of ECs. In addition, attachment of VSMCs to ECs was significantly reduced in cells in which Akt1 was silenced. Mural cell coverage of retinal vasculature was reduced in mice lacking Akt1. Finally, mice lacking Akt1 showed severe retinal hemorrhage compared to the wild-type. These results suggest that the regulation of EC function and mural cell coverage by Akt1 is important for blood vessel maturation during angiogenesis., Competing Interests: The authors declare no conflict of interest.

Formononetin is one of the main active components of traditional Chinese medicine red clover (Trifolium pratense L). Prior studies have demonstrated that formononetin is a typical phytoestrogen and exhibits an estrogen-like effect that protects the cardiovascular system with minimal side effects. In this study, we further investigated the role of formononetin in human umbilical vein endothelial cells (HUVECs) and its potential molecular mechanisms. We founded that formononetin promotes proliferation and migration of HUVECs as assessed by the MTT and wound healing assays. Meanwhile, insulin-like growth factor 1 (IGF-1) and intercellular adhesion molecule 1 (ICAM-1) protein levels were increased in a dose-dependent manner. Our findings illustrated that formononetin exhibits a protective effect on HUVECs, and the molecular mechanisms may correlate with IGF-1R and ICAM-1, as well as upregulation of vascular endothelial growth factor (VEGF) and activation of extracellular signal-regulated kinase (ERK)., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Natural flavonoids are ubiquitous in dietary plants and vegetables and have been proposed to have antiviral, antioxidant, cardiovascular protective, and anticancer effects. Volume-regulated anion channels (VRACs), which are essential for cell volume regulation, have been proposed to play a key role in cell proliferation and migration, apoptosis, transepithelial transport, and cancer development. In this study, we screened a group of 53 structurally related natural flavonoids and three synthetic flavonoids for their inhibitory activities on VRAC currents. A whole-cell patch technique was used to record VRAC currents in the human embryonic kidney (HEK) 293 and human umbilical vein endothelial (HUVEC) cells. The 5'-bromo-2-deoxyuridine (BrdU) assay technique was used to investigate cell proliferation. At 100 μM, 34 of 53 compounds significantly inhibited hypotonic extrasolution-induced VRAC currents by > 50% in HEK293 cells. Among these compounds, luteolin, baicalein, eupatorin, galangin, quercetin, fisetin, karanjin, Dh-morin, genistein, irisolidone, and prunetin exhibited the highest efficacy for VRAC blockade (the mean inhibition > 80%) with IC 50 s of 5-13 μM and E max s of about 87-99%. We also studied the effects of three synthetic flavonoids on VRAC currents in HEK293 cells. Flavoxate showed high inhibition efficacy toward VRAC currents (IC 50  = 2.3 ± 0.3 μM; E max  = 91.8% ± 2.7%). Finally, these flavonoids inhibited endogenous VRAC currents and cell proliferation in endothelial cells. This study demonstrates that natural and synthetic flavonoids are potent VRAC current inhibitors, and VRAC inhibition by flavonoids might be responsible for their anti-angiogenic effects.

We investigated the role of exosomes derived from maternal and umbilical cord blood in the regulation of angiogenesis. We report here that both maternal exosomes (MEs) and umbilical exosomes (UEs) significantly enhance HUVEC proliferation, migration, and tube formation. Importantly, ME-treated HUVECs (MEXs) displayed significantly increased migration, but not proliferation or tube formation, compared with UE-treated HUVECs (UEXs). We found that the expression of a subset of migration-related microRNAs (miRNAs), including miR-210-3p, miR-376c-3p, miR-151a-5p, miR-296-5p, miR-122-5p, and miR-550a-5p, among others, were significantly increased or decreased in UEs, and this altered expression was likely correlated with the differential regulation of HUVEC migration. We also found that the mRNA expression of hepatocyte growth factor (HGF) was up-regulated in MEXs and UEXs and, moreover, that inhibiting HGF partially abolished the enhanced cell migration induced by UEs. Our results suggest that both MEs and UEs greatly enhanced endothelial cell (EC) functions and differentially regulated EC migration, which was mostly attributed to the different expression profiles of exosomal miRNA. These findings highlight the importance of exosomes in the regulation of angiogenesis during pregnancy. Exosomal miRNAs, in particular, may be of great significance for the regulation of angiogenesis in maintaining normal pregnancy.-Jia, L., Zhou, X., Huang, X., Xu, X., Jia, Y., Wu, Y., Yao, J., Wu, Y., Wang, K. Maternal and umbilical cord serum-derived exosomes enhance endothelial cell proliferation and migration.

MiRNAs are considered as a novel class of biomarkers or treatment targets for cardiovascular diseases. Hsa-miR-6086, a novel mi-RNA, was reported to be downregulated during the differentiation of human embryonic stem cells into endothelial cells (ECs). Interestingly, CDH5 (cadherin 5), encoding a classical cadherin of the cadherin superfamily, is a cellular marker of ECs and has been reported to be a target of hsa-miR-6086. However, the role of hsa-miR-6086 in ECs is virtually unknown. Herein, we report that hsa-miR-6086 was markedly induced by TNFα stimulation in human umbilical vein endothelial cells (HUVECs), whereas CDH5 expression was greatly reduced. Importantly, TNFα-induced suppression of CDH5 expression was largely prevented by inhibiting hsa-miR-6086, and hsa-miR-6086 mimic greatly decrease CDH5 expression in HUVECs, suggesting that the induction of hsa-miR-6086 is responsible for CDH5 downregulation by TNFα. In addition, restoration of CDH5 expression level by either inhibiting hsa-miR-6086 or exogenously expressing CDH5 cDNA that is not affected by hsa-miR-6086 protected HUVECs against TNFα-induced apoptosis and cell growth inhibition. Taken together, our study reveals that hsa-miR-6086 is induced by TNFα and mediates TNFα-induced HUVEC growth inhibition through downregulating CDH5 expression. Hence, hsa-miR-6086 might be a new target for treating TNFα-induced endothelial dysfunction., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Growth factors hold great promise for regenerative therapies. However, their clinical use has been halted by poor efficacy and rapid clearance from tissue, necessitating the delivery of extremely high doses to achieve clinical effectiveness which has raised safety concerns. Thus, strategies to either enhance growth factor activity at low doses or to increase their residence time within target tissues are necessary for clinical success. In this study, we generated multivalent conjugates (MVCs) of basic fibroblast growth factor (bFGF), a key growth factor involved in angiogenesis and wound healing, to hyaluronic acid (HyA) polymer chains. Multivalent bFGF conjugates (mvbFGF) were fabricated with minimal non-specific interaction observed between bFGF and the HyA chain. The hydrodynamic radii of mvbFGF ranged from ∼50 to ∼75 nm for conjugation ratios of bFGF to HyA chains at low (10 : 1) and high (30 : 1) feed ratios, respectively. The mvbFGF demonstrated enhanced bioactivity compared to unconjugated bFGF in assays of cell proliferation and migration, processes critical to angiogenesis and tissue regeneration. The 30 : 1 mvbFGF outperformed the 10 : 1 conjugate, which could be due to either FGF receptor clustering or interference with receptor mediated internalization and signal deactivation. This study simultaneously investigated the role of both protein to polymer ratio and multivalent conjugate size on their bioactivity, and determined that increasing the protein-to-polymer ratio and conjugate size resulted in greater cell bioactivity.

