Damage In Human Umbilical Vein Endothelial Cells Research Articles

Antioxidant protein peroxiredoxin 6 suppresses the vascular inflammation, oxidative stress and endothelial dysfunction in angiotensin II-induced endotheliocyte.

Cardiovascular disease (CVD) states are associated with endothelial dysfunction (ED) and increased production of ROS in endothelial cells. The present study aimed to explore the protective effects of antioxidant protein peroxiredoxin 6 (PRDX6) on angiotensin II (AngII)‑induced human umbilical vein endothelial cell (HUVEC) dysfunction. To investigate cell viability, levels of inflammatory molecules and proteins were assayed using the CCK-8 assay and evaluated by ELISA and Western blot. NO and ROS levels were determined by Griess assay and the fluorescent probe DCFH-DA. Cell migration capacity was assessed by Transwell assay. AngII decreased cell viability and PRDX6, upregulated the expression levels of TNF-α, IL-6, IL-1β, LDH and MDA, stimulated ROS production, and reduced NO synthase, the expressions of eNOS, MnSOD, ICAM-1, VCAM-1, and activated the MAPK family of signaling proteins. However, the stimulatory effects of AngII on HUVECs were remarkably suppressed by PRDX6. Furthermore, mercaptosuccinate (MS; PRDX6 inhibitor) had similar effects as AngII in aggravating HUVECs damage. Conversely, these adverse events caused by AngII and MS were obviously reversed by ML3404 and SP600125. The present study indicated that PRDX6 overexpression inactivated p38 MAPK and JNK pathway through decrease AngII-induced inflammation, oxidative stress and endothelial dysfunction leading to attenuation of endothelial cell damage.

Protective Effect of Simplicillium sp. Ethyl Acetate Extract against High Glucose-Induced Oxidative Stress in HUVECs.

This study aimed at investigating the cytoprotective effect of an ethyl acetate extract of insect fungi against high glucose- (HG-) induced oxidative damage in human umbilical vein endothelial cells (HUVECs). An insect fungus strain termed CH180672 (CH) was found for protecting HUVECs from HG-induced damage. In this study, CH was identified as Simplicillium sp. based on a phylogenetic analysis of ITS‐rDNA sequences. Ethyl acetate extract (EtOAc) of this strain (CH) was subjected to the following experiments. Cell viability was examined with the MTT method. To evaluate the protection of CH, intracellular reactive oxygen species (ROS), malondialdehyde (MDA) levels, and the activities of antioxidant enzymes were measured and the expression of oxidation-associated proteins was assessed. In the current study, it has been found that CH can increase the survival rate of HUVECs induced by HG. Additionally, we found that HG-induced nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) signal decreased and increased the intracellular ROS and MDA generation in HUVECs. However, CH treatment strongly promoted the translocation of Nrf2 and its transregulation on HO-1 and ultimately inhibited the high level of ROS and MDA induced by HG. The regulatory ability of CH was similar to Nrf2 agonist bardoxolone, while the effect was abolished by ML385, suggesting that Nrf2 mediated the inhibition of CH on HG-induced oxidative stress in HUVECs. Taken together, CH can improve HG-induced oxidative damage of HUVECs, and its mechanism may be related to the regulation of the Nrf2/HO-1 pathway.

Vanadium compounds induced damage of human umbilical vein endothelial cells and the protective effect of berberine.

