Effect of tumor suppressor PTEN gene on apoptosis and cell cycle of human airway smooth muscle cells

Tumor necrosis factor-alpha (TNFα) plays a crucial role in inflammatory diseases such as rheumatoid arthritis and postmenopausal osteoporosis. Recently, it has been demonstrated that hydrogen gas, known as a novel antioxidant, can exert therapeutic anti-inflammatory effect in many diseases. In this study, we investigated the effect of treatment with hydrogen molecule (H₂) on TNFα-induced cell injury in osteoblast. The osteoblasts isolated from neonatal rat calvariae were cultured. It was found that TNFα suppressed cell viability, induced cell apoptosis, suppressed Runx2 mRNA expression, and inhibited alkaline phosphatase activity, which was reversed by co-incubation with H₂. Incubation with TNFα-enhanced intracellular reactive oxygen species (ROS) formation and malondialdehyde production increased NADPH oxidase activity, impaired mitochondrial function marked by increased mitochondrial ROS formation and decreased mitochondrial membrane potential and ATP synthesis, and suppressed activities of antioxidant enzymes including SOD and catalase, which were restored by co-incubation with H₂. Treatment with H₂ inhibited TNFα-induced activation of NFκB pathway. In addition, treatment with H₂ inhibited TNFα-induced nitric oxide (NO) formation through inhibiting iNOS activity. Treatment with H₂ inhibited TNFα-induced IL-6 and ICAM-1 mRNA expression. In conclusion, treatment with H₂ alleviates TNFα-induced cell injury in osteoblast through abating oxidative stress, preserving mitochondrial function, suppressing inflammation, and enhancing NO bioavailability.     

Sphingosine-1-phosphate (S1P) displays sustained S1P1 receptor agonism and signaling through S1P lyase-dependent receptor recycling

The sphingosine-1-phosphate (S1P) type 1 receptor (S1P₁R) is a novel therapeutic target in lymphocyte-mediated autoimmune diseases. S1P₁ receptor desensitization caused by synthetic S1P₁ receptor agonists prevents T-lymphocyte egress from secondary lymphoid organs into the circulation. The selective S1P₁ receptor agonist ponesimod, which is in development for the treatment of autoimmune diseases, efficiently reduces peripheral lymphocyte counts and displays efficacy in animal models of autoimmune disease. Using ponesimod and the natural ligand S1P, we investigated the molecular mechanisms leading to different signaling, desensitization and trafficking behavior of S1P₁ receptors. In recombinant S1P₁ receptor-expressing cells, ponesimod and S1P triggered Gαi protein-mediated signaling and β-arrestin recruitment with comparable potency and efficiency, but only ponesimod efficiently induced intracellular receptor accumulation. In human umbilical vein endothelial cells (HUVEC), ponesimod and S1P triggered translocation of the endogenous S1P₁ receptor to the Golgi compartment. However, only ponesimod treatment caused efficient surface receptor depletion, receptor accumulation in the Golgi and degradation. Impedance measurements in HUVEC showed that ponesimod induced only short-lived Gαi protein-mediated signaling followed by resistance to further stimulation, whereas S1P induced sustained Gαi protein-mediated signaling without desensitization. Inhibition of S1P lyase activity in HUVEC rendered S1P an efficient S1P₁ receptor internalizing compound and abrogated S1P-mediated sustained signaling. This suggests that S1P lyase – by facilitating S1P1 receptor recycling – is essential for S1P-mediated sustained signaling, and that synthetic agonists are functional antagonists because they are not S1P lyase substrates.     

Systemic induction of H2O2 in pea seedlings pretreated by wounding and exogenous jasmonic acid

Pea seedlings (Pisum sativum L.) were used as materials to test the timings and compartments of hydrogen peroxide (H₂O₂) triggered by wounding and exogenous jasmonic acid (JA). The results showed that H₂O₂ could be systemically induced by wounding and exogenous JA. H₂O₂ increased within 1 h and reached the peak 3–5 h after wounding in either the wounded leaves or the unwounded leaves adjacent to the wounded ones and the inferior leaves far from the wounded ones. After this, H₂O₂ decreased and recovered to the control level 12 h after wounding. The activities of antioxidant enzymes, however, were rapidly increased by wounding. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, could significantly inhibit H₂O₂ burst that was mediated by wounding and exogenous JA. Assay of H₂O₂ subcellular location showed that H₂O₂ in response to wounding and exogenous JA was predominantly accumulated in plasma membrane, cell wall and apoplasmic space. Numerous JA (gold particles) was found via immunogold electron microscopy to be located in cell wall and phloem zones of mesophyll cell after wounding.     