Adropin, a peptide hormone expressed in liver and brain, is known to improve insulin resistance and endothelial dysfunction. Serum levels of adropin are negatively associated with the severity of coronary artery disease. However, it remains unknown whether adropin could modulate atherogenesis. We assessed the effects of adropin on inflammatory molecule expression and human THP1 monocyte adhesion in human umbilical vein endothelial cells (HUVECs), foam cell formation in THP1 monocyte-derived macrophages, and the migration and proliferation of human aortic smooth muscle cells (HASMCs) in vitro and atherogenesis in Apoe -/- mice in vivo. Adropin was expressed in THP1 monocytes, their derived macrophages, HASMCs, and HUVECs. Adropin suppressed tumor necrosis factor α-induced THP1 monocyte adhesion to HUVECs, which was associated with vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 downregulation in HUVECs. Adropin shifted the phenotype to anti-inflammatory M2 rather than pro-inflammatory M1 via peroxisome proliferator-activated receptor γ upregulation during monocyte differentiation into macrophages. Adropin had no significant effects on oxidized low-density lipoprotein-induced foam cell formation in macrophages. In HASMCs, adropin suppressed the migration and proliferation without inducing apoptosis via ERK1/2 and Bax downregulation and phosphoinositide 3-kinase/Akt/Bcl2 upregulation. Chronic administration of adropin to Apoe -/- mice attenuated the development of atherosclerotic lesions in the aorta, with reduced the intra-plaque monocyte/macrophage infiltration and smooth muscle cell content. Thus, adropin could serve as a novel therapeutic target in atherosclerosis and related diseases.

Heparan sulfate proteoglycans, HSPGs, modulate major transformations of cancer cells, leading to tumor growth, invasion and metastasis. HSPGs also regulate neo-angiogenesis which prompts cancer progression and metastatic spread. A different aspect of heparin and analogs is their prominent role in the coagulation of blood. The interplay between coagulation and metastasis is being actively studied: anticoagulants such as heparin-derivatives have anticancer activity and procoagulants, such as thrombin, positively modulate proliferation, migration and invasion. The branched peptide NT4 binds to HSPGs and targets selectively cancer cells and tissues. For this, it had been extensively investigated in the last years and it proved to be efficient as chemotherapeutic and tumor tracer in in vivo models of cancer. We investigated the effects of the branched peptide in terms of modulation of angiogenesis and invasiveness of cancer cells. NT4 proved to have a major impact on endothelial cell proliferation, migration and tube formation, particularly when induced by FGF2 and thrombin. In addition, NT4 had important effects on aggressive tumor cells migration and invasion and it also had an anticoagulant profile.The peptide showed very interesting evidence of interference with tumor invasion pathways, offering a cue for its development as a tumor-targeting drug, and also for its use in the study of links between coagulation and tumor progression involving HSPGs.

Drug eluting stents (DES) have been extensively applied nowadays and reduce the incidence of in-stent restenosis (ISR) greatly as compared with bare metal stents (BMS). However, the development of DES is hindered by the risk of late stent thrombosis (LST) due to delayed re-endothelialization, while endothelialization is an important process related to ISR and LST after implantation. 316L is a traditional stent material without bioactivity and have a high risk of ISR. Cu is recognized for angiogenesis stimulation in these years. Hence a copper bearing 316L stainless steel (316L-Cu) was prepared and evaluated about its effect on endothelialization in this paper. Compared with traditional 316L, it was proved that 316L-Cu increased the proliferation of co-cultured human umbilical vein endothelial cells (HUVECs) at first day. Moreover, HUVECs stretched better on the surface of 316L-Cu. It also improved the expression of angiogenesis related genes and tube formation ability in vitro. 316L-Cu-BMS, DES and 316L-BMS were implanted in swine to evaluate the re-endothelialization ability in vivo. And 316L-Cu-BMS showed the best effect on endothelialization with good biosafety. Consequently, 316L-Cu is a kind of promising BMS material for coronary field.

Tumor growth and metastasis are angiogenesis dependent. Angiogenic growth involves endothelial cell proliferation, migration, and invasion. Ephrin-B2 is a ligand for Eph receptor tyrosine kinases and is an important mediator in vascular endothelial growth factor-mediated angiogenesis. However, research offer controversial information regarding effects of ephrin-B2 on vascular endothelial cells. In this paper, proteome analyses showed that ephrin-B2/Fc significantly activates multiple signaling pathways related to cell proliferation, survival, and migration and suppresses apoptosis and cell death. Cytological experiments further confirm that ephrin-B2/Fc stimulates endothelial cell proliferation, triggers dose-dependent migration, and suppresses cell apoptosis. Results demonstrate that soluble dose-dependent ephrinB2 can promote proliferation and migration and inhibit apoptosis of human umbilical vein endothelial cells. These results also suggest that ephrinB2 prevents ischemic disease and can potentially be a new therapeutic target for treating angiogenesis-related diseases and tumors.

The aim of this study was to investigate, for the first time, the effects of using adipose-derived mesenchymal stem cells (AD-MSCs) transfected with an episomal plasmid encoding fibroblast growth factor 1 (FGF1) (AD-MSCs FGF1 ), in providing the microenvironment required for angiogenic proliferation. The isolated rat AD-MSCs were positive for mesenchymal (CD29 and CD90) and negative for hematopoietic (CD34 and CD45) surface markers. Adipogenic and osteogenic differentiation of the AD-MSCs also occurred in the proper culture media. The presence of FGF1 in the conditioned medium from the AD-MSCs FGF1 was confirmed by Western blotting. G418 and PCR were used for selection of transfected cells and confirmation of the presence of FGF1 mRNA, respectively. Treatment with the AD-MSC FGF1 -conditioned medium significantly increased the NIH-3T3 cell proliferation and human umbilical vein endothelial cell (HUVEC) tube formation compared to conditioned medium from nontransfected AD-MSCs (p < 0.001). In conclusion, the AD-MSCs FGF1 efficiently secreted functional FGF1, which promoted angiogenic proliferation. Using AD-MSCs FGF1 may provide a useful strategy in cell therapy, which can merge the beneficial effects of stem cells with the positive biological effects of FGF1 in various disorders, especially tissue defects, neurodegenerative, cardiovascular and diabetes endocrine pathologies, which remain to be tested in preclinical and clinical studies.

Emerging evidence has demonstrated an important role of microRNAs (miRNAs) in the pathogenesis of cerebral infarction. In the present study, a down-regulation of microRNA-433 (miR-433) is identified in hypoxia-induced human umbilical vein vascular endothelial cells (HUVECs) as well as in rat neurons, and is found to be negatively regulated cell proliferation and migration. Moreover, the expression of miR-433 is inversely correlated with the expression of hypoxia-inducible factor 1 alpha (HIF-1α), which has been shown to play critical role in responding to hypoxia conditions. Overexpression or knockdown of miR-433 responsively alters both mRNA and protein levels of HIF-1α and its downstream genes, vascular endothelial growth factor, Glut-1, and Angpt2. In a luciferase reporter system, miR-433 down-regulates the luciferase activity of HIF-1α 3'-UTR, and these effects are abolished by a mutation in the putative miR-433-binding site. Further investigation confirms that knockdown of HIF-1α blocked the stimulatory effect of anti-miR-433, while overexpression of HIF-1α reversed the inhibitory effects of pre-miR-433 on proliferation and migration of HUVEC and neurons. Taken together, our findings indicate that miR-433 plays an important role in response to hypoxia, inhibiting HUVEC and neuron proliferation and migration by down-regulating HIF-1α.

