Fumonisin B1 Inhibits Cell Proliferation and Decreases Barrier Function of Swine Umbilical Vein Endothelial Cells.

The fumonisins are a group of common mycotoxins found around the world that mainly contaminate maize. As environmental toxins, they pose a threat to human and animal health. Fumonisin B1 (FB1) is the most widely distributed and the most toxic. FB1 can cause pulmonary edema in pigs. However, the current toxicity mechanism of fumonisins is still in the exploratory stage, which may be related to sphingolipid metabolism. Our study is designed to investigate the effect of FB1 on the cell proliferation and barrier function of swine umbilical vein endothelial cells (SUVECs). We show that FB1 can inhibit the cell viability of SUVECs. FB1 prevents cells from entering the S phase from the G1 phase by regulating the expression of the cell cycle-related genes cyclin B1, cyclin D1, cyclin E1, Cdc25c, and the cyclin-dependent kinase-4 (CDK-4). This results in an inhibition of cell proliferation. In addition, FB1 can also change the cell morphology, increase paracellular permeability, destroy tight junctions and the cytoskeleton, and reduce the expression of tight junction-related genes claudin 1, occludin, and ZO-1. This indicates that FB1 can cause cell barrier dysfunction of SUVECs and promote the weakening or even destruction of the connections between endothelial cells. In turn, this leads to increased blood vessel permeability and promotes exudation. Our findings suggest that FB1 induces toxicity in SUVECs by affecting cell proliferation and disrupting the barrier function.

Effect of maternal oxytocin on umbilical venous and arterial blood flows during physiological-based cord clamping in preterm lambs.

BACKGROUND: Delayed umbilical cord clamping (UCC) after birth is thought to cause placental to infant blood transfusion, but the mechanisms are unknown. It has been suggested that uterine contractions force blood out of the placenta and into the infant during delayed cord clamping. We have investigated the effect of uterine contractions, induced by maternal oxytocin administration, on umbilical artery (UA) and venous (UV) blood flows before and after ventilation onset to determine whether uterine contractions cause placental transfusion in preterm lambs. METHODS AND FINDINGS: At ~128 days of gestation, UA and UV blood flows, pulmonary arterial blood flow (PBF) and carotid arterial (CA) pressures and blood flows were measured in three groups of fetal sheep during delayed UCC; maternal oxytocin following mifepristone, mifepristone alone, and saline controls. Each successive uterine contraction significantly (p<0.05) decreased UV (26.2±6.0 to 14.1±4.5 mL.min-1.kg-1) and UA (41.2±6.3 to 20.7 ± 4.0 mL.min-1.kg-1) flows and increased CA pressure and flow (47.1±3.4 to 52.8±3.5 mmHg and 29.4±2.6 to 37.3±3.4 mL.min-1.kg-1). These flows and pressures were partially restored between contractions, but did not return to pre-oxytocin administration levels. Ventilation onset during DCC increased the effects of uterine contractions on UA and UV flows, with retrograde UA flow (away from the placenta) commonly occurring during diastole. CONCLUSIONS: We found no evidence that amplification of uterine contractions with oxytocin increase placental transfusion during DCC. Instead they decreased both UA and UV flow and caused a net loss of blood from the lamb. Uterine contractions did, however, have significant cardiovascular effects and reduced systemic and cerebral oxygenation.

6-Nitrodopamine is released by human umbilical cord vessels and modulates vascular reactivity.

AIMS: Human umbilical cord vessels (HUCV) release dopamine and nitric oxide (NO). This study aims to verify whether HUCV release nitrocatecholamines such as 6-nitrodopamine (6-ND). MAIN METHODS: Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to identify 6-ND release from HUCV rings incubated in Krebs-Henseileit's solution. Vascular reactivity of HUCV rings was tested (with and without endothelium integrity) by suspension of the rings in an organ bath under isometric tension and application of 6-ND and other known mediators. KEY FINDINGS: LC-MS/MS revealed a basal release of 6-ND from endothelium intact from both human umbilical artery (HUA) and vein (HUV). The endothelium intact release was inhibited by the pre-treatment with NO synthesis inhibitor L-NAME (100 µM). In contrast to dopamine, noradrenaline and adrenaline, 6-ND did not contract HUCV, even in presence of L-NAME or ODQ. 6-ND (10 µM) produced a rightward shift of the concentration-response curves to dopamine (pA2: 5.96 in HUA and 5.72 in HUV). Contractions induced by noradrenaline and adrenaline were not affected by pre-incubation with 6-ND (10 µM). In U-46619 (10 nM) pre-contracted endothelium intact tissues, 6-ND and the dopamine D2-receptor antagonist haloperidol induced concentration-dependent relaxations of HUA and HUV. Incubation with the dopamine D1-receptor antagonist SCH-23390 (10 nM) abolished relaxation induced by fenoldopam but did not affect those induced by 6-ND. SIGNIFICANCE: 6-ND is released by HUCV and acts as a selective dopamine D2-receptor antagonist in this tissue. This represents a novel mechanism by which NO may modulate vascular reactivity independently of cGMP production.

