Zinc oxide nanoparticles induce migration and adhesion of monocytes to endothelial cells and accelerate foam cell formation

Metal oxide nanoparticles are widely used in industry, cosmetics, and biomedicine. However, the effects of exposure to these nanoparticles on the cardiovascular system remain unknown. The present study investigated the effects of nanosized TiO₂ and ZnO particles on the migration and adhesion of monocytes, which are essential processes in atherosclerogenesis, using an in vitro set-up of human umbilical vein endothelial cells (HUVECs) and human monocytic leukemia cells (THP-1). We also examined the effects of exposure to nanosized metal oxide particles on macrophage cholesterol uptake and foam cell formation. The 16-hour exposure to ZnO particles increased the level of monocyte chemotactic protein-1 (MCP-1) and induced the migration of THP-1 monocyte mediated by increased MCP-1. Exposure to ZnO particles also induced adhesion of THP-1 cells to HUVECs. Moreover, exposure to ZnO particles, but not TiO₂ particles, upregulated the expression of membrane scavenger receptors of modified LDL and increased cholesterol uptake in THP-1 monocytes/macrophages. In the present study, we found that exposure to ZnO particles increased macrophage cholesterol uptake, which was mediated by an upregulation of membrane scavenger receptors of modified LDL. These results suggest that nanosized ZnO particles could potentially enhance atherosclerogenesis and accelerate foam cell formation.     

NME1 suppression of endometrial stromal cells promotes angiogenesis in the endometriotic milieu via stimulating the secretion of IL-8 and VEGF

Nonmetastatic gene 23-H1 (NME1, also known as nm23-H1) is a wide-spectrum tumor metastasis suppressor gene that plays an important role in suppressing the proliferation, adhesion and invasion of endometrial stromal cells (ESCs). The present study is undertaken to explore the mechanism by which NME1 in ESCs from endometriosis modulates the angiogenesis and herein participates in the pathogenesis of endometriosis. The expression of NME1 in the primary ESCs from normal endometrium without endometriosis was higher than that from eutopic endometrium and ectopic lesion with endometriosis. Silencing NME1 stimulated the secretion of angiogenic factors interleukin-8 (IL-8) and vascular-endothelial growth factor (VEGF) of the eutopic ESCs from women with endometriosis, and these effects could be abrogated by MAPK/ERK1/2 or AKT inhibitor. In addition, the supernatant of NME1-silenced ESCs increased the expression of angiogenesis-relative molecules CD62E and CD105, and promoted angiogenesis of human umbilical vein endothelial cells (HUVECs). Anti-human IL-8 or VEGF neutralizing antibody reversed the effect on angiogenesis of HUVECs induced by NME1-silenced ESCs. Our current results suggest that the abnormal lower expression of NME1 in ESCs secrete more IL-8 and VEGF through activation of MAPK/ERK1/2 and AKT signal pathways, up-regulate the level of CD62E and CD105, and finally lead to numerous angiogenesis of vascular endothelial cells in the endometriotic milieu, which is beneficial to the origin and development of endometriosis.     

Protection effect of [Gly14]-Humanin from apoptosis induced by high glucose in human umbilical vein endothelial cells

AimsHumanin (HN) is known for its anti-apoptotic functions in neuronal cells. In this study, we sought to investigate the protective effect of [Gly14]-Humanin (HNG) in high glucose (HG)-induced apoptosis of human umbilical vein endothelial cells (HUVECs).Methods3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to examine cell viability, DNA chromatin morphology was assessed using Hoechst 33342 staining, and the generation of intracellular reactive oxygen species (ROS) was assessed using the fluorescent probe dichlorofluorescein diacetate (DCFH-DA). The expression of poly ADP-ribose polymerase (PARP), the pro-apoptotic protein bax and the anti-apoptotic protein bcl-2 were examined using western blot analysis. The mRNA level of bax and bcl-2 were detected by quantitative Real-Time PCR.ResultsCompared with treatment with HG 72 h, pretreatment with HNG for 3 h significantly increased cell viability (P < 0.001), reduced nuclear fluorescence of HUVECs (P < 0.05), the levels of cleaved PARP (P < 0.05), ROS formation (P < 0.05) and the ratio of bax/bcl-2 (P < 0.05) compared with treatment with HG for 72 h. Quantitative Real-Time PCR showed that mRNA level of bax reduced (P < 0.05) and mRNA level of bcl-2 increased (P < 0.05) after pretreatment with HNG.ConclusionsOur results imply that HNG can protect HUVECs from apoptosis induced by HG through the bax/bcl-2 pathway.     