Objective: To investigate the effects of polyunsaturated fatty acids (PUFA) ω-3 and ω-6, and their middle metabolites PGE2 and PGE3 on angiogenesis formation of gastric cancer, and to explore associated mechanism., Methods: The effects of ω-3, ω-6, PGE2, PGE3 on the proliferation and migration of human umbilical vein endothelial cell (HUVEC) were measured by proliferation and migration assay respectively. The angiogenesis assay in vivo was used to measure the effects of ω-3, ω-6, PGE2 and PGE3 on neovascularization. In all the assays, groups without ω-3, ω-6, PGE2 and PGE3 were designed as the control., Results: With the increased concentration of ω-6 from 1 μmol/L to 10 μmol/L, the proliferation ability of HUVECs enhanced, and the number of migration cells also increased from 28.2±3.0 to 32.8±2.1, which was higher than control group (21.2±3.2) respectively (both P<0.05). With the increased concentration of ω-3 from 1 μmol/L to 10 μmol/L, the proliferation ability of HUVECs was inhibited, and the number of migration cells decreased from 15.8±2.0 to 11.0±2.1, which was lower than control group (22.1±3.0) respectively (both P<0.05). In the angiogenesis assay, compared with control group (standard number: 43 721±4 654), the angiogenesis ability of HUVECs was significantly enhanced by ω-6 in concentration-dependent manner (1 μmol/L group: 63 238±4 795, 10 μmol/L group: 78 166±6 123, all P<0.01). Meanwhile, with the increased concentration of ω-3 from 1 μmol/L to 10 μmol/L, the angiogenesis ability was significantly decreased from 30 129±3 102 to 20 012±1 541(all P<0.01). The proliferation and migration ability of HUVECs were significantly promoted by ω-6 metabolites PGE2 (P<0.05) in a concentration-dependent manner. In contrast, ω-3 metabolites PGE3 significantly inhibited the proliferation and migration ability of HUVECs in a concentration-dependent manner (all P<0.05). After rofecoxib (a COX-2 specific inhibitor) inhibited the expression of COX-2, the expression level of PGE2 was significantly decreased in a dose-dependent manner. In co-culture system, whose gastric cancer cells expressed positive COX-2, ω-6 could increase angiogenesis of gastric cancer cells(P<0.01), but ω-3 could inhibit such angiogenesis(P<0.01). In co-culture system, whose gastric cancer cells did not express COX-2, ω-3 could inhibit the angiogenesis of gastric cancer cells (P<0.05), but ω-6 had no effect on angiogenesis., Conclusions: The PUFA ω-6 can enhance the angiogenesis via the promotion of proliferation and migration of HUVECs, and COX-2 and PGE2 may play an important role in this process, whereas, the ω-3 can inhibit the angiogenesis through its middle metabolites PGE3 to inhibit the proliferation and migration of HUVECs. Results of this experiment may provide a new approach to inhibit and prevent the spread of gastric cancer.

Epigenetics, including DNA methylation, is one way for a cell to respond to the surrounding environment. Traditionally, DNA methylation has been perceived as a quite stable modification; however, lately, there have been reports of a more dynamic CpG methylation that can be affected by, for example, long-term culturing. We recently reported that methylation in the enhancer of the gene encoding the key fibrinolytic enzyme tissue-type plasminogen activator (t-PA) was rapidly erased during cell culturing. In the present study we used sub-culturing of human umbilical vein endothelial cells (HUVECs) as a model of environmental challenge to examine how fast genome-wide methylation changes can arise. To assess genome-wide DNA methylation, the Infinium HumanMethylation450 BeadChip was used on primary, passage 0, and passage 4 HUVECs. Almost 2% of the analyzed sites changed methylation status to passage 4, predominantly displaying hypomethylation. Sites annotated as enhancers were overrepresented among the differentially methylated sites (DMSs). We further showed that half of the corresponding genes concomitantly altered their expression, most of them increasing in expression. Interestingly, the stroke-related gene HDAC9 increased its expression several hundredfold. This study reveals a rapid hypomethylation of CpG sites in enhancer elements during the early stages of cell culturing. As many methods for methylation analysis are biased toward CpG rich promoter regions, we suggest that such methods may not always be appropriate for the study of methylation dynamics. In addition, we found that significant changes in expression arose in genes with enhancer DMSs. HDAC9 displayed the most prominent increase in expression, indicating, for the first time, that dynamic enhancer methylation may be central in regulating this important stroke-associated gene.

The aim of this study was to investigate the effects of interleukin (IL)-22 on proliferation function and inflammatory mediator production and barrier function of human umbilical vein endothelial cells (HUVECs). The expression of mRNA was detected by RT-PCR. The proliferation ability of cells was evaluated using a cell counting kit assay. Real-time quantitative PCR and Western blot were used to detect the expression of inflammatory mediators. The endothelial barrier permeability was assessed by measuring permeability to FITC-labeled dextran. The distribution of occludin was detected by immunofluorescence. IL-22R1 mRNA expression was noted in HUVECs. IL-22 could enhance the proliferation ability of HUVECs and suppress lipopolysaccharide (LPS)-induced proliferation inhibition in these cells. IL-22 also enhanced the production of CCL2 and CCL20 by HUVECs. Besides, IL-22 could improve barrier function and decrease LPS-induced increased cellular permeability and inhibit the LPS-induced destruction of occludin in HUVECs. IL-22 may play a protective role in the development of vasculitis.

In an attempt to enhance endothelial cell capture and promote the vascularization of engineered tissue, we biosynthesized and characterized the recombinant fusion protein consisting of human vascular endothelial-cadherin extracellular domain and immunoglobulin IgG Fc region (hVE-cad-Fc) to serve as a bioartificial extracellular matrix. The hVE-cad-Fc protein naturally formed homodimers and was used to construct hVE-cad-Fc matrix by stably adsorbing on polystyrene plates. Atomic force microscop assay showed uniform hVE-cad-Fc distribution with nanorod topography. The hVE-cad-Fc matrix markedly promoted human umbilical vein endothelial cells (HUVECs) adhesion and proliferation with fibroblastoid morphology. Additionally, the hVE-cad-Fc matrix improved HUVECs migration, vWF expression, and NO release, which are closely related to vascularization. Furthermore, the hVE-cad-Fc matrix activated endogenous VE-cadherin/β-catenin proteins and effectively triggered the intracellular signals such as F-actin stress fiber, p-FAK, AKT, and Bcl-2. Taken together, hVE-cad-Fc could be a promising bioartificial matrix to promote vascularization in tissue engineering.

Background Aims: The amniotic fluid is a new source of multipotent stem cells with therapeutic potential for human diseases. In agreement with the regulatory requirement to reduce and possibly to avoid animal-derived reagents in the culture of cells intended for cell therapy, bovine serum, the most common supplement in the culture medium, was replaced by human platelet-derived growth factors., Methods: We tested a new culture medium to expand monolayers of human amniotic fluid stem cells (hAFSC) for clinical use. The AFSC were isolated by c-Kit selection and expanded in media supplemented with either bovine serum or a human platelet lysate (Lyset)., Results: We compared proliferation kinetics, colony-forming unit percentage, multilineage differentiation, immunophenotypic characterization and inhibition of peripheral blood mononuclear cell proliferation of the two AFSC cell cultures and we found no significant differences. Moreover, the karyotype analysis of the cells expanded in the presence of the platelet lysate did not present cytogenetic abnormalities and in vitro and in vivo studies revealed no cell tumorigenicity., Conclusions: Platelet derivatives represent a rich source of growth factors that can play a safety role in the homeostasis, proliferation and remodeling of tissue healing. We propose human platelet extracts as a preferential alternative to animal serum for the expansion of stem cells for clinical applications., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Objective: To study the impact of intense picosecond pulsed electric field (psPEF) on the biological behaviors of human umbilical venous endothelial cells (HUVECs) in vitro., Methods: Intense psPEF with constant parameters (pulse duration, 800 ps; recurrence rate, 3 Hz; the number of pulses, 2000) and three-level electric field strength (200, 300 and 400 KV/cm) were performed on HUVECs. CCK-8 assay was used to detect the cell proliferation of HUVECs treated with psPEF for 2, 4, 6, 8, 10, 12, 14, 16 hours. The effect of psPEF on HUVEC migration was measured by wound healing method and Transwell(TM) assay. The apoptosis and cell cycle were detected by flow cytometry., Results: The death rate of HUVECs went up with the increase of the amount of pulses, and the maximum inhibitory rate was at 12 hours after the treatment with psPEF. Intense psPEF inhibited dose-dependently the migration of HUVECs. What's more, the treatment with psPEF arrested cell cycle in G2/M phase and increased the apoptosis rate., Conclusion: Intense psPEF inhibits the migration and proliferation of HUVECs as well as induces cell apoptosis and arrests the cell cycle in G2/M phase.