Increased Interleukin-11 and Stress-Related Gene Expression in Human Endothelial and Bronchial Epithelial Cells Exposed to Silver Nanoparticles.

This article aimed to identify and distinguish the various responses to silver nanoparticles (NPs) of endothelial and epithelial cells. We also assessed the significantly increased gene expression levels, as shown by microarray analysis. We evaluated the median lethal dose of NPs in each cell line and found that each value was different. We also confirmed the toxicity of 5 nm silver NPs. Meanwhile, cell death was not observed in cells exposed to 100 nm silver NPs at a high concentration. We verified that 5 nm silver NPs affected the variation in gene expression in cells through microarray analysis and observed a noticeable increase in interleukin (IL)-8 and IL-11 gene expression in early stages. This study showed noticeable variation in the expression of oxidative stress-related genes in early stages. Microarray results showed considerable variation in cell death-, apoptosis-, and cell survival-related gene expression. Of note, IL-11 gene expression was particularly increased following the exposure of endothelial and epithelial cells to 5 nm silver NPs. In conclusion, this study demonstrated that intracellular genes specifically responded to silver NPs in respiratory epithelial cells and endothelial cells. Among cytokine genes, IL-11 expression was noticeably increased. Additionally, we confirmed that NP toxicity was affected by NP size and dose.

Protocol for Cryopreservation of Endothelial Monolayers.

One of the major challenges in the preservation of complex tissues is the cryosensitivity of the endothelium, the single layer of cells lining blood vessels, corneas, and other tissues. The increasing importance of endothelial monolayers in tissue-engineered constructs for transplantation and research warrants the need to develop protocols for the successful cryopreservation of cells in monolayers. In this chapter, we describe a recently published cryopreservation protocol that we developed based on examination of various factors that influence the post-thaw recovery of endothelial monolayers. To efficiently investigate cryopreservation protocol parameters, we employed an interrupted slow-cooling procedure (graded freezing) that allows dissecting loss of cell viability into contributions from slow-cooling injury and rapid-cooling injury. Our optimized protocol involves culturing cells on Rinzl plastic coverslips, using a combination of a penetrating cryoprotectant (5% dimethyl sulfoxide) and a non-penetrating cryoprotectant (6% hydroxyethyl starch), addition of 2% chondroitin sulfate, controlled cooling at 0.2 °C/min or 1 °C/min, and removal of cryoprotectant immediately after thaw. The protocol has been validated for human umbilical vein and porcine corneal endothelial cell monolayers.

Cryopreservation of Fern Spores and Pollen.

Fern spores and pollen are haploid plant germplasm of microscopic nature that can be used to regenerate full plants through germination (fern spores) or to fertilize seed-bearing plants through breeding programs (pollen). Due to their short life span in conventional storage (i.e., dry at -20 °C), the use of cryopreservation has been indicated for long-term ex situ conservation. While fern spores of most species and pollen from many seeded plants tolerate desiccation and can be stored dry at liquid nitrogen temperatures, some pollen is desiccation sensitive, and cryopreservation protocols require controlled drying and cooling and some level of cryoprotection. In this chapter we describe the cryopreservation process for fern spores used in the Millennium Seed Bank of Royal Botanic Gardens, Kew, including some details of the fern spores harvest and cleaning methods. In addition, two protocols for pollen cryopreservation are described, one generic for desiccation-tolerant pollen that can be used for multiple species and one specific for a desiccation sensitive pollen (Zea mays).

Complex Method of CT and Morphological Examination of Placental Angioarchitechtonics.