Heterophilic interactions between cell adhesion molecule L1 and α<Subscript>v</Subscript> β<Subscript>3</Subscript>-integrin induce HUVEC process extension <Emphasis Type="Italic">in vitro</Emphasis> and angiogenesis <Emphasis Type="Italic">in vivo</Emphasis>

Cell adhesion molecule L1 was implicated in angiogenic processes, tumor formation and metastasis. Here, we provide evidence that the sixth Ig-like domain of L1 (L1Ig6) interacts with _{v 3} to induce process extension of human umbilical vein endothelial cells (HUVECs) in vitro and angiogenesis in vivo. HUVECs formed network-like structures on full-length L1 or L1Ig6 substrates comparable to structures found on matrigel. In the presence of mab _{v 3} or cyclic RGD, apoptosis was induced. In fibrin matrices where L1Ig6 was covalently incorporated, HUVECs formed multicellular and hollow processes through interactions between cell-surface _{v 3} and RGD-sites of matrix-immobilized L1Ig6. No such processes were induced by L1Ig6 having non-functional RDG-sites, or in the presence of mab _{v 3} or cyclic RGD. In those matrices, increased apoptosis was found. Co-immunoprecipitation of L1 or L1Ig6 with _{v 3} suggests close interactions. Furthermore, L1Ig6 stimulated HUVECs showed increased tyrosine phosphorylation of _{v 3} and phosphorylation of MAP kinases (ERK1 and ERK2) but not AKT indicating specific activation of _v and _{v 3} followed by activation of downstream kinases. Application of L1Ig6-modified fibrin matrices on CAMs induced 50–60% increased _v and _v3 protein expression and in vivo angiogenesis indicated by ~50% increased mean vascular length density. The results demonstrate angiogenic potential of L1Ig6 involving ligation and activation of _v3     

GLP-1 promotes angiogenesis in human endothelial cells in a dose-dependent manner,through the Akt,Src and PKC pathways

Introduction

Novel anti-diabetic medications that mimic or augment the physiological actions of GLP-1 improve cardiovascular risk factors in diabetics and GLP-1 has been proposed to have a beneficial role in the cardiovascular system. GLP-1 may have a direct cardioprotective role by decreasing infarct size and protecting from ischemia–reperfusion injury while prolonging survival in rodent models. The mechanisms underlying these observations remain largely unknown. In vitro studies suggest that GLP-1 may promote endothelial cell proliferation, but no study to date has evaluated a potential direct effect of GLP-1 on angiogenesis.

Specific Aim

To evaluate whether GLP-1 affects angiogenesis in humans and to elucidate underlying molecular mechanisms.

Material and Methods

We utilized a 3D culture system where spherules of human umbilical vein endothelial cells (HUVECs) embedded in a collagen scaffold were treated with escalating doses of human recombinant GLP-1 (50–2000 nmol/L) and the formation of new vessels was observed and quantified. Signaling inhibitors were utilized to identify molecular pathways through which GLP-1 promotes angiogenesis.

Results

We demonstrate that GLP-1 promotes angiogenesis in a dose-dependent manner. The maximum effect on angiogenesis was observed at a GLP-1 dose of 500 nmol/L, while increased angiogenesis occurred in response to doses ranging from 200 nmol/L to 1000 nmol/L. Pre-treatment of the system with Akt inhibitor IV, Bisindolylmaleimide (PKC inhibitor) and src inhibitor I resulted in a significant decrease of the GLP-1 induced angiogenesis.