Background Aims: Cord blood (CB) hematopoietic stem cell transplantation has often been limited by the scarcity of stem cells. Therefore, the number of CB hematopoietic stem/progenitor cells (HSPCs) should be increased while maintaining the stem cell characteristics., Methods: We designed an ex vivo culture system using endothelial progenitor cells (EPCs) as stroma to determine the capacity of expanding CB-HSPCs in a defined medium, the effect on engraftment of the expanded cells in a mouse model and the underlying mechanism., Results: After 7 days of culture, compared with those cultured with cytokines alone (3.25 ± 0.59), CD34+ cells under contact and non-contact co-culture with EPCs were expanded by 5.38 ± 0.61 (P = 0.003) and 4.06 ± 0.43 (P = 0.025)-fold, respectively. Direct cell-to-cell contact co-culture with EPCs resulted in more primitive CD34+ CD38- cells than stroma-free culture (156.17 ± 21.32 versus 79.12 ± 19.77-fold; P = 0.010). Comparable engraftment of day 7 co-cultured HSPCs with respect to HSPCs at day 0 in nonobese diabetic-severe combined immunodeficiency disease (NOD/SCID) mice was measured as a percentage of chimerism (13.3% ± 11.0% versus 16.0% ± 14.3%; P = 0.750). EPCs highly expressed interleukin 6 (IL6) and angiopoietin 1 (ANGPT1), the hematopoietic- related cytokines. A higher transcriptional level of WNT5A genes in EPCs and co-cultured HSPCs suggests that the activation of Wnt signaling pathway may play a role in HSPCs' expansion ex vivo., Discussion: These data demonstrated that EPCs improve the CD34+ population but do not compromise the repopulating efficacy of the amplified HSPCs, possibly via cytokine secretion and Wnt signaling pathway activation., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Objective: To explore the effect of cryptotanshinone (CTS) on vascular endothelial growth factor (VEGF) expression in U2OS osteosarcoma cells, with a focus on angiogenesis., Methods: U2OS osteosarcoma cells cultured in vitro were divided into (20, 40, 80, 160) μmol/L CTS treated groups and control group. Real-time quantitative PCR was performed to detect the expression of VEGF mRNA in U2OS osteosarcoma cells. The VEGF protein level was determined using Western blotting and immunofluorescence cytochemistry. Cell proliferation ability was detected by CCK-8 assay. The tube formation assay in vitro was used to observe the angiogenesis ability., Results: CTS inhibited the levels of VEGF mRNA and protein in a dose-dependent manner in U2OS osteosarcoma cells obviously. CCK-8 assay indicated that CTS inhibited the proliferation of U2OS osteosarcoma cells. The tube formation assay in vitro revealed that CTS inhibited the angiogenesis ability., Conclusion: CTS could down-regulate the expression of VEGF and inhibit angiogenesis in U2OS osteosarcoma cells.

Purposes: ER520, a derivative of indenoisoquinoline, is a patented compound. This study was designed to screen its biological properties and to evaluate its antineoplastic and antiangiogenic effect., Methods: Western blot was employed to monitor the ERα and ERβ protein expression in human breast cancer MCF-7 cells and endometrial carcinoma Ishikawa cells. MTT assay was employed to determine cell proliferation. Cell adhesion, scratch and Transwell assay were utilized to estimate the ability of cellular adhesion, migration and invasion. ELISA kit was applied to detect the VEGF products in culture medium. In addition, the inhibitory effect of ER520 on the vessel-like construction of HUVEC cells and the angiogenesis of chicken embryos was investigated. The efficiency of ER520 on tumor growth in nude mice was also assessed., Results: ER520 inhibited the expression of ERα in MCF-7 and Ishikawa cells, while it increased ERβ protein level. ER520 also suppressed the proliferation of MCF-7 and Ishikawa cells. Due to its remarkably negative role in cell adhesion, migration and invasion, ER520 showed a potential ability of inhibiting tumor metastasis. Meanwhile, ER520 reduced the VEGF secretion of MCF-7 and Ishikawa cells, prevented the formation of VEGF-stimulated tubular structure and the cell migration of HUVEC cells, and inhibited the angiogenesis of chicken chorioallantoic membrane. Animal experiment also demonstrated that ER520 could frustrate the in vivo tumor growth and the inhibitory ratio was 48.5 % compared with control group., Conclusion: Our findings indicate that ER520 possesses the competence to be a candidate against breast cancer and angiogenesis.

Introduction: Vascular graft materials in clinical use, such as polytetrafluoroethylene (PTFE) and Dacron, do not endothelialise and have low patency rates. The importance of an endothelial cell layer on the luminal surface of a vascular graft is well-known with surface topography and chemistry playing an important role. The aim of this study was to investigate the potential of plasma treatment and topographical structures on the luminal graft surface to enhance the self-endothelialisation potential of a nanocomposite vascular graft., Methods: POSS-PCU is a polycarbonate urea urethane (PCU) with a nanoparticle, polyhedral oligomeric silsesquioxane (POSS) incorporated within it. Planar, microgrooved, and nanopit patterned polymer films were fabricated using photolithography, electron beam lithography, reactive ion etching, and replication by solvent casting. Films were then exposed to oxygen plasma treatment at different powers for a fixed time (40 W, 60 W, 80 W/60 seconds). Effects of plasma treatment were assessed using scanning electron microscopy, atomic force microscopy and water contact angle analysis. Human umbilical vein endothelial cell (HUVEC) proliferation and morphology were characterised using immunostaining, live/dead staining, and Coomassie blue staining., Results: Successful embossing of the micro- and nanostructures was confirmed. Oxygen plasma treatment of the different samples showed that increasing power significantly increased the hydrophilicity of the samples (p < .0001). Improved HUVEC adhesion was seen on plasma modified compared with untreated samples (p < .0001). Coomassie blue staining showed that after 5 days, cells started to form monolayers and live/dead staining showed the cells were viable. Immunostaining showed that HUVECs expressed nitric oxide synthase on all topographies with focal adhesions appearing more pronounced on nanopit surfaces, showing retention of morphology and function., Conclusion: These encouraging results indicate a future important role for plasma treatment and nanotopography in the development of endothelialised vascular grafts., (Copyright © 2014 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.)

Nano-Mg(OH)2 is efficiently used in pollutant adsorption and removal due to its high adsorption capability, low-cost, and recyclability. A recent research from our group showed that Mg(OH)2 nanoflakes are not evidently internalized by cancer cells and are not cytotoxic. But the biocompatibility and potential toxicity of nano-Mg(OH)2 in a normal biological system are largely unclear. Nanoparticles could affect the function of endothelial cells, and endothelial dysfunction represents an early sign of lesion within the vasculature. Here, we applied the human umbilical vein vascular endothelial cells (HUVECs) as an in vitro model of the endothelium to study the cytotoxicity of nano-Mg(OH)2. Our results showed that nano-Mg(OH)2 at 200 μg/ml impaired proliferation and induced dysfunction of HUVECs, but did not result in cell necrosis and apoptosis. Transmission electron microscopy images and immunofluorescence results showed that the nano-Mg(OH)2 could enter HUVECs through caveolin-1-mediated endocytosis. Nano-Mg(OH)2 at high concentrations decreased the level of caveolin-1 and increased the activity of endothelial nitric oxide synthase (eNOS), thus leading to the production of excess nitric oxide (NO). In this work, we provide the cell damage concentrations of nano-Mg(OH)2 nanoparticles, and we propose a mechanism of injury induced by nano-Mg(OH)2 in HUVECs.