Intermolecular disulfide bond formation in the NEMO dimer requires Cys54 and Cys347

NEMO is an essential regulatory component of the IκB kinase (IKK) complex, which controls activation of the NF-κB signaling pathway. Herein, we show that NEMO exists as a disulfide-bonded dimer when isolated from several cell types and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. Treatment of cells with hydrogen peroxide (H₂O₂) induces further formation of NEMO dimers. Disulfide bond-mediated formation of NEMO dimers requires Cys54 and Cys347. The ability of these residues to form disulfide bonds is consistent with their location in a NEMO dimer structure that we generated by molecular modeling. We also show that pretreatment with H₂O₂ decreases TNFα-induced IKK activity in NEMO-reconstituted cells, and that TNFα has a diminished ability to activate NF-κB DNA binding in cells reconstituted with NEMO mutant C54/347A. This study implicates NEMO as a target of redox regulation and presents the first structural model for the NEMO protein.     

Synthesis and biological activity of halophenols as potent antioxidant and cytoprotective agents

A series of new bromophenols and chlorophenols were prepared by a practical route. The in vitro antioxidative activity of the halophenols was evaluated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay, and their cytoprotective activity was also tested on hydrogen peroxide (H₂O₂)-induced injury in human umbilical vein endothelial cells (HUVEC). All halophenols tested displayed moderate to good DPPH radical-scavenging activity, and two bromophenols, 2,3′-dibromo-4,5,6′-trihydroxydiphenylmethanone (16c) and 2,3-dibromo-4,5-dihydroxydiphenylmethanone (17c) exhibited high protective activity against H₂O₂-induced injury in HUVEC with EC₅₀ values of 0.4 and 0.8 μM, respectively. The preliminary structure–activity relationships of these compounds were also investigated in order to determine the essential structures required for their bioactivities.     

Hydrogen peroxide stimulates exocytosis of von Willebrand factor in human umbilical vein endothelial cells

The aim of our research was to study the influence of hydrogen peroxide on the exocytosis of von Willebrand factor (vWF) in human umbilical vein endothelial cells (HUVEC). We have found that H₂O₂ at a non-toxic concentration (100 μM) increases the amount of vWF secreted by HUVEC by 43 ± 14% over control (p < 0.03) and elevates total exposition of vWF on cell surface by 89 ± 5% (p < 0.01). Analysis of immunofluorescent images of HUVEC with CellProfiler program revealed that the average number of antigen positive structures on the single cell surface increases from 11.4 ± 0.16 in control up to 17.5 ± 0.21 after incubation with H₂O₂ (p < 0.01). vWF is exposed on the cell surface as dots with the average sizes around 1–3 μm. H₂O₂ causes an increase in the number of these dots and the appearence of the strings of vWF which are absent in control HUVEC. It is suggested that H₂O₂ may serve as a messenger which stimulates vWF exocytosis.     

Hydrogen sulfide reduces cell adhesion and relevant inflammatory triggering by preventing ADAM17‐dependent TNF‐α activation