Conclusions

This is the first study to demonstrate that GLP-1 promotes angiogenesis in a HUVEC three dimensional in vitro model. This effect requires pharmacological doses and is mediated through the Akt, PKC and src pathways.     

OSU-A9 inhibits angiogenesis in human umbilical vein endothelial cells via disrupting Akt–NF-κB and MAPK signaling pathways

Since the introduction of angiogenesis as a useful target for cancer therapy, few agents have been approved for clinical use due to the rapid development of resistance. This problem can be minimized by simultaneous targeting of multiple angiogenesis signaling pathways, a potential strategy in cancer management known as polypharmacology. The current study aimed at exploring the anti-angiogenic activity of OSU-A9, an indole-3-carbinol-derived pleotropic agent that targets mainly Akt–nuclear factor-kappa B (NF-κB) signaling which regulates many key players of angiogenesis such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). Human umbilical vein endothelial cells (HUVECs) were used to study the in vitro anti-angiogenic effect of OSU-A9 on several key steps of angiogenesis. Results showed that OSU-A9 effectively inhibited cell proliferation and induced apoptosis and cell cycle arrest in HUVECs. Besides, OSU-A9 inhibited angiogenesis as evidenced by abrogation of migration/invasion and Matrigel tube formation in HUVECs and attenuation of the in vivo neovascularization in the chicken chorioallantoic membrane assay. Mechanistically, Western blot, RT-PCR and ELISA analyses showed the ability of OSU-A9 to inhibit MMP-2 production and VEGF expression induced by hypoxia or phorbol-12-myristyl-13-acetate. Furthermore, dual inhibition of Akt–NF-κB and mitogen-activated protein kinase (MAPK) signaling, the key regulators of angiogenesis, was observed. Together, the current study highlights evidences for the promising anti-angiogenic activity of OSU-A9, at least in part through the inhibition of Akt–NF-κB and MAPK signaling and their consequent inhibition of VEGF and MMP-2. These findings support OSU-A9's clinical promise as a component of anticancer therapy.     

Drug-Eluting Beads Loaded with Antiangiogenic Agents for Chemoembolization: In Vitro Sunitinib Loading and Release and In Vivo Pharmacokinetics in an Animal Model

PurposeThe combination of embolic beads with a multitargeted tyrosine kinase inhibitor that inhibits tumor vessel growth is suggested as an alternative and improvement to the current standard doxorubicin-eluting beads for use in transarterial chemoembolization. This study demonstrates the in vitro loading and release kinetics of sunitinib using commercially available embolization microspheres and evaluates the in vitro biologic efficacy on cell cultures and the resulting in vivo pharmacokinetics profiles in an animal model.Materials and MethodsDC Bead microspheres, 70–150 µm and 100–300 µm (Biocompatibles Ltd., Farnham, United Kingdom), were loaded by immersion in sunitinib solution. Drug release was measured in saline in a USP-approved flow-through apparatus and quantified by spectrophotometry. Activity after release was confirmed in cell culture. For pharmacokinetics and in vivo toxicity evaluation, New Zealand white rabbits received sunitinib either by intraarterial injection of 100–300 µm sized beads or per os. Plasma and liver tissue drug concentrations were assessed by liquid chromatography–tandem mass spectroscopy.ResultsSunitinib loading on beads was close to complete and homogeneous. A total release of 80% in saline was measured, with similar fast-release profiles for both sphere sizes. After embolization, drug plasma levels remained below the therapeutic threshold (< 50 ng/mL), but high concentrations at 6 hours (14.9 µg/g) and 24 hours (3.4 µg/g) were found in the liver tissue.ConclusionsDC Bead microspheres of two sizes were efficiently loaded with sunitinib and displayed a fast and almost complete release in saline. High liver drug concentrations and low systemic levels indicated the potential of sunitinib-eluting beads for use in embolization.     