We propose an original method of complex assessment of the placental angioarchitechtonics based on computed tomography (CT) and morphological examination. A prerequisite condition of successful examination and assessment of the placental angioarchitechtonics is the pre-preparative stage including clearing of the placental and umbilical cord vessels from blood clots by placement of placenta into 10% hypertonic NaCl solution and then on a hygroscopic substrate. The major stage of this method is injection of contrast staining mixtures into the umbilical vessels followed by CT. The concentration of radiocontrast agent in water solution of gouache should be 70% for arteries and 15% for veins. The volumes of mixtures for contrast staining should be calculated according to the weight of the placenta. The contrast staining mixture was first injected into the catheterized unpaired umbilical vein, and then into both umbilical arteries. Each injection of the contrast staining mixture was visually inspected; then branching of the stained vessel was photographed and scanned by CT. The CT scans were used to construct 3D models of placental vessels and spectral color maps, which made it possible to examine the peculiarities of placental angioarchitechtonics, to identify and evaluate anastomoses of placental vessels, and to establish the type of these anastomoses.

The activity of IKCa and BKCa channels contributes to insulin-mediated NO synthesis and vascular tone regulation in human umbilical vein.

Insulin regulates the l-arginine/nitric oxide (NO) pathway in human umbilical vein endothelial cells (HUVECs), increasing the plasma membrane expression of the l-arginine transporter hCAT-1 and inducing vasodilation in umbilical and placental veins. Placental vascular relaxation induced by insulin is dependent of large conductance calcium-activated potassium channels (BKCa), but the role of KCa channels on l-arginine transport and NO synthesis is still unknown. The aim of this study was to determine the contribution of KCa channels in both insulin-induced l-arginine transport and NO synthesis, and its relationship with placental vascular relaxation. HUVECs, human placental vein endothelial cells (HPVECs) and placental veins were freshly isolated from umbilical cords and placenta from normal pregnancies. Cells or tissue were incubated in absence or presence of insulin and/or tetraethylammonium, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole, iberiotoxin or NG-nitro-l-arginine methyl ester. l-Arginine uptake, plasma membrane polarity, NO levels, hCAT-1 expression and placenta vascular reactivity were analyzed. The inhibition of intermediate-conductance KCa (IKCa) and BKCa increases l-arginine uptake, which was related with protein abundance of hCAT-1 in HUVECs. IKCa and BKCa activities contribute to NO-synthesis induced by insulin but are not directly involved in insulin-stimulated l-arginine uptake. Long term incubation (8 h) with insulin increases the plasma membrane hyperpolarization and hCAT-1 expression in HUVECs and HPVECs. Insulin-induced relaxation in placental vasculature was reversed by KCa inhibition. The results show that the activity of IKCa and BKCa channels are relevant for both physiological regulations of NO synthesis and vascular tone regulation in the human placenta, acting as a part of negative feedback mechanism for autoregulation of l-arginine transport in HUVECs.

Toxicity of cooking oil fume derived particulate matter: Vitamin D3 protects tubule formation activation in human umbilical vein endothelial cells.

Cooking oil fumes-derived PM2.5 (COFs-derived PM2.5) is the main source of indoor pollution. Exposure to COFs-derived PM2.5 can cause oxidative stress and affect angiogenesis. Here we investigated the roles of vitamin D3 (VD3) in protecting tubule formation injury induced by COFs-derived PM2.5, and the roles of ROS/NLRP3/VEGF signaling pathway in the effects. Human umbilical vein endothelial cells (HUVECs) were exposed to 0 (1‰ DMSO), 1000 nmol/l VD3, 100 µg/ml PM2.5, and 1000 nmol/l VD3 + 100 µg/ml PM2.5, respectively. Cell viability and tube formation, as well as protein and mRNA levels were measured. The results showed that exposure of COFs-derived PM2.5 dose-and time-dependently reduced the viability of HUVECs, increased the levels of mitochondrial and intracellular ROS, and changed the mitochondrial membrane potential level. While co-incubation with VD3 rescued these adverse effects. Both Western blot and real-time PCR (RT-PCR) showed that the expressions of NLRP3, caspase-1, Interleukin (IL)-1ß, and IL-18 in COFs-derived PM2.5 exposure group increased significantly, which could be effectively decreased by co-incubation with VD3. COFs-derived PM2.5 exposure could also reduce the expression of VEGF, while co-incubating HUVECs with VD3 evidently up-regulated the protein level of VEGF in HUVECs. In addition, COFs-derived PM2.5 could also inhibit the tube formation of HUVECs in vitro, which could be effectively rescued by the co-incubation of VD3. Our study proved that COFs-derived PM2.5 could damage the tubule formation of HUVECs in vitro, which could be effectively rescue by co-incubation with VD3, in which processes the ROS/NLRP3/VEGF signaling pathway played a crucial role. It provides a new theoretical basis for further study on the toxicity of PM2.5 to umbilical cord blood vessels.