This study was conducted to investigate the damage caused by vanadium compounds and to explore the protective effects of berberine (BBR) in human umbilical vein endothelial cells (HUVECs). BBR is a biologically active small molecule found in Coptis rhizome, a remedy used in traditional Chinese medicine to treat diabetes. BBR has also been shown to lower blood glucose in diabetic patients. MTT assay was performed to observe the influence of bis(acetylacetonato)-oxidovanadium [VO(acac)2] or sodium metavanadate (NaVO3) and BBR on viability of HUVECs. The monolayer permeability of the HUVECs was assessed by measuring the transendothelial electrical resistance (TER). The endothelial nitric oxide synthase (eNOS) activity was detected by ELISA. Flow cytometry was performed to detect the generation of reactive oxygen species (ROS). The results showed that the viability of HUVECs was decreased by treatment with vanadium compounds 50-400μM in a concentration-dependent manner, while 0.01-1μM BBR effectively protected HUVECs from the inhibitory effects of vanadium compounds on cell viability. Also 100 and 200μM VO(acac)2 induced high permeability and decreased eNOS activity in HUVECs. While 0.01-1μM BBR showed no improvement in the permeability, and failed to reverse the VO(acac)2-induced changes of eNOS activity, but BBR treatment increased the eNOS activity in control cells. The addition of 200μM VO(acac)2 significantly induced ROS generation in HUVECs, while 0.01 or 0.1 μM BBR reversed the change of ROS. In summary, BBR has protective effects in HUVECs damage induced by vanadium compounds, which is not mediated by eNOS, but related to reduced intracellular ROS.

The size-dependent genotoxic potentials of titanium dioxide nanoparticles to endothelial cells.

Despite intensive research activities, there are still many major knowledge gaps over the potential adverse effects of titanium dioxide nanoparticles (TiO2 -NPs), one of the most widely produced and used nanoparticles, on human cardiovascular health and the underlying mechanisms. In the present study, alkaline comet assay and cytokinesis-block micronucleus test were employed to determine the genotoxic potentials of four sizes (100, 50, 30, and 10 nm) of anatase TiO2 -NPs to human umbilical vein endothelial cells (HUVECs) in culture. Also, the intracellular redox statuses were explored through the measurement of the levels of reactive oxygen species (ROS) and reduced glutathione (GSH) with kits, respectively. Meanwhile, the protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) were also detected by western blot. The results showed that at the exposed levels (1, 5, and 25 μg/mL), all the four sizes of TiO2 -NPs could elicit an increase of both DNA damage and MN frequency in HUVECs in culture, with a positive dose-dependent and negative size-dependent effect relationship (T100 < T50 < T30 < T10). Also, increased levels of intracellular ROS, but decreased levels of GSH, were found in all the TiO2 -NP-treated groups. Intriguingly, a very similar manner of dose-dependent and size-dependent effect relationship was observed between the ROS test and both comet assay and MN test, but contrary to that of GSH assay. Correspondingly, the levels of Nrf2 protein were also elevated in the TiO2 -NP-exposed HUVECs, with an inversely size-dependent effect relationship. These findings indicated that induction of oxidative stress and subsequent genotoxicity might be an important biological mechanism by which TiO2 -NP exposure would cause detrimental effects to human cardiovascular health.

Plasma from Volunteers Breathing Helium Reduces Hypoxia-Induced Cell Damage in Human Endothelial Cells\u2014Mechanisms of Remote Protection Against Hypoxia by Helium

PurposeRemote ischemic preconditioning protects peripheral organs against prolonged ischemia/reperfusion injury via circulating protective factors. Preconditioning with helium protected healthy volunteers against postischemic endothelial dysfunction. We investigated whether plasma from helium-treated volunteers can protect human umbilical vein endothelial cells (HUVECs) against hypoxia in vitro through release of circulating of factors.MethodsHealthy male volunteers inhaled heliox (79% helium, 21% oxygen) or air for 30 min. Plasma was collected at baseline, directly after inhalation, 6 h and 24 h after start of the experiment. HUVECs were incubated with either 5% or 10% of the plasma for 1 or 2 h and subjected to enzymatically induced hypoxia. Cell damage was measured by LDH content. Furthermore, caveolin 1 (Cav-1), hypoxia-inducible factor (HIF1α), extracellular signal-regulated kinase (ERK)1/2, signal transducer and activator of transcription (STAT3) and endothelial nitric oxide synthase (eNOS) were determined.ResultsPrehypoxic exposure to 10% plasma obtained 6 h after helium inhalation decreased hypoxia-induced cell damage in HUVEC. Cav-1 knockdown in HUVEC abolished this effect.ConclusionsPlasma of healthy volunteers breathing helium protects HUVEC against hypoxic cell damage, possibly involving circulating Cav-1.