H₂S is the third endogenous gaseous mediator, after nitric oxide and carbon monoxide, possessing pleiotropic effects, including cytoprotection and anti‐inflammatory action. We analyzed, in an in vitro model entailing monocyte adhesion to an endothelial monolayer, the changes induced by H₂S on various potential targets, including cytokines, chemokines, and proteases, playing a crucial role in inflammation and cell adhesion. Results show that H₂S prevents the increase in monocyte adhesion induced by tumor necrosis factor‐α (TNF‐α). Under these conditions, downregulation of monocyte chemoattractant protein‐1 (MCP‐1), chemokine C‐C motif receptor 2, and increase of cluster of differentiation 36 could be detected in monocytes. In endothelial cells, H₂S treatment reduces the increase in MCP‐1, inter‐cellular adhesion molecule‐1, vascular cell adhesion molecule‐1, and of a disintegrin and metalloproteinase metallopeptidase domain 17 (ADAM17), both at the gene expression and protein levels. Cystathionine γ‐lyase and 3‐mercaptopyruvate sulfurtransferase, the major H₂S forming enzymes, are downregulated in endothelial cells. In addition, H₂S significantly reduces activation of ADAM17 by PMA in endothelial cells, with consequent reduction of both ADAM17‐dependent TNF‐α ectodomain shedding and MCP‐1 release. In conclusion, H₂S is able to prevent endothelial activation by hampering endothelial activation, triggered by TNF‐α. The mechanism of this protective effect is mainly mediated by down‐modulation of ADAM17‐dependent TNF‐converting enzyme (TACE) activity with consequent inhibition of soluble TNF‐α shedding and its relevant MCP‐1 release in the medium. These results are discussed in the light of the potential protective role of H₂S in pro‐inflammatory and pro‐atherogenic processes, such as chronic renal failure. J. Cell. Biochem. 114: 1536–1548, 2013. © 2013 Wiley Periodicals, Inc.     

Ginsenoside Rb1 Prevents H2O2-Induced HUVEC Senescence by Stimulating Sirtuin-1 Pathway

Purposes

We have previously reported that Ginsenoside Rb1 may effectively prevent HUVECs from senescence, however, the detailed mechanism has not demonstrated up to now. Recent studies have shown that sirtuin-1 (Sirt1) plays an important role in the development of endothelial senescence. The purpose of this study was to explore whether Sirt1 is involved in the action of Ginsenoside Rb1 regarding protection against H₂O₂-induced HUVEC Senescence.

Methods and Results

Senescence induced by hydrogen peroxide (H₂O₂) in human umbilical vein endothelial cells (HUVECs) was examined by analyzing plasminogen activator inhibitor-1 (PAI-1) expression, cell morphology, and senescence-associated beta-galactosidase (SA-β-gal) activity. The results revealed that 42% of control-treated HUVECs were SA-β-gal positive after treatment by 60 µmol/L H₂O₂, however, this particular effect of H₂O₂ was decreased more than 2-fold (19%) in the HUVECs when pretreated with Rb1 (20 µmol/L) for 30 min. Additionally, Rb1 decreased eNOS acetylation, as well as promoted more NO production that was accompanied by an increase in Sirt1 expression. Furthermore, upon knocking down Sirt1, the effect of Rb1 on HUVEC senescence was blunted.

Conclusions

The present study indicated that Ginsenoside Rb1 acts through stimulating Sirt1 in order to protect against endothelial senescence and dysfunction. As such, Sirt1 appears to be of particular importance in maintaining endothelial functions and delaying vascular aging.     

Mechanistic insight of platelet apoptosis leading to non-surgical bleeding among heart failure patients supported by continuous-flow left ventricular assist devices

Vascular endothelial cells are highly sensitive to oxidative stress, and this is one of the mechanisms by which widespread endothelial dysfunction is induced in most cardiovascular diseases and disorders. However, how these cells can survive in oxidative stress environments remains unclear. Salidroside, a traditional Chinese medicine, has been shown to confer vascular protective effects. We aimed to understand the role of autophagy and its regulatory mechanisms by treating human umbilical vein endothelial cells (HUVECs) with salidroside under oxidative stress. HUVECs were treated with salidroside and exposed to hydrogen peroxide (H₂O₂). The results indicated that salidroside exerted cytoprotective effects in an H₂O₂-induced HUVEC injury model and suppressed H₂O₂-induced apoptosis of HUVECs. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased oxidative stress-induced HUVEC apoptosis, while the autophagy activator rapamycin induced anti-apoptosis effects in HUVECs. Salidroside increased autophagy and decreased apoptosis of HUVECs in a dose-dependent manner under oxidative stress. Moreover, 3-MA attenuated salidroside-induced HUVEC autophagy and promoted apoptosis, whereas rapamycin had no additional effects compared with salidroside alone. Salidroside upregulated AMPK phosphorylation but downregulated mTOR phosphorylation under oxidative stress; however, administration of compound C, an AMPK inhibitor, abrogated AMPK phosphorylation and increased mTOR phosphorylation and apoptosis compared with salidroside alone. These results suggest that autophagy is a protective mechanism in HUVECs under oxidative stress and that salidroside might promote autophagy through activation of the AMPK pathway and downregulation of mTOR pathway.     