At the cardiovascular level, nitric oxide (NO) controls smooth muscle functions, maintains vascular integrity, and exerts an antihypertensive effect. Metal-nonoates are a recently discovered class of NO donors, with NO release modulated through the complexation of the N-aminoethylpiperazine N-diazeniumdiolate ligand to metal ions, and thus representing a significant innovation with respect to the drugs traditionally used. In this study, we characterized the vascular protective effects of the most effective compound of this class, Ni(PipNONO)Cl, compared with the commercial N-diazeniumdiolate group derivate, diethylenetriamine/nitric oxide (DETA/NO). Ni(PipNONO)Cl induced a concentration-dependent relaxation of precontracted rat aortic rings. The ED50 was 0.67 µM, compared with 4.3 µM obtained with DETA/NO. When tested on cultured microvascular endothelial cells, Ni(PipNONO)Cl exerted a protective effect on the endothelium, promoting cell proliferation and survival in the picomolar range. The administration of Ni(PipNONO)Cl to vascular smooth muscle cells reduced the cell number, promoting their apoptosis at a high concentration (10 µM). Inhibition of smooth muscle cell migration, a hallmark of atherosclerosis, was accompanied by cytoskeletal rearrangement and loss of lamellipodia. When added to isolated platelets, Ni(PipNONO)Cl significantly reduced ADP-induced aggregation. Since atherosclerosis is accompanied by an inflammatory environment, cultured endothelial cells were exposed to interleukin (IL)-1β. In the presence of IL-1β, Ni(PipNONO)Cl inhibited cyclooxygenase-2 and inducible nitric oxide synthase upregulation, and reduced endothelial permeability and the platelet and monocyte adhesion markers CD31 and CD40 at the plasma membrane. Overall, these data indicate that Ni(PipNONO)Cl exerts vascular protective effects relevant for vascular dysfunction and prevention of atherosclerosis and thrombosis., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Introduction: Mesenchymal stem cells (MSCs) transplantation has been demonstrated to be an effective strategy for the treatment of cardiovascular disease. However, the low survival rate of MSCs at local diseased tissue reduces the therapeutic efficacy. We therefore investigated the influence of MicroRNA-378 (miR-378) transfection on MSCs survival and vascularization under hypoxic-ischemic condition in vitro., Methods: MSCs were isolated from bone marrow of Sprague-Dawley rats and cultured in vitro. The third passage of MSCs were divided into the miR-378 group and control group. For the miR-378 group, cells were transfected with miR-378 mimic. Both groups experienced exposure to hypoxia (1% O2) and serum deprivation for 24 hours, using normoxia (20% O2) as a negative control during the process. After 24 hours of reoxygenation (20% O2), cell proliferation and apoptosis were evaluated. Expressions of apoptosis and angiogenesis related genes were detected. Both groups were further co-cultured with human umbilical vein endothelial cells to promote vascular differentiation for another 6 hours. Vascular density was assessed thereafter., Results: Compared with the control group, MSCs transfected with miR-378 showed more rapid growth. Their proliferation rates were much higher at 72 h and 96 h under hypoxic condition (257.33% versus 246.67%, P <0.01; 406.84% versus 365.39%, P <0.05). Cell apoptosis percentage in the miR-378 group was significantly declined under normoxic and hypoxic condition (0.30 ± 0.10% versus 0.50 ± 0.10%, P <0.05; 0.60 ± 0.40% versus 1.70 ± 0.20%, P <0.01). The miR-378 group formed a larger number of vascular branches on matrigel. BCL2 level was decreased accompanied with an upregulated expression of BAX in the two experimental groups under the hypoxic environment. BAX expression was reduced in the miR-378 group under the hypoxic environment. In the miR-378 group, there was a decreased expression of tumor necrosis factor-α on protein level and a reduction of TUSC-2 under normoxic environment. Their expressions were both downregulated under hypoxic environment. For the angiogenesis related genes, enhanced expressions of vascular endothelial growth factorα, platelet derived growth factor-β and transforming growth factor-β1 could be detected both in normoxic and hypoxic-ischemic conditions., Conclusion: MiR-378 transfection could effectively promote MSCs survival and vascularization under hypoxic-ischemic condition in vitro.

Although preclinical work with rapalogs suggests potential in the treatment of gastric cancer, they have been less successful clinically. In this study, we report the impact of the investigational drug PP242, a potent and selective small-molecule active-site TORC1/2 kinase inhibitor, on tumor growth and metastasis. The antiproliferative effect of PP242 was assessed using the Cell Counting Kit-8 assay. The migration and invasion potential were analyzed using wound-healing and transwell assays, respectively. The Matrigel capillary tube formation assay was performed to mimic in-vivo angiogenesis. Immunoblotting and immunofluorescence were used to observe protein levels and distribution of actin fibers. Finally, p-mammalian target of rapamycin (mTOR) expression was detected on gastric cancer tissues using immunohistochemistry. First, PP242 potently inhibited cell proliferation in gastric cancer cell lines and in human endothelial cells in vitro at the IC50 ranged from 50 to 500 nmol/l. Then, an inhibitory effect of PP242 on metastasis was observed in gastric cancer cell AGS, along with the cytoskeletal rearrangements and suppression of the phosphorylation of PI3K downstream factors including AKT, mTOR, and P70S6K. Furthermore, PP242 was found to decrease the tube formation and migration of human umbilical vein endothelial cells. Using immunohistochemistry, we found that p-mTOR staining was observed in 41.8% (82/196) of gastric cancer tissues and correlated with depth of mural invasion, lymph node metastasis, tumor node metastasis stage, and vascular invasion. These results show that PP242 suppresses cell proliferation and angiogenesis of gastric cancer through inhibition of the PI3K/AKT/mTOR pathway, which might be an effective novel therapeutic candidate against gastric cancer in the future.

Background: Luminal, estrogen receptor-positive breast cancers represent more than 70% of cases. Despite initial good prognoses one third of Luminal cancers eventually recur locally or at distant sites and exhibit hormone resistance. Here we demonstrate that factors elaborated by malignant stromal cells can induce Luminal tumor cells proliferation and promote angiogenesis and hormone independence. We recently isolated a malignant mouse mammary gland stromal cell line named BJ3Z that increases proliferation and angiogenesis in estrogen-free xenografted Luminal MCF-7 breast cancer cells., Methods: BJ3Z and Normal mouse mammary Fibroblasts (NMFs) were expression profiled using microarray assays. Messenger RNA levels were confirmed by RT-PCR and by immunohistochemistry (IHC). Breast cancer MCF-7, BT-474, BT-20 and MDA-MB-231cell lines and stromal BJ3Z and NMFs were grown for in vitro assays: breast cancer cell lines were treated with stromal cells conditioned media, for three-dimensional (3D) mono and co-cultures in Matrigel, proliferation was measured by Bromo-deoxyuridine (BrdU) incorporation using IHC. Tubule formation in vitro, a proxy for angiogenesis, was assessed using 3D cultured Human Umbilical cord Vascular Endothelial Cells (HUVEC)., Results: We show that under estrogen-free conditions, BJ3Z cells but not NMFs increase proliferation of co-cultured Luminal but not basal-like human breast cancer cells in 2D or as 3D Matrigel colonies. Gene expression profiling, RT-PCR analysis and IHC of colony-derived BJ3Z cells and NMFs shows that Platelet Derived Growth Factor ligands (PDGF-A and -B) are elaborated by BJ3Z cells but not NMFs; while PDGF receptors are present on NMFs but not BJ3Z cells. As a result, in colony co-culture assays, BJ3Z cells but not NMFs increase MCF-7 cell proliferation. This can be mimicked by direct addition of PDGF-BB, and blocked by the PDGF receptor inhibitor Imatinib Mesylate. Both normal and malignant stromal cells enhance angiogenesis in an in vitro model. This effect is also due to PDGF and is suppressed by Imatinib., Conclusions: We provide evidence that Luminal breast cancer cells can be targeted by the PDGF signaling pathway leading to estrogen-independent proliferation and angiogenesis. We speculate that stroma-directed therapies, including anti-PDGFR agents like Imatinib, may be useful in combination with other therapies for treatment of luminal cancers.

Background: Bronchopulmonary dysplasia and emphysema are life-threatening diseases resulting from impaired alveolar development or alveolar destruction. Both conditions lack effective therapies. Angiogenic growth factors promote alveolar growth and contribute to alveolar maintenance. Endothelial colony-forming cells (ECFCs) represent a subset of circulating and resident endothelial cells capable of self-renewal and de novo vessel formation. We hypothesized that resident ECFCs exist in the developing lung, that they are impaired during arrested alveolar growth in experimental bronchopulmonary dysplasia, and that exogenous ECFCs restore disrupted alveolar growth., Methods and Results: Human fetal and neonatal rat lungs contain ECFCs with robust proliferative potential, secondary colony formation on replating, and de novo blood vessel formation in vivo when transplanted into immunodeficient mice. In contrast, human fetal lung ECFCs exposed to hyperoxia in vitro and neonatal rat ECFCs isolated from hyperoxic alveolar growth-arrested rat lungs mimicking bronchopulmonary dysplasia proliferated less, showed decreased clonogenic capacity, and formed fewer capillary-like networks. Intrajugular administration of human cord blood-derived ECFCs after established arrested alveolar growth restored lung function, alveolar and lung vascular growth, and attenuated pulmonary hypertension. Lung ECFC colony- and capillary-like network-forming capabilities were also restored. Low ECFC engraftment and the protective effect of cell-free ECFC-derived conditioned media suggest a paracrine effect. Long-term (10 months) assessment of ECFC therapy showed no adverse effects with persistent improvement in lung structure, exercise capacity, and pulmonary hypertension., Conclusions: Impaired ECFC function may contribute to arrested alveolar growth. Cord blood-derived ECFC therapy may offer new therapeutic options for lung diseases characterized by alveolar damage., (© 2014 American Heart Association, Inc.)