Toxic effects of Cr(VI) on the bovine hemoglobin and human vascular endothelial cells: Molecular interaction and cell damage

Hexavalent chromium [Cr(VI)] is the main harmful component in the atmosphere released by chemical industry. The study was conducted to assess Cr(VI) inducing cardiovascular diseases (CVDs) in vitro by investigating the effects of Cr(VI) on bovine hemoglobin (BHb) and human umbilical vein endothelial cells (HUVECs). Multi-spectroscopic techniques and molecular docking method were used to determine the interaction of Cr(VI) and BHb. Fluorescence spectra results showed that the quenching constant (Ksv) decreased with temperature raise, indicating that Cr(VI) quenches BHb fluorescence through static quenching mechanism. The number of binding sites was 1.14 (310 K), enthalpy and entropy changes revealed the interaction of Cr(VI) and BHb was driven by hydrogen bonds. The results of synchronous fluorescence and circular dichroism (CD) spectra suggested that Cr(VI) could change BHb conformation and influence the microenvironment of Trp and Tyr residues. Moreover, in order to study Cr(VI) induced HUVECs damage, inflammatory factors were detected at the mRNA level, JNK and p38 MAPK pathways were analyzed. The results shown that Cr(VI) could induce mRNA expression of NLRP3, ICAM-1, VCAM-1, TNF-α and IL-1β, and increased intracellular ROS. Furthermore, Cr(VI) could induce oxidative stress in HUVECs, and then activate JNK and p38 MAPK pathways, ultimately lead to apoptosis of HUVECs by activating mitochondrial apoptosis pathways. These results suggested that Cr(VI) might bring about CVDs by both changing the BHb conformation and inducing HUVECs damage.

Protective Effect of Hot Water Extract of Loliolus Beka Gray Meat Against Palmitate-Induced HUVEC Damage.

Endothelial dysfunction is a critical factor in the development of diabetes-mediated cardiovascular complications. Free fatty acids (FFA), such as palmitate, which are elevated in diabetes and obesity, have been shown to mediate endothelial dysfunction, perhaps related to oxidative stress and inflammation. Taurine ameliorates endothelial dysfunction induced by diabetes. However, there has been no reports on the effect of Loliolus beka gray meat extracts, which contain large amounts of taurine. Here, we investigated the protective effect of a hot water extract of Loliolus beka gray meat (LBM), on palmitate-induced cell damage in human umbilical vein endothelial cells (HUVEC). The LBM extract was found to inhibit palmitate-induced cytotoxicity and DNA damage. In addition, the LBM extract reduced the level of reactive oxygen species (ROS) and nitric oxide (NO), as well as pro-inflammatory cytokines in HUVEC. These results suggest that the LBM extract protects against palmitate-induced cytotoxicity in HUVECs. Therefore, potential therapeutic and/or inhibitors of vascular disease may be derived from the LBM extract.

Undescribed C-geranylflavonoids isolated from the fruit peel of Paulownia catalpifolia T. Gong ex D.Y. Hong with their protection on human umbilical vein endothelial cells injury induced by hydrogen peroxide

Six undescribed C-geranylated flavonoids, including five C-geranylflavanones named as paucatalinones F - J, one C-geranylflavonol named as paucatalinone K, along with seven known geranylated flavanones, were isolated from the fruit peel of Paulownia catalpifolia T. Gong ex D.Y. Hong. Their structures were elucidated distinctly according to their UV, IR, MS, NMR, and CD data. Among them, two compounds were substituted with unusual modified geranyl groups, namely paucatalinone F with an oxygenated cyclogeranyl substituent and paucatalinone H with a terminal pyranoid geranyl substituent. Furthermore, the protective effects on human umbilical vein endothelial cells (HUVECs) injury induced by H2O2 were evaluated, and paucatalinone F showed the most potential activity. The bioactive results suggested that the geranyl substituent may be an important factor for restraining oxidative HUVECs damage and Paulownia C-geranylated flavonoids might have the potential for preventing cardiovascular complications.