Amifostine has antiangiogenic properties in vitro by changing the redox status of human endothelial cells

Amifostine is a broad-spectrum cytoprotective agent, selective for normal tissues. It is a pro-drug metabolised to the free thiol WR-1065 that may act as a scavenger of free radicals, generated in tissues exposed to chemotherapeutic agents or radiation. WR-1065 can be further oxidized to its symmetric disulfide WR-33278 or degraded to hydrogen peroxide (H₂O₂). Both WR-1065 and WR-33278 resemble endogenous polyamines. Although amifostine is used in some cases in the clinic, there are only few studies concerning its actions at the cellular level. We have previously shown that amifostine inhibits angiogenesis in vivo, affecting the expression of several angiogenic genes. In the present work, we studied the effect of amifostine on human umbilical vein endothelial cell (HUVEC) functions in vitro, in order to further clarify its mechanism(s) of action. Amifostine increased HUVEC proliferation, an effect that was reversed by the intracellular H₂O₂ scavenger sodium pyruvate, agents that increase intracellular cAMP levels and L-valine. On the other hand, amifostine decreased HUVEC migration, an effect that was reversed by L-valine or L-arginine but not sodium pyrouvate. The decrease in migration was in line with decreased tube formation on matrigel and decreased amounts of metalloproteinase-2 released into the culture medium of HUVEC. Finally, amifostine reduced tyrosine nitration of the cytoskeletal proteins actin and α-tubulin in a time dependent manner. This last action could be due to the reduced production of nitric oxide (NO) or to other not yet identified mechanisms. Collectively, our results suggest that amifostine acts on endothelial cells through pathways that affect the redox status of the cells, either by producing H₂O₂ or by modulating NO production.     

Curcumin attenuates endothelial cell oxidative stress injury through Notch signaling inhibition

Previous studies have demonstrated that Notch signaling pathway plays a regulatory role in cellular oxidative stress injury (OSI). In this study, our aim was to explore the role of the Notch signaling pathway in hydrogen peroxide (H₂O₂)-induced OSI and the protective effect of curcumin during (H₂O₂)-induced injury in human umbilical vein endothelial cells (HUVECs). DAPT, a specific inhibitor of the Notch signaling pathway, and Notch1 siRNA were used to study Notch activity. Further, HUVECs were exposed to H₂O₂ in the absence or presence of curcumin. DAPT and Notch1 siRNA significantly inhibited OSI and the expression of Notch1 and Hes1. Curcumin conferred a protective effect on the HUVECs against H₂O₂, which was evidenced by improved cell viability, adhesive ability and migratory ability and a decreased apoptotic index, decreased production of reactive oxygen species (ROS) and a reduction in several biochemical parameters. Immunofluorescence and Western blotting analyses demonstrated that H₂O₂ treatment upregulated the expression of Notch1, Hes1, Caspase3, Bax and cytochrome c downregulated the expression of Bcl2, and treatment with curcumin reversed these effects. We demonstrated for the first time that the inhibition of Notch signaling pathway imparts a protective effect against endothelial OSI. The protective effects of curcumin against OSI are at least in part dependent on Notch1 inhibition.     