Objectives: Because angiogenesis is essential for tumor growth and metastasis, the development of antiangiogenic agents is an urgent issue in cancer treatment. Zerumbone, a component of subtropical ginger, has been shown to exhibit anticancer activities in various cancer cells; however, little is known about its biological mechanisms against angiogenesis in pancreatic cancer (PaCa). Here, we evaluated the effects of zerumbone on PaCa angiogenesis., Methods: The cytotoxicity of zerumbone in PaCa was measured using premix WST-1 cell proliferation assays. The influence of zerumbone on the angiogenic factors vascular endothelial growth factor and interleukin 8 was measured using the reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assays. Changes in nuclear factor-κB (NF-κB) activities were measured using NF-κB transcription factor assays. We also examined the effects of zerumbone on PaCa-induced angiogenesis using angiogenesis assays., Results: Zerumbone inhibited mRNA expression and protein secretion of angiogenic factors and NF-κB activity. Tube formation in human umbilical vein endothelial cells was enhanced by coculture with PaCa cells, and these enhancements were significantly inhibited by zerumbone treatment., Conclusions: Zerumbone blocked the PaCa-associated angiogenesis through the inhibition of NF-κB and NF-κB-dependent proangiogenic gene products.

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is over-expressed in pancreatic cancer cells, and it is associated with the progression of pancreatic cancer. We tested a single domain antibody (sdAb) targeting CEACAM6, 2A3, which was isolated previously from a llama immune library, and an Fc conjugated version of this sdAb, to determine how they affect the pancreatic cancer cell line BxPC3. We also compared the effects of the antibodies to gemcitabine. Gemcitabine and 2A3 slowed down cancer cell proliferation. However, only 2A3 retarded cancer cell invasion, angiogenesis within the cancer mass and BxPC3 cell MMP-9 activity, three features important for tumour growth and metastasis. The IC50s for 2A3, 2A3-Fc and gemcitabine were determined as 6.5μM, 8μM and 12nM, respectively. While the 2A3 antibody inhibited MMP-9 activity by 33% compared to non-treated control cells, gemcitabine failed to inhibit MMP-9 activity. Moreover, 2A3 and 2A3-Fc inhibited invasion of BxPC3 by 73% compared to non-treated cells. When conditioned media that were produced using 2A3- or 2A3-Fc-treated BxPC3 cells were used in a capillary formation assay, the capillary length was reduced by 21% and 49%, respectively. Therefore 2A3 is an ideal candidate for treating tumours that over-express CEACAM6., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Introduction: Stem cells are being investigated as catalysts of tissue regeneration to either directly replace or promote cellularity lost as a result of traumatic injury or degenerative disease. In many reports, despite low numbers of stably integrated cells, the transient presence of cells delivered or recruited to sites of tissue remodeling globally benefits functional recovery. Such findings have motivated the need to determine how paracrine factors secreted from transplanted cells may be capable of positively impacting endogenous repair processes and somatic cell responses., Methods: Embryonic stem cells were differentiated as embryoid bodies (EBs) in vitro and media conditioned by EBs were collected at different intervals of time. Gene and protein expression analysis of several different growth factors secreted by EBs were examined by polymerase chain reaction and enzyme-linked immunosorbent assay analysis, respectively, as a function of time. The proliferation and migration of fibroblasts and endothelial cells treated with EB conditioned media was examined compared with unconditioned and growth media controls., Results: The expression of several growth factors, including bone morphogenic protein-4, insulin-like growth factors and vascular endothelial growth factor-A, increased during the course of embryonic stem cell (ESC) differentiation as EBs. Conditioned media collected from EBs at different stages of differentiation stimulated proliferation and migration of both fibroblasts and endothelial cells, based on 5-bromo-2'-deoxyuridine incorporation and transwell assays, respectively., Conclusions: Overall, these results demonstrate that differentiating ESCs express increasing amounts of various growth factors over time that altogether are capable of stimulating mitogenic and motogenic activity of exogenous cell populations.

Tumor angiogenesis is a complicated process based upon a sequence of interactions between tumor and vessel endothelial cells. Tumor conditioned medium has been widely used to stimulate endothelial cells in vitro angiogenesis. This work was aimed to investigate the effects of gold nanoparticles (GNPs) on angiogenesis in hepatic carcinoma-conditioned endothelial cells. Human umbilical vein endothelial cells (HUVECs) were cultured with conditioned medium (CM) from the human hepatocarcinoma cell line HepG2 (HepG2-CM), and then treated with different concentrations of GNPs. The effects of GNPs on the viability, migration and active VEGF level of HUVECs were investigated by MTT assay, wound healing assay and transwell chamber assay, and ELISA assay, respectively. The data showed that GNPs significantly inhibited HUVECs proliferation and migration induced by HepG2-CM, and also reduced the levels of active VEGF in the co-culture system. Then, the alterations in morphology and ultrastructure of HUVECs detected by atomic force microscopy (AFM) showed that there appeared obvious pseudopodia, larger membrane particle sizes and much rougher surface in HUVECs after HepG2-CM treatment, which were all reversed after GNPs treatment. Changes in cytoskeleton of HUVECs determined by immunocytochemistry demonstrated that GNPs treatment remarkably inhibited the activation effect of HepG2-CM on HUVECs, which was associated with the disruption of actin filaments induced by GNPs. This study indicates that GNPs can significantly inhibit HepG2-CM activated endothelial cell proliferation and migration through down-regulation of VEGF activity and disruption of cell morphology, revealing the potential applications of GNPs as antiangiogenic agent for the treatment of hepatic carcinoma., (Copyright © 2014. Published by Elsevier Ltd.)

Objectives: Proteinase inhibitors act as a defensive system against predators e.g. insects, in plants. Bauhinia bauhinioides kallikrein inhibitor (BbKI) is a serine proteinase inhibitor, isolated from seeds of Bauhinia bauhinioides and is structurally similar to plant Kunitz-type inhibitors but lacks disulfide bridges. In this study we evaluated the antiproliferative effect of BbKI on endothelial cells and its impact on changes in membrane potential and intracellular calcium., Materials and Methods: HUVEC proliferation was significantly reduced by incubation with BbKI 50 and 100 µM 12% and 13%. Furthermore, BbKI (100 µM) exposure caused a significant increase in intracellular Ca2+ concentration by 35% as compared to untreated control., Results: The intracellular rise in calcium was not affected by the absence of extracellular calcium. BBKI also caused a significant change in the cell membrane potential but the antiproliferative effect was independent of changes in membrane potential., Conclusions: BBKI has an antiproliferative effect on HUVEC, which is independent of the changes in membrane potential, and it causes an increase in intracellular Ca2+.