Baicalin alleviates hyperglycemia-induced endothelial impairment 1 via Nrf2.

Baicalin is the major component found in Scutellaria baicalensis root, a widely used herb in traditional Chinese medicine, which exhibits strong anti-inflammatory, anti-viral and anti-tumor activities. The present work was devoted to elucidate the molecular and cellular mechanisms underlying the protective effects of Baicalin against diabetes-induced oxidative damage, inflammation and endothelial dysfunction. Diabetic mice, induced by streptozotocin (STZ), were treated with intraperitoneal Baicalin injections. Human umbilical vein endothelial cells (HUVECs) were cultured either in normal glucose (NG, 5.5 mM) or high glucose (HG, 33 mM) medium in the presence or absence of Baicalin for 72 h. We observed an obvious inhibition of hyperglycemia-triggered oxidative damage and inflammation in HUVECs and diabetic aortal vasculature by Baicalin, along with restoration of hyperglycemia-impaired nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway activity. However, the protective effects of Baicalin almost completely abolished in HUVECs transduced with shRNA against Nrf2, but not with nonsense shRNA. Mechanistic studies demonstrated that HG decreased Akt and GSK3B phosphorylation, restrained nuclear export of Fyn and nuclear localization of Nrf2, blunted Nrf2 downstream target genes, and subsequently induced oxidative stress in HUVECs. However, those destructive cascade, were well prevented by Baicalin in HUVECs. Furthermore, LY294002 and ML385 (inhibitor of PI3K and Nrf2) attenuated Baicalin mediated Nrf2 activation and the ability of facilitates angiogenesis in vivo and ex vivo. Taken together, the endothelial protective effect of Baicalin under hyperglycemia condition could be partly attributed to its role in downregulating reactive oxygen species (ROS) and inflammation via the Akt/GSK3B/Fyn-mediated Nrf2 activation.

Protocatechuic Acid Ameliorated Palmitic-Acid-Induced Oxidative Damage in Endothelial Cells through Activating Endogenous Antioxidant Enzymes via an Adenosine-Monophosphate-Activated-Protein-Kinase-Dependent Pathway.

Protocatechuic acid (PCA, 3,4-dihydroxybenzoic acid), the main metabolite of anthocyanins, is widely distributed in fruits and vegetables and has been reported to possess a strong antioxidant activity. Herein, we aimed to investigate the protective effect of PCA against high palmitic-acid (PA)-induced oxidative damage and the underling molecular mechanisms in human umbilical vein endothelial cells (HUVECs). PCA reduced the levels of intracellular reactive oxygen species and malondialdehyde and increased the activities of endogenous antioxidant enzymes, including superoxide dismutase, glutathione peroxidase 1, and heme oxygenase 1 (HO-1). Metabolomic analysis showed that PCA affected numerous metabolites, especially some of which were related with energy metabolism. PCA also upregulated the phosphorylation of adenosine-monophosphate-activated protein kinase (AMPK) at Thr172 through activating liver kinase B1 and then promoted the expression of p-Nrf2 and HO-1. Moreover, PCA reversed the decreased expression of peroxisome proliferator-activated receptor γ coactivator 1α and significantly increased the mitochondrial density. Collectively, these results demonstrated that PCA attenuated PA-induced oxidative damage in HUVECs via an AMPK-dependent pathway.

D-Fagomine Attenuates High Glucose-Induced Endothelial Cell Oxidative Damage by Upregulating the Expression of PGC-1α.

d-Fagomine, an analogue of 1-deoxynojirimycin (DNJ), has been shown to have hypoglycemic activity. This study is aimed at investigating if d-fagomine could attenuate high glucose-induced oxidative stress in human umbilical vein endothelial cells (HUVECs) and elucidate the underlying mechanism. Our results showed that d-fagomine reduced intracellular reactive oxygen species (ROS) production and malondialdehyde (MDA) levels. It also reversed the decrease of superoxide dismutases (SOD) and glutathione reductase (GR) activity, suggesting an inhibitory effect of d-fagomine on oxidative damage in HUVECs. d-Fagomine restored the loss of mitochondrial membrane potential, implying its protective role on mitochondrial function. In addition, d-fagomine activated the AMPK signaling pathway through LKB1, increased the expression of SIRT1 and PGC-1α, and attenuated the inhibitory effect on SIRT1 and PGC-1α activity caused by AMPK and SIRT1 inhibitor. d-Fagomine attenuated high glucose-induced oxidative stress in HUVECs through the AMPK/SIRT1/PGC-1α pathway.