Cyclic nucleotide-dependent relaxation in human umbilical vessels.

Umbilical vessels have a low sensitivity to dilate, and this property is speculated to have physiological implications. We aimed to investigate the different relaxing responses of human umbilical arteries (HUAs) and veins (HUVs) to agonists acting through the cAMP and cGMP pathways. Vascular rings were suspended in organ baths for isometric force measurement. Following precontraction with the thromboxane prostanoid (TP) receptor agonist U44069, concentration-response curves to the nitric oxide (NO) donor sodium nitroprusside (SNP), the soluble guanylate cyclase (sGC) stimulator BAY 41-2272, the adenylate cyclase (AC) activator forskolin, the ß-adrenergic receptor agonists isoproterenol (ADRB1), salmeterol (ADRB2), and BRL37344 (ADRB3), and the phosphodiesterase (PDE) inhibitors milrinone (PDE3), rolipram (PDE4), and sildenafil (PDE5) were performed. None of the tested drugs induced a relaxation higher than 30% of the U44069-induced tone. Rings from HUAs and HUVs showed a similar relaxation to forskolin, SNP, PDE inhibitors, and ADRB agonists. BAY 41-2272 was significantly more efficient in relaxing veins than arteries. ADRB agonists evoked weak relaxations (< 20%), which were impaired in endothelium-removed vessels or in the presence of the NO synthase inhibitor L-NAME, sGC inhibitor ODQ. PKA and PKG inhibitors impaired ADBR1-mediated relaxation but did not affect ADRB2-mediated relaxation. ADRB3-mediated relaxation was impaired by PKG inhibition in HUAs and by PKA inhibition in HUVs. Although HUA and HUV rings were relaxed by BRL37344, immunohistochemistry and RT-qPCR analysis showed that, compared to ADRB1 and ADRB2, ADRB3 receptors are weakly or not expressed in umbilical vessels. In conclusion, our study confirmed the low relaxing capacity of HUAs and HUVs from term infants. ADRB-induced relaxation is partially mediated by endothelium-derived NO pathway in human umbilical vessels.

In vitro profiling of endothelial volatile organic compounds under resting and pro-inflammatory conditions.

INTRODUCTION: The evaluation of volatile organic compounds(VOCs) emitted by human body offers a unique tool to set up new non-invasive devices for early diagnosis and long-lasting monitoring of most human diseases. However, their cellular origin and metabolic fate have not been completely elucidated yet, thus limiting their clinical application. Endothelium acts as an interface between blood and surrounding tissues. As such, it adapts its physiology in response to different environmental modifications thus playing a role in the pathogenesis of many metabolic and inflammatory diseases. OBJECTIVES: Since endothelium specifically reshapes its physiologic functions upon environmental changes the objective of this study was to evaluate if and how pro-inflammatory stimuli affect VOC metabolism in endothelial cell in culture. METHODS: Gas chromatography with mass spectrometric detection was applied to profile VOCs in the headspace of cultured endothelial cells (EC) in the absence or presence of the pro-inflammatory stimulus lipopolysaccharide (LPS). RESULTS: We observed that, under resting conditions, EC affected the amount of 58 VOCs belonging to aldehyde, alkane and ketone families. Among these, LPS significantly altered the amount of 15 VOCs. ROC curves show a perfect performance (AUC = 1) for 10 metabolites including 1-butanol, 3-methyl-1-butanol and 2-ethyl-1-hexanol. DISCUSSION: The emission and uptake of the aforementioned VOCs disclose potential unexplored metabolic pathways for EC that deserve to be investigated. Overall, we identified new candidate VOC potentially exploitable, upon experimental confirm in in vivo model of disease, as potential biomarkers of sepsis and pro-inflammatory clinical settings.

Arsenic trioxide inhibits angiogenesis in vitro and in vivo by upregulating FoxO3a.