Deoxynivalenol inhibits proliferation and induces apoptosis in human umbilical vein endothelial cells

Deoxynivalenol (DON) is a stable mycotoxins found in cereals infected by certain fungal species and causes adverse health effects in animals and human such as vomiting, diarrhea and reproductive toxicity. In this study, we investigated the toxic and apoptotic effects of DON in human umbilical vein endothelial cells (HUVECs), a good model for studying inflammation. The results show that DON significantly inhibited the viability of HUVECs. DON could also inhibit the proliferation of HUVECs through G2/M phase arrest in cell cycle progression. Moreover, oxidative stress induced by DON was indicated by observations of increased levels of reactive oxygen species (ROS). In addition, DON also causes mitochondrial damage by decreasing the mitochondrial membrane potential and inducing apoptosis by up-regulation of apoptosis-related genes like caspase-3, caspase-9, and Bax genes, and down-regulation of Bcl-2 gene. These results together suggest that DON could induce cell cycle arrest, oxidative stress, and apoptosis in HUVECs.     

MiR-495通过下调PCNA抑制人血管内皮细胞增殖的作用研究

探讨MicroRNA-495（miR-495）对人血管内皮细胞增殖的作用及其分子作用机制。方法 选择人脐静脉内皮细胞（HUVECs）,利用lipofectamine2000转染试剂,将 miR-495 mimics（模拟物）或 miR-495 inhibitors（抑制物） 转染HUVECs,分别以mimics control和inhibitors control作为对照。通过MTT实验分析过表达或干扰miR-495对HUVECs细胞增殖的影响,进而通过生物信息学软件预测,双荧光素酶报告基因系统,荧光定量PCR及Western blot,预测并验证miR-495靶基因,最后通过功能互补实验,观察提高或敲低PCNA（proliferating cell nuclear antigen）表达对HUVECs细胞增殖的影响。结果 体外细胞增殖实验发现,过表达miR-495可明显抑制HUVECs细胞的增殖,而干扰miR-495则可促进HUVECs细胞的增殖。PCNA是miR-495的靶基因,过表达miR-495可明显下调HUVECs细胞中PCNA mRNA及蛋白表达水平。功能互补实验显示,敲低PCNA的表达亦可降低HUVECs细胞的增殖,反之促进HUVECs细胞的增殖。结论 MiR-495可抑制HUVECs细胞的增殖, 其作用机制主要通过下调PCNA的表达而实现。     

Ginsenoside Rb1 prevents homocysteine-induced endothelial dysfunction via PI3K/Akt activation and PKC inhibition

Hyperhomocysteinemia (HHcy), a risk factor for cardiovascular disease, is associated with endothelial dysfunction. Ginsenoside Rb1, the major active constituent of ginseng, potently attenuates homocysteine (Hcy)-induced endothelial damage. However, the underlying mechanism remains unknown. In this study, we have investigated the effect of Ginsenoside Rb1 on Hcy-induced endothelial dysfunction and its underlying signal pathway in vivo and in vitro. Ginsenosides prevented Hcy-induced impairment of endothelium-dependent relaxation and Rb1 reversed Hcy-induced reduction of NO production in a dose-dependent manner as detected by nitrate reductase method. Rb1 activated serine-1177 phosphorylation of endothelial nitric oxide synthase (eNOS) and serine-473 phosphorylation of Akt, while inhibited threonine-495 phosphorylation of eNOS as detected by western blotting. Rb1-induced phosphorylation of serine-1177 was significantly inhibited by wortmannin, PI3K inhibitor or SH-5, an Akt inhibitor, and partially reversed by Phorbol 12-myristate 13-acetate (PMA), a PKC activator. PMA also stimulated phosphorylation of threonine-495 which was inhibited by Rb1. Here we show for the first time that Rb1 prevents Hcy-induced endothelial dysfunction via PI3K/Akt activation and PKC inhibition. These findings demonstrate a novel mechanism of the action of Rb1 that may have value in prevention of HHcy associated cardiovascular disease.