Mechanism of endogenous digitalis-like factor‑induced vascular endothelial cell damage in patients with severe preeclampsia.

Although endogenous digitalis‑like factor (EDLF) is associated with the development of various physical disorders, the role in preeclampsia remains unclear. This study investigated the effects of EDLF on vascular endothelial cell damage in patients with preeclampsia and the potential mechanisms. From July2014 to July2015, 120 singleton pregnancy cases underwent a prenatal examination, inpatient delivery and had normal blood pressure were included in the study, either as patients with severe preeclampsia or the control patients. Serum EDLF levels were compared in these two groups, and an invitro hypoxic trophocyte‑induced vascular endothelial cell damage model was established to explore the changes in hypoxic trophocyte EDLF level and the subsequent effects on human umbilical vein endothelial cells(HUVECs). Nuclear factor‑κB(NF‑κB) p65 gene expression was silenced in hypoxic trophocytes, and EDLF levels and HUVEC damage were subsequently assessed. Serum EDLF levels were significantly higher in the severe preeclampsia cases than in the controls at the same gestational week (P<0.001). EDLF levels in hypoxic trophocytes increased with the increasing co‑culture duration. Damage to the biofunctions of HUVECs co‑cultured with hypoxic trophocytes also increased with co‑culture duration. However, silencing of NF‑κBp65 in the hypoxic trophocytes reduced the EDLF levels. AnnexinA2 was highly expressed in HUVECs, and no biofunctions were significantly damaged (P<0.05) compared with the group without receiving NF‑κBp65 silencing. Serum EDLF levels were significantly higher in patients with severe preeclampsia compared with the controls. The results of the current study indicate that NF‑κBp65 has a role in regulating EDLF production in hypoxic trophocytes.

Effect of β-arrestin on damage of human umbilical vein endothelial cell induced by angiotensin II.

β-arrestin (ARRB2) is a member of arrestin family and a negative regulatory protein of G-coupling receptor, which is closely associated with the pathogenesis of several autoimmune diseases. This study aimed to investigate the mechanism of the effect of ARRB2 on the damage of human umbilical vein endothelial cells (HUVECs), which is induced by angiotensin II (Ang II). ARRB2 at different concentration was used to interfere with the damage of HUVECs induced by Ang II or RNA interference technology to interfere with the expression of HUVECs followed by addition of Ang II to culture for 24 hours. Nitrate reduction method was used to measure the content of nitric oxide (NO) and radioimmunoassay was used to measure endothelin-1; Western blot assay was used to detect the expression of B-cell lymphoma-2 (Bcl-2), and flow cytometry was used to detect the intracellular level of reactive oxygen (ROS) and apoptosis of HUVECs. Our study found that ARRB2 could significantly reduce the generation and release of ROS, endothelin-1 (ET-1), lactic dehydrogenase (LDH) of HUVECs induced by Ang II and promote the generation of NO, superoxide dismutase (SOD) and scavenging in a dose-dependent manner. On the contrary, when expression of ARRB2 was disturbed by siRNA, increased generation and release of ROS, ET-1, and LDH were observed with reduced generation of NO, SOD and scavenging. In addition, ARRB2 could reverse the apoptosis of HUVECs induced by Ang II and was related to upregulate the expression of Bax. ARRB2 could protect the damage of HUVECs induced by Ang II and the mechanism was associated with upregulation of the expression of apoptosis and anti-apoptosis protein of Bcl-2.