Arsenic trioxide (As2O3) has been used clinically for the treatment of acute promyelocytic leukemia and some solid tumors. However, the mechanisms of its anti-tumor effects are still elusive. Angiogenesis is a key process for tumor initiation, and increasing evidence has supported the role of anti-angiogenesis caused by arsenic in tumor suppression, although the detailed mechanism is not well understood. In the present study, we found that As2O3 significantly inhibited the angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro, and this was mediated by the upregulation of FoxO3a. Knockdown of FoxO3a could restore the angiogenic ability of HUVECs. Moreover, vascular endothelial cell-specific knockout of FoxO3a in mice could disrupt the anti-angiogenesis effect of As2O3 and endow the tumors with resistance to As2O3 treatments. Our results revealed a new mechanism by which As2O3 suppresses angiogenesis and tumor growth.

Promoter methylation changes and vascular dysfunction in pre-eclamptic umbilical vein.

BACKGROUND: Hypertension is one of primary clinical presentations of pre-eclampsia. The occurrence and progress of hypertension are closely related to vascular dysfunction. However, information is limited regarding the pathological changes of vascular functions in pre-eclamptic fetuses. Human umbilical cord vein was used to investigate the influence of pre-eclampsia on fetal blood vessels in this study. RESULTS: The present study found that the vasoconstriction responses to arginine vasopressin (AVP) and oxytocin (OXT) were attenuated in the pre-eclamptic umbilical vein as compared to in normal pregnancy, which was related to the downregulated AVP receptor 1a (AVPR1a), OXT receptor (OXTR), and protein kinase C isoform ß (PKCß), owing to the deactivated gene transcription, respectively. The deactivated AVPR1a, OXTR, and PKCB gene transcription were respectively linked with an increased DNA methylation within the gene promoter. CONCLUSIONS: To the best of our knowledge, this study first revealed that a hyper-methylation in gene promoter, leading to relatively reduced patterns of AVPR1a, OXTR, and PKCB expressions, which was responsible for the decreased sensitivity to AVP and OXT in the umbilical vein under conditions of pre-eclampsia. The data offered new and important information for further understanding the pathological features caused by pre-eclampsia in the fetal vascular system, as well as roles of epigenetic-mediated gene expression in umbilical vascular dysfunction.

Adiponectin is protective against endoplasmic reticulum stress-induced apoptosis of endothelial cells in sepsis.

Endoplasmic reticulum (ER) stress is a critical molecular mechanism involved in the pathogenesis of sepsis. Hence, strategies for alleviating this stress may be essential for preventing cardiovascular injuries under sepsis. Adiponectin is secreted by adipocytes and its levels are decreased in sepsis. The purpose of this study was to investigate the protective effects of adiponectin treatment on endothelial cells and its mechanism. Male Wistar rats underwent cecal ligation and puncture (CLP) before being treated with adiponectin (72 and 120 µg/kg). The levels of malondialdehyde (MDA) in plasma, histological structure, and apoptosis of endothelial cells were evaluated. In vitro, human umbilical vein endothelial cells (HUVECs) were treated with adiponectin at 10 and 20 µg/mL for 24 h after stimulation by lipopolysaccharide (LPS). The levels of reactive oxygen species (ROS), ultrastructure, rate of apoptosis, the expression of inositol-requiring enzyme 1α (IRE1α) protein, and its downstream molecules (78 kDa glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP), and caspase-12) were detected. The results showed that the levels of MDA and ROS induced by CLP or LPS stimulation were increased. Furthermore, endothelial cell apoptosis was increased under sepsis. The IRE1α pathway was initiated, as evidenced by activated IRE1α, increased GRP78, and up-regulated CHOP and caspase-12 in HUVECs. Following treatment with adiponectin, the number of apoptotic endothelial cells was markedly decreased. These findings demonstrated that treatment with adiponectin decreased apoptosis of endothelial cells caused by sepsis by attenuating the ER stress IRE1α pathway activated by oxidative stress.