Background: Tumors may develop resistance to specific angiogenic inhibitors via activation of alternative pathways. Therefore, multiple angiogenic pathways should be targeted to achieve significant angiogenic blockade. In this study we investigated the effects of a combined application of the angiogenic inhibitors endostatin and tumstatin in a model of human glioblastoma multiforme., Results: Inhibitors released by stably transfected porcine aortic endothelial cells (PAE) showed anti-angiogenic activity in proliferation and wound-healing assays with endothelial cells (EC). Interestingly, combination of endostatin and tumstatin (ES + Tum) also reduced proliferation of glioma cells and additionally induced morphological changes and apoptosis in vitro. Microencapsulated PAE-cells producing these inhibitors were applied for local therapy in a subcutaneous glioblastoma model. When endostatin or tumstatin were applied separately, in vivo tumor growth was inhibited by 58% and 50%, respectively. Combined application of ES + Tum, in comparison, resulted in a significantly more pronounced inhibition of tumor growth (83%). cDNA microarrays of tumors treated with ES + Tum revealed an up-regulation of prolactin receptor (PRLR). ES + Tum-induced up-regulation of PRLR in glioma cells was also found in in vitro. Moreover, exogenous PRLR overexpression in vitro led to up-regulation of its ligand prolactin and increased proliferation suggesting a functional autocrine growth loop in these cells., Conclusion: Our data indicate that integrin-targeting factors endostatin and tumstatin act additively by inhibiting glioblastoma growth via reduction of vessel density but also directly by affecting proliferation and viability of tumor cells. Treatment with the ES + Tum-combination activates the PRLR pro-proliferative pathway in glioblastoma. Future work will show whether the prolactin signaling pathway represents an additional target to improve therapeutic strategies in this entity.

Background: We have recently reported that Origanummajorana exhibits anticancer activity by promoting cell cycle arrest and apoptosis of the metastatic MDA-MB-231 breast cancer cell line. Here, we extended our study by investigating the effect of O. majorana on the migration, invasion and tumor growth of these cells., Results: We demonstrate that non-cytotoxic concentrations of O. majorana significantly inhibited the migration and invasion of the MDA-MB-231 cells as shown by wound-healing and matrigel invasion assays. We also show that O. majorana induce homotypic aggregation of MDA-MB-231 associated with an upregulation of E-cadherin protein and promoter activity. Furthermore, we show that O. majorana decrease the adhesion of MDA-MB-231 to HUVECs and inhibits transendothelial migration of MDA-MB-231 through TNF-α-activated HUVECs. Gelatin zymography assay shows that O. majorana suppresses the activities of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9). ELISA, RT-PCR and Western blot results revealed that O. majorana decreases the expression of MMP-2, MMP-9, urokinase plasminogen activator receptor (uPAR), ICAM-1 and VEGF. Further investigation revealed that O. majorana suppresses the phosphorylation of IκB, downregulates the nuclear level of NFκB and reduces Nitric Oxide (NO) production in MDA-MB-231 cells. Most importantly, by using chick embryo tumor growth assay, we also show that O. majorana promotes inhibition of tumor growth and metastasis in vivo., Conclusion: Our findings identify Origanummajorana as a promising chemopreventive and therapeutic candidate that modulate breast cancer growth and metastasis.

Aim: The aim of this study was to investigate the effects of bemiparin, nadroparin, enoxaparin, and heparin on viability of human umbilical vein endothelial cells (HUVEC)., Methods: Cultivation of HUVEC was performed in an incubator having 5 % CO(2) at 37 °C and with predefined supplemented medium, until cell monolayers attained confluence which occurred after 7 days. The assays were performed in the exponential growth phase of the cells. The cell viability was assessed using the cleavage of tetrazolium salts added to the culture medium. Heparin sodium, enoxaparin sodium, bemiparin sodium, and nadroparin calcium with concentrations of 100, 10, and 1 IU/100 μL were used for the proliferation assay in which cells were incubated for 24, 48, and 72 h with these drugs. The experiments were conducted in four replicates., Results: Among the study drugs with maximal concentration used in the experiments (100 IU/100 μL), heparin was found to be associated with the lowest viability score in 24 and 48 h, while bemiparin showed the lowest at 72 h. Bemiparin 100 IU/100 μL was significantly associated with lower viability score than that of bemiparin 10 IU/100 μL and bemiparin 1 IU/100 μL at every time interval. Among gradual concentrations of enoxaparin, however, concentration of 1 IU/100 μL was associated with the lowest viability scores at every time point. Heparin 1 IU/100 μL, nadroparin 100 IU/100 μL, and enoxaparin 100 IU/100 μL groups had the highest viability score after 72 h of incubation., Conclusion(s): Among low molecular weight heparins (LMWHs), 100 IU/100 μL concentration of bemiparin was associated with a more pronounced effect on reducing viability of HUVEC after 72 h of incubation, while nadroparin 100 IU/100 μL and enoxaparin 100 IU/100 μL showed the least effects. LMWHs differ both from each other and heparin with respect to their effects on cellular viability of HUVEC in dose- and time-dependent manner.

Over the past 10 years, a great deal has been learned about the fundamental biology and therapeutic application of bone marrow-derived human mesenchymal stem cells (MSCs). Intravenous administration of these cells is the preferred route for therapeutic delivery of MSCs. Vascular endothelial cells (ECs) are the first cell type that MSCs encounter following IV administration. However, little is known about the biological consequences of interactions between MSCs and ECs, and if any therapeutic benefit results from this interaction. We show that MSCs exert potent stabilizing effects on ECs using an in vitro coculture system. Such effects include decreased EC proliferation and the reduction of EC vascular network formation in matrigel. Interestingly, these effects appear to require EC-MSC contact and result in enhanced colocalization of VE-Cadherin and β-catenin at the cell membrane. Disruption of the VE-Cadherin/β-catenin interaction abrogates the observed effects. As a functional in vivo correlate, we show that intravenously administered MSCs strongly inhibit angiogenesis in a matrigel plug assay. Taken together, these results identify a novel mechanism of action of MSCs that involves a contact-dependent EC interaction. These findings are relevant to intravenous use of MSCs and provide insight into further optimizing therapeutic strategies involving MSCs.

Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1 and 2 (Epn1/2) die at embryonic day 10 and exhibit an abnormal vascular phenotype. To examine the angiogenic role of endothelial epsins, we generated mice with constitutive or inducible deletion of Epn1/2 in vascular endothelium. These mice exhibited no abnormal phenotypes under normal conditions, suggesting that lack of endothelial epsins 1 and 2 did not affect normal blood vessels. In tumors, however, loss of epsins 1 and 2 resulted in disorganized vasculature, significantly increased vascular permeability, and markedly retarded tumor growth. Mechanistically, we show that VEGF promoted binding of epsin to ubiquitinated VEGFR2. Loss of epsins 1 and 2 specifically impaired endocytosis and degradation of VEGFR2, which resulted in excessive VEGF signaling that compromised tumor vascular function by exacerbating nonproductive leaky angiogenesis. This suggests that tumor vasculature requires a balance in VEGF signaling to provide sufficient productive angiogenesis for tumor development and that endothelial epsins 1 and 2 negatively regulate the output of VEGF signaling. Promotion of excessive VEGF signaling within tumors via a block of epsin 1 and 2 function may represent a strategy to prevent normal angiogenesis in cancer patients who are resistant to anti-VEGF therapies.

Psoriasin (S100A7), originally identified in psoriasis, is a calcium-binding protein belonging to the multigenic S100 family. In high-grade ductal carcinoma in situ, psoriasin was identified as one of the most abundant transcripts. We have previously shown that psoriasin was induced by reactive oxygen species (ROS). Moreover, the downregulation of psoriasin by short hairpin RNA (shRNA) led to the reduced expression of vascular endothelial growth factor (VEGF) and inhibited tumor growth in vivo. The aim of the present study was to investigate whether psoriasin could have direct effects on endothelial cells. In this study we demonstrated that psoriasin increased VEGF expression in mammary epithelial cells. The treatment of endothelial cells with recombinant psoriasin increased proliferation comparable to that of recombinant VEGF protein. No change in proliferation was seen when endothelial cells were infected with psoriasin-expressing adenoviruses, suggesting that the proliferative effect of psoriasin was mediated by a specific receptor. Treatment with sRAGE, targeting the receptor for advanced glycation end products (RAGE), thus inhibited endothelial cell proliferation and tube formation enhanced by recombinant psoriasin. We showed that VEGF expression was not induced by hydrogen peroxide, when psoriasin was silenced by shRNA, which led to the hypothesis that psoriasin induces ROS. Indeed, psoriasin was shown to induce ROS in both endothelial and epithelial cells. Moreover, sRAGE inhibited the psoriasin-dependent generation of ROS in endothelial cells. Finally, treatment with antioxidant Bcl-2 protein abolished the effect of psoriasin on endothelial cell proliferation. Our data suggest that psoriasin expression in mammary epithelial cells leads to increased endothelial cell proliferation in a paracrine manner through RAGE. Psoriasin may therefore play a role in breast cancer progression by promoting oxidative stress response and angiogenesis.