Protective effects of Paeoniflorin against AOPP-induced oxidative injury in HUVECs by blocking the ROS-HIF-1α/VEGF pathway

BackgroundPaeoniflorin, a monoterpene glycoside, exerts protective vascular effects, showing good antioxidant properties. However, whether Paeoniflorin has protective effect against the oxidative damage induced by advanced oxidation protein products (AOPPs) in Human umbilical vein endothelial cells (HUVECs) is unknown, as is the underlying mechanism. PurposeThe present study was designed to investigate the effect of Paeoniflorin on oxidative damage of HUVECs and elucidate its underlying molecular mechanisms. MethodsThe fluorescence intensity of 2′, 7′-dichlorofluorescein-diacetate (DCFH-DA) staining was detected for intracellular reactive oxygen species (ROS) production. The increases mitochondrial membrane potential (MMP) was measured via flow cytometry and confocal microscopy using MitoTracker® Deep Red/ MitoTracker® Green staining. The intracellular adenosine triphosphate (ATP) was measured by ATP Determination Kit according to the manufacturer's protocol. Nox2, Nox4, hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and nuclear factor-κB (NF-κB) p65 expressions were detected by western blot. ResultsOur results showed that Paeoniflorin increases MMP and ATP levels of HUVECs induced by AOPPs, and attenuates NF-κB p65 expression on HUVECs might mainly result from its antioxidant capability by suppressing ROS production. Moreover, we also found that Paeoniflorin can suppress HIF-1α and VEGF protein expression through a decrease of ROS production via down-regulation of Nox2/Nox4 expression in HUVECs. AOPP-induced RAGE mRNA up-regulation was blocked by Paeoniflorin treatment in HUVECs. ConclusionOur results provided the first experimental that Paeoniflorin protects against AOPP-induced oxidative damage in HUVECs, mainly through a mechanism involving a decrease in ROS production by the inhibition of Nox2/Nox4 and RAGE expression; restored ATP depletion and mitochondria dysfunction via ROS suppression; and down-regulated HIF-1α/VEGF, possibly via the ROS-NF-κB axis.

Endothelial BMP4 Regulates Leukocyte Diapedesis and Promotes Inflammation

Leukocyte recruitment is a fundamental event in the response of the innate immune system to injury. This process is promoted in part by the opening of endothelial cell adherens junctions that allows leukocyte extravasation through gaps between adjacent endothelial cells. VE-cadherin is a key component of endothelial cell adherens junctions and a negative regulator of leukocyte emigration. Accumulating evidence implicates bone morphogenetic protein (BMP) 4 as a critical regulator in vascular biology, but its role in leukocyte extravasation in vitro and in vivo has not been investigated so far. To assess the impact of BMP4 on leukocyte emigration in vivo, we used the thioglycollate-induced peritonitis model. C57BL/6 mice were intraperitoneally (i.p.) injected with recombinant BMP4 in addition to thioglycollate. Compared to solvent-treated controls, we observed higher accumulation of leukocytes in the peritoneal lavage of BMP4-treated mice indicating that BMP4 promotes leukocyte diapedesis into the inflamed peritoneal cavity. Endothelial cell-specific deletion of BMP4 in mice markedly diminished leukocyte diapedesis following thioglycollate administration suggesting that endothelial BMP4 is required for leukocyte recruitment. Consistent with these in vivo results, transwell migration assays with human umbilical vein endothelial cells (HUVECs) in vitro revealed that recombinant BMP4 enhanced leukocyte transmigration through the endothelial monolayer. Conversely, silencing of endothelial BMP4 by siRNA dampened leukocyte diapedesis in vitro. Mechanistic studies showed that loss of BMP4 improved endothelial junction stability by upregulation of VE-cadherin expression in vitro and in vivo. Vice versa, treatment of HUVECs with recombinant BMP4 decreased expression of VE-cadherin and impaired endothelial junction stability shown by Western blotting and immunocytochemistry. Finally, severe endothelial damage in HUVECs in response to serum of patients collected 24h after survived cardiac arrest was accompanied by increase in leukocyte migration in transwell assays and activation of the BMP pathway most probably by upregulation of endothelial BMP4 RNA and protein expression. Collectively, the present study provides novel evidence that endothelial BMP4 controls leukocyte recruitment through a VE-cadherin-dependent mechanism and that BMP4-induced inflammation might be involved in the pathogenesis of endothelial cell damage following successful resuscitation after cardiac arrest.