Bio-Effects of TiO2 Nanoparticles on Human Colorectal Cancer and Umbilical Vein Endothelial Cell Lines

Background: Due to the possible biomedical potential of nanoparticles, titanium dioxide nanoparticles (TiO2 NPs) have received great attention in cancer research. Although selectivity of cytotoxicity with TiO2 NPs in various cells is clinically significant comparisons of cancer and non-cancer cells have been limited. Therefore, we here studied exposure to TiO2 NPs in colorectal cancer cells (CRCs) and human umbilical vein endothelial cells (HUVECs). Methods: After characterization of TiO2 NPs, culture and treatment of cells (HCT116, HT29 and HUVEC), viability was assessed by MTT assay and in terms of morphological features. Acridine orange (AO) and propidium iodide (PI) assays were carried out to estimate the incidence of apoptosis. The RT-PCR method was also employed to evaluate the expression of P53, Bax, Bcl-2 and Caspase 3. Results: Exposure to increasing concentrations of TiO2 NPs enhanced overall cell survival of HCT116 cells and reduced the Bcl-2 and Caspase 3 expression while the ratio of Bax/Bcl-2 was down-regulated. TiO2 NPs at 400 and 50 µg/ml concentrations suppressed cell proliferation and induced apoptosis of HT29 cells and also up-regulated P53 and Bax at the mRNA level, enhanced the Bax/Bcl-2 ratio and eventually up-regulated Caspase 3 mRNA. Although, inhibition of cell proliferation in HUVECs was seen at 200 and 400 µg/ml TiO2 NPs, it was not marked. Conclusion: TiO2 NPs have selective bio-effects on exposed cells with dose- and cell-dependent influence on viability. Cell proliferation in HCT116 as a metastatic colorectal cancer cell line appeared to be stimulated via multiple signaling pathways, with promotion of apoptosis in less metastatic cells at 50 and 400 µg/ml concentrations. This was associated with elevated P53, Bax and Caspase 3 mRNA and reduced Bcl-2 expression. However, TiO2 NPs did not exert any apparent significant effects on HUVECs as hyperproliferative angiogenic cells.

Homocysteine induces vascular inflammatory response via SMAD7 hypermethylation in human umbilical vein smooth muscle cells.

Homocysteine (Hcy) can induce atherosclerosis through the inflammatory response and DNA methylation disorder. Our recent study has reported a novel epigenetic modified gene related to atherosclerosis -SMAD7. To further understand the pathogenesis of atherosclerosis, the current study was designed to investigate an inflammatory role of Hcy in human umbilical vein smooth muscle cells (HUVSMCs) through interfering with SMAD7 methylation. Using MALDI-TOF MS, we found that Hcy increased DNA methylation levels of SMAD7 promoter in a dose and time-dependent manner in HUVSMCs. Meanwhile, both SMAD7 mRNA and protein levels were decreased along with the increase of Hcy concentrations and treating time. Decreased SMAD7 levels led to up regulation of pro-inflammatory cytokines (TNF-α and IL-1ß) expression in HUVSMCs. Furthermore, we found that activation of NF-κB pathway was the mechanism by which reduced Smad7 levels enhanced vascular inflammation. Thus, Hcy is able to activate NF-κB-mediated vascular inflammatory response via inducing hypermethylation of SMAD7 promoter in HUVSMCs. The in vitro findings supplement our recent clinical study that SMAD7 methylation as a novel marker in atherosclerosis and further elucidate the role of Hcy in atherogenesis.

Free fatty acids mediates human umbilical vein endothelial cells inflammation through toll-like receptor-4.

OBJECTIVE: To investigate the role of Toll-like receptor-4 (TLR4) in the free fatty acids (FFAs) induced human umbilical vein endothelial cells (HUVECs) inflammation and to explore the underlying mechanisms. MATERIALS AND METHODS: HUVECs and HEK293 cell lines were obtained from Shanghai Type Culture Collection. Cell counting kit-8 (CCK8) and flow cytometry (FCM) were performed to examine the cell viability and apoptosis rate of HUVECs induced by FFAs treatments with or without infection of toll-like receptor-4 interference (TLR4i) adenovirus. Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the inflammatory cytokines release. Quantitative polymerase chain reaction (qPCR) and Western Blot (WB) were used to test the molecular mechanisms of inflammation. RESULTS: FFAs induced inflammatory responses in HUVECs via modulating the TLR4 receptor complex. TLR4i adenovirus interference increased cell viability and decreased cell apoptosis rate. FFAs treatments significantly increased the expressions of inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), C-C motif chemokine ligand 5 (CCL5) and CXC chemokine ligand 10 (CXCL10), while TLR4i adenovirus interference significantly reduced these cytokines levels. TLR4-mediated myeloid differential protein-88 (MyD88) expression activating the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and inhabiting kappa B kinase-beta (IKK-ß). TLR4i adenovirus interference decreased the expressions of these genes at both mRNA level and protein level. CONCLUSIONS: TLR4 mediates FFAs induced inflammatory responses in HUVECs. TLR4 interference in HUVECs significantly reduces the inflammatory cytokines expression, decreases the cell apoptosis rate and increases cell viability.