Proliferation and migration of vascular smooth muscle cells (VSMCs) are critical events in the initiation and development of restenosis upon percutaneous transluminal coronary angioplasty (PTCA). Polyphenols have been suggested to ameliorate post-angioplasty restenosis. Salvianolic A (SalA) is one of the most abundant polyphenols extracted from salvia. In this study, we investigated the effect of salvianolic A (SalA) on the migration and proliferation of VSMCs. We found a preferential interaction of SalA with cellular systems that rely on the PDGF signal, but not on the EGF and bFGF signal. SalA inhibits PDGF-BB induced VSMC proliferation and migration in the concentration range from 0.01 to 0.1 μM. The inhibition of SalA on VSMC proliferation is associated with cell cycle arrest. We also found that SalA inhibits the PDGFRβ-ERK1/2 signaling cascade activated by PDGF-BB in VSMCs. In addition, SalA does not influence the proliferation of endothelial cells, the synthesis of NO and eNOS protein expression. Our results suggest that SalA inhibits migration and proliferation of VSMCs induced by PDGF-BB via the inhibition of the PDGFRβ-ERK1/2 cascade, but that it does not constrain endothelial cell proliferation and nitric oxide biosynthesis. Thus, the present study suggests a novel adjunct pharmacological strategy to prevent angioplasty-related restenosis.

Many studies suggest that far-infrared (FIR) therapy can reduce the frequency of some vascular-related diseases. The non-thermal effect of FIR was recently found to play a role in the long-term protective effect on vascular function, but its molecular mechanism is still unknown. In the present study, we evaluated the biological effect of FIR on vascular endothelial growth factor (VEGF)-induced proliferation in human umbilical vein endothelial cells (HUVECs). We found that FIR ranging 3∼10 µm significantly inhibited VEGF-induced proliferation in HUVECs. According to intensity and time course analyses, the inhibitory effect of FIR peaked at an effective intensity of 0.13 mW/cm(2) at 30 min. On the other hand, a thermal effect did not inhibit VEGF-induced proliferation in HUVECs. FIR exposure also inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinases in HUVECs. FIR exposure further induced the phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) and NO generation in VEGF-treated HUVECs. Both VEGF-induced NO and reactive oxygen species generation was involved in the inhibitory effect of FIR. Nitrotyrosine formation significantly increased in HUVECs treated with VEGF and FIR together. Inhibition of phosphoinositide 3-kinase (PI3K) by wortmannin abolished the FIR-induced phosphorylation of eNOS and Akt in HUVECs. FIR exposure upregulated the expression of PI3K p85 at the transcriptional level. We further found that FIR exposure induced the nuclear translocation of promyelocytic leukemia zinc finger protein (PLZF) in HUVECs. This induction was independent of a thermal effect. The small interfering RNA transfection of PLZF blocked FIR-increased PI3K levels and the inhibitory effect of FIR. These data suggest that FIR induces the nuclear translocation of PLZF which inhibits VEGF-induced proliferation in HUVECs.

Aim: To investigate the action of salvianolic acid A (SalA) on angiotensin II (Ang II)-induced proliferation of human umbilical vein endothelial cells (HUVECs) and the possible signaling pathways mediating this action., Methods: Cell proliferation was examined with MTT assay. The expression levels of Src phosphorylation (phospho-Src), Akt phosphorylation (phospho-Akt), and NADPH oxidase 4 (Nox4) in HUVECs were determined by Western blot. The production of reactive oxygen species (ROS) was estimated using fluorescence-activated cell sorting (FACS)., Results: SalA (6.25-50 μmol/L) did not affect the viability of HUVECs. Treatment of HUVECs with Ang II (1 μmol/L) markedly increased the cell viability; pretreatment of HUVECs with SalA (12.5, 25 and 50 μmol/L) prevented Ang II-induced increase of the cell viability in a concentration-dependent manner. Treatment of HUVECs with Ang II (1 μmol/L) markedly up-regulated the protein expression levels of phospho-Src, phospho-Akt (473) and Nox4; pretreatment of HUVECs with SalA (12.5, 25 and 50 μmol/L) blocked all the effects in a concentration-dependent manner. Treatment of HUVECs with Ang II (1 μmol/L) dramatically increased ROS production in HUVECs; pretreatment of HUVECs with SalA (12.5, 25 and 50 μmol/L) blocked the ROS production in a concentration-dependent manner., Conclusion: SalA inhibits Ang II-induced proliferation of HUVECs via reducing the expression levels of phospho-Src and phospho-Akt (473), thereby attenuating the production of ROS.

A major clinical problem of high relevance in the cardiovascular field is late stent thrombosis after implantation of drug eluting stents (DES). Clinical widely used DES currently utilize durable polymer coatings, which can induce persistent arterial wall inflammation and delayed vascular healing resulting in an impaired endothelialization. In this study we explored the interaction of smooth muscle cells (SMC) and human umbilical vein endothelial cells (HUVEC) with electrospun scaffolds prepared from resorbable polyetheresterurethane (PDC) and poly(p-dioxanone) (PPDO), as well as polyetherimide (PEI), which can be surface modified, in comparison to poly(vinylidene fluoride-co-hexafluoropropene) (PVDF) as reference material, which is established as coating material of DES in clinical applications. Our results show that adhesion could be improved for HUVEC on PDC, PPDO and PEI compared to PVDF, whereas almost no SMC attached to the scaffolds indicating a cell-specific response of HUVEC towards the different fibrous structures. Proliferation and apoptosis results revealed that PPDO and PEI have no significant negative influence on vitality and cell cycle behaviour compared to PVDF. Hence, they represent promising candidates for temporary blood vessel support that induce HUVEC attachment and prevent SMC proliferation.

Objective: To observe the effect of recombinant periostin protein on the proliferation, apoptosis and migration of human umbilical vein endothelial cells (HUVECs) in acidic environment., Methods: HUVECs were divided into three groups: normal control (pH 7.31±0.03), acidic control (pH 6.95±0.05) and experimental groups [(periostin 100 μg/L, (pH 6.95±0.05)]. The cellular proliferation,apoptosis and migration were measured by Cell Counting Kit-8(CCK-8) assay, Annexin V-FITC with Flow Cytometer(at 60 h after periostin was given) and Transwell chamber assay (at 20 h after periostin was given)., Results: In CCK-8 assay, the optical density values D450 nm (at 72 h after periostin was given) of normal control, acidic control and experimental groups were 0.71± 0.05, 0.62±0.04, 0.69±0.02, respectively. In the same order, the total apoptotic rate (%) of the three groups were 13.06±1.35, 16.95±0.46, 12.97±1.60, respectively; the numbers of migrated cells of the three groups were 67.45±11.88,44.89±8.67,64.60±9.63, respectively. Compared with normal control, the D(450 nm) value and the number of migrated cells of acidic control reduced 12.68% (P<0.01) and 33.44% (P<0.01) respectively. Otherwise, the total apoptotic rate (%) increased by 29.79% (P<0.05). Compared with acidic control, the D(450 nm) value and the number of migrated cells of experimental group increased by 11.29% (P<0.01) and 43.90% (P<0.01), respectively. Meantime, the total apoptotic rate (%) reduced 23.48% (P<0.05)., Conclusion: In acidic environment, the HUVECs' abilities of proliferation and migration were lower; while, the apoptotic value was raised. The recombinant periostin protein could counteract the acidic influence by enhancing the cellular abilities of proliferation and migration. Periostin could make endothelial cells reisist acidosis and keep growing to form blood vessels so that the local histiocytes could get enough nutrients for survival.