MiR-34a/sirtuin-1/foxo3a is involved in genistein protecting against ox-LDL-induced oxidative damage in HUVECs

The antioxidant activity of genistein is associated with preventing atherosclerosis; however, the underlying mechanisms are not fully understood. In this study, human umbilical vein endothelial cells (HUVECs) were pretreated with genistein at different concentrations (10nM, 100nM and 1000nM) for 6h and then exposed to ox-LDL (50mg/L) for another 24h.Results showed that genistein restrained reactive oxygen species (ROS) and malondialdehyde (MDA) production, and ameliorated the inhibitory effect on superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione peroxidase (GPx) activity elicited by ox-LDL stimulation. The effects of genistein were correlated with the upregulation of sirtuin-1 via inhibiting miR-34a, and were abolished by sirtuin-1 siRNA or miR-34a mimic. Moreover, the antioxidation of genistein was associated with miR-34a/sirtuin-1-mediated nuclear translocation and deacetylation of foxo3a, accompanying with the enhanced expressions of MnSOD and CAT. The present study suggests that miR-34a/sirtuin-1/foxo3a might play an important role in genistein reversing ox-LDL-induced oxidative damage in HUVECs.

Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways

BackgroundAtherosclerosis is a common cardiovascular disease that causes myocardial infarction, heart failure, and stroke. Increased oxidized low density lipoprotein (ox-LDL) in the sub-endothelium is the characteristic origin of atherogenesis. Klotho, an anti-aging protein, has been reported to protect against atherosclerosis and ameliorate endothelial dysfunction in vivo. The aim of this study is to investigatethe anti-oxidative activity of Klothoin ox-LDL-treated human umbilical vein endothelial cells (HUVECs).MethodsAfter pre-treatment with 200 pMKlotho for 1 h, HUVECs were stimulated with 50 μg/ml ox-LDL for 24 h. Reactive oxygen species (ROS) and superoxide dismutase (SOD) levels were analyzed in the cells. Nitric oxide (NO) concertation was measured in the medium supernatant. Related proteins or genes were detected with Western blot or real time PCR, respectively, in the cell lysates.ResultsInitially, oxidative damage in HUVECs was established by adding 50 μg/mL ox-LDL, which resulted in decreased cellular viability, SOD/Cu/Zn-SOD and endothelial NO synthase (eNOS) expression and NO production, as well as increased malondialdehyde (MDA) levels, ROS production, inducible NO synthase (iNOS), phosphatidyl inositol-3 kinase (PI3K), protein kinase B (Akt), gp91 phox, and lectin-like ox-LDL receptor (LOX-1) expression in HUVECs. Pre-incubation with recombinant Klotho (200 pM) significantly prevented all of these alterations. These results suggest that Klotho can attenuate ox-LDL-induced oxidative stress in HUVECs through upregulating oxidative scavengers (SOD and NO) viaactivating the PI3K/Akt/eNOS pathway and depressing LOX-1expression.ConclusionsThese results suggest that Klotho has a potential therapeutic effect on attenuating endothelial dysfunction and ameliorating atherosclerosis.

Protective effects of physiological testosterone on advanced glycation end product‑induced injury in human endothelial cells.