ROS-Autophagy pathway mediates monocytes-human umbilical vein endothelial cells adhesion induced by apelin-13.

Apelin is the endogenous ligand of APJ receptor. Both monocytes (MCs) and human umbilical vein endothelial cells (HUVECs) express apelin and APJ, which play important roles in the physiological processes of atherosclerosis. Our previous research indicated that apelin-13 promoted MCs-HUVECs adhesion. Here, we further explore the mechanism responsible for MCs-HUVECs adhesion induced by apelin-13. Apelin-13 promoted reactive oxygen species (ROS) generation and NOX4 expression in HUVECs. Apelin-13 inducedautophagy, increased proteins beclin1 and LC3-II/I expression and induced autophagy flux in HUVECs, which was blocked by NAC, catalase and DPI. Autophagy flux induced by apelin-13 was inhibited by NAC and catalase but not hydroxychloroquine (HCQ). NAC, catalase, and DPI prevented apelin-13 induced ICAM-1 expression in HUVECs. Rapamycin enhanced MCs-HUVECs adhesion that was reversed by NAC, catalase, and DPI. Down-regulation of beclin1 and LC3 by siRNA blocked MCs-HUVECs adhesion. Apelin-13 induced atherosclerotic plaque and increased NOX4, LC3-II/I expression in ApoE-/-(HFD) mouse model. Our results demonstrated that apelin-13 induced MCs-HUVECs adhesion via a ROS-autophagy pathway.

The long non-coding RNA MALAT1 activates Nrf2 signaling to protect human umbilical vein endothelial cells from hydrogen peroxide.

The potential effect of the long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) against hydrogen peroxide (H2O2)-induced oxidative injury in endothelial cells was tested. We show that forced-expression of MALAT1 using a lentiviral vector ("LV-MALAT1") significantly attenuated H2O2-induced death and apoptosis of human umbilical vein endothelial cells (HUVECs). Conversely, knocking down of MALAT1 by targeted siRNA exacerbated H2O2-induced HUVEC injury. For the mechanism study, we show that LV-MALAT1 induced Keap1 downregulation, leading to nuclear-factor-E2-related factor 2 (Nrf2) stabilization and activation. Critically, Nrf2 shRNA almost completely abolished LV-MALAT1-mediated HUVEC protection against H2O2. Significantly, H2O2-induced oxidative stress, lipid peroxidation and DNA damages in HUVECs were attenuated by LV-MALAT1, but were intensified with MALAT1 siRNA. In summary, we identified a novel signaling axis involving MALAT1, Keap1 and Nrf2, which in turn protects HUVECs from oxidative injury.

Aberrant promoter methylation of multiple genes in VSMC proliferation induced by Hcy.

Vascular smooth muscle cell (VSMC) proliferation is a primary pathological event in atherosclerosis (AS), and homocysteine (Hcy) is an independent risk factor for AS. However, the underlying mechanisms are still lagging. Studies have used the combination of methylation of promoters of multiple genes to diagnose tumors, thus the aim of the current study was to investigate the role of methylation status of several genes in VSMCs treated with Hcy. CpG islands were identified in the promoters of platelet­derived growth factor (PDGF), p53, phosphatase and tensin homologue on chromosome 10 (PTEN) and mitofusin 2 (MFN2). Hypomethylation was observed to occur in the promoter region of PDGF, hypermethylation in p53, PTEN and MFN2, and hypomethylation in two global methylation indicators, aluminium (Alu) and long interspersed nucleotide element­1 (Line­1). This was accompanied by an increase in the expression of PDGF, and reductions of p53, PTEN and MFN2, both in mRNA and protein levels. An elevation of S­adenosylmethionine (SAM) and a reduction of S­adenosylhomocysteine (SAH) and the SAM/SAH ratio were also identified. In conclusion, Hcy impacted methylation the of AS­associated genes and global methylation status that mediate the cell proliferation, which may be a character of VSMCs treated with Hcy. The data provided evidence for mechanisms of VSMCs proliferation in AS induced by Hcy and may provide a new perspective for AS induced by Hcy.