The effect of testosterone, a sex steroid, on endothelial cells is controversial as it is uncertain if it has a protective effect on them. Whether physiological testosterone can inhibit the deleterious effects of advanced glycation end products (AGEs) on endothelial cells remains to be elucidated. The present study focused on elucidating the effect of testosterone on the injury of endothelial cells induced by AGEs. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and treated with AGEs in the presence or absence of various concentrations of testosterone. The cell viability in each group was measured using an MTS assay. Early-stage apoptosis was detected using flow cytometry with Annexin V-fluorescein isothiocyanate/propidium iodide double staining, and the expression levels of apoptosis-associated proteins, B cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and caspase-3, were determined using western blot analysis. Oxidative stress and pro-inflammatory parameters in the medium were evaluated using an enzyme-linked immunosorbent assay. The MTS results showed that AGEs significantly decreased the proliferation of HUVECs, whereas a physiological concentration of testosterone alleviated this damage. Physiological concentrations of testosterone protected the HUVECs from AGE-induced apoptosis, mediated by caspase-3 and Bax/Bcl-2. In addition, treatment of the HUVECs with AGEs caused a significant decrease in anti-oxidative parameters, but increased the concentrations of malondialdehyde and tumor necrosis factor-α. The activation of Janus kinase 2 and signal transducer and activator of transcription 3 was significantly increased by incubation with AGEs. However, pre-incubation with a physiological concentration of testosterone attenuated these changes. Therefore, the data obtained in the present study established the potential role of physiological testosterone in ameliorating AGE-induced damage in HUVECs.

The effects of microRNA-34a regulating Notch-1/NF-κB signaling pathway on lipopolysaccharide-induced human umbilical vein endothelial cells.

Notch-1/NF-κB signaling plays a key role in the cecal ligation and puncture (CLP)-induced sepsis. This study aims to investigate the intervention effects of microRNA-34a (miR-34a) lentivirus regulating Notch-1/NF-κB signaling pathway on lipopolysaccharide (LPS)-induced human umbilical vein endothelial cells (HUVEC). HUVEC were divided into four groups as the following: they were infected with negative control lentivirus (NC group) or miR-34a lentivirus (OE group); LPS (1 μg/mL) was added on the third day on the basis of NC group and OE group for 24 hours (NC+LPS group or OE+LPS group). The levels of TNF-α, IL-1β, IL-6, and IL-10 in the cell supernatants, and the mRNA and protein expression of Notch-1 and NF-κB in the HUVEC were evaluated. After 24 hours, the levels of TNF-α, IL-1β, IL-6 in the cell supernatants and the protein expression of NF-κB from NC+LPS group were significantly higher than those of NC group, but IL-10 level and the protein expression of Notch-1 in NC+LPS group were the opposite. After intervention of miR-34a lentivirus, the cell supernatants TNF-α and the protein expression of NF-κB in OE+LPS group after 24 hours markedly decreased compared to NC+LPS group. While the cell supernatants IL-1β and IL-6 and the mRNA expression of NF-κB slightly decreased in OE+LPS group, IL-10 and the mRNA and protein expression of Notch-1 were the opposite. miR-34a regulating Notch-1/NF-κB signaling pathway can reduce the HUVEC damage caused by LPS stimulation.

Effect of endotoxin on mitochondrial dynamics in human umbilical vein endothelial cells

Objective To investigate the effect of endotoxin on mitochondrial dynamics in human umbilical vein endothelial cells (HUVECs). Methods HUVECs were cultured in vitro and seeded in the culture plate and randomly divided into 2 groups (n=54 each) using a random number table: control group (group C) and endotoxin group(group E). Lipopolysaccharides(LPS) with the final concentration of 10 μg/ml was added in group E. At 2, 8 and 24 h of incubation with LPS, the cell viability was detected using the methyl thiazolyl tetrazolium assay, the intracellular ATP content was determined by the phosphomolybdic acid colorimetric method, and the expression of the mitochondrial fission protein dynamin-related protein 1 (DRP1) was detected by Western blot. Results Compared with group C, the cell viability and ATP content were significantly decreased, and the expression of DRP1 was significantly up-regulated at each time point of incubation with LPS in group E (P 0.05). Compared with the values at 8 h of incubation with LPS, the ATP content was significantly increased, and the expression of DRP1 was significantly down-regulated at 24 h of incubation with LPS (P 0.05). Conclusion The mechanism underlying endotoxin-induced mitochondrial damage in HUVECs is associated with up-regulation of DRP1 expression and promotion of excessive mitochondrial fission. Key words: Endotoxemia; Blood vessels; Endothelial cells; Mitochondria