Novel role of silent information regulator 1 in acute endothelial cell oxidative stress injury

Silent information regulator 1 (SIRT1), a class III histone deacetylase, retards aging and plays roles in cellular oxidative stress injury (OSI). However, the biological context in which SIRT1 promotes oxidative injury is not fully understood. Here, we show that SIRT1 essentially mediates hydrogen peroxide (H₂O₂)-induced cytotoxicity in human umbilical vein endothelial cell (HUVEC). In HUVECs, SIRT1 protein expression was significantly increased in a dose-dependent manner after H₂O₂ treatment, whereas the acetylation levels of the NF-κB p65 subunit and p53 were decreased. EX527 (a specific SIRT1 inhibitor) conferred protection to the HUVECs against H₂O₂, as indicated by an improved cell viability, adhesion, an enhanced migratory ability, a decreased apoptotic index, decreased reactive oxygen species (ROS) production and reductions in several biochemical parameters. Immunofluorescence and Western blot analyses demonstrated that H₂O₂ treatment up-regulated SIRT1, phosphorylated-JNK (p-JNK), p-p38MAPK, and p-ERK expression. EX527 pretreatment reversed these effects on SIRT1, p-JNK, and p-p38MAPK but further increased the p-ERK levels. Similar results were confirmed in SIRT1 siRNA experiments. In summary, SIRT1 signaling pathway inhibition imparts protection against acute endothelial OSI, and modulation of MAPKs (JNK, p38MAPK, and ERK) may be involved in the protective effect of SIRT1 inhibition.     

Proliferative and secretory activity of human umbilical endothelial cells cultivated under various hypoxia conditions

In this study, we examined the impact of 3-day hypoxia of different degrees on the viability, proliferation, and secretory activity of endothelial cells from human umbilical vein (HUVEC). A gas mixture of three components was used (%): 1) 10 O₂, 5 CO₂, and 85 Ar; 2) 5 O₂, 5 CO₂, and 80 Ar; and 3) 1 O₂, 5 CO₂, and 94 Ar. Cells cultivated in a CO₂ incubator in atmospheric oxygen (21% O₂) served as control. It was found that 3-day HUVEC cultivation at 1% O₂ increased NO synthesis; enhanced secretion of endothelin-1, IL-6, IL-8, TNF-alpha, sVCAM-1, sE-cadherin, sE-selectin, VEGF-A, and bFGF; and inhibited proliferation. HUVEC cultivated under 10% O₂ and 5% O₂ exhibited the lowest level of basal secretion of these substances and increased proliferative activity. These cells cultivated under conditions of atmospheric oxygen for 3 days displayed activated secretion of NO, IL-6, IL-8, and von Willebrand factor; the activation was higher than at 10% O₂ and 5% CO₂. Thus, the gaseous medium with reduced oxygen content (5%) is a more physiological condition for HUVEC cultivation. An increase in the amount of oxygen up to the atmospheric level causes endotheliocyte activation; the cells exhibit the features of endothelial dysfunction. Oxygen content reduced to 1% induces endothelial dysfunction and reduced proliferative potential.     

Synthesis of a series of caffeic acid phenethyl amide (CAPA) fluorinated derivatives: Comparison of cytoprotective effects to caffeic acid phenethyl ester (CAPE)

A series of catechol ring-fluorinated derivatives of caffeic acid phenethyl amide (CAPA) were synthesized and screened for cytoprotective activity against H₂O₂ induced oxidative stress in human umbilical vein endothelial cells (HUVEC). CAPA and three fluorinated analogs were found to be significantly cytoprotective when compared to control, with no significant difference in cytoprotection between caffeic acid phenethyl ester (CAPE) and CAPA.     

H2O2-mediated permeability II: importance of tyrosine phosphatase and kinase activity

We previously reportedthat exposure of endothelial cells to H₂O₂results in a loss of cell-cell apposition and increased endothelialsolute permeability. The purpose of this study was to determine howtyrosine phosphorylation and tyrosine phosphatases contribute tooxidant-mediated disorganization of endothelial cell junctions. Wefound that H₂O₂ caused a rapid decrease in total cellular phosphatase activity that facilitates a compensatory increase in cellular phosphotyrosine residues.H₂O₂ exposure also results in increasedendothelial monolayer permeability, which was attenuated by pp60, aninhibitor of src kinase. Inhibition of protein tyrosinephosphatase activity by phenylarsine oxide (PAO) demonstrated a similarpermeability profile compared with H₂O₂,suggesting that tyrosine phosphatase activity is important inmaintaining a normal endothelial solute barrier. Immunofluorescence shows that H₂O₂ exposure caused a loss ofpan-reactive cadherin and -catenin from cell junctions that was notblocked by the src kinase inhibitor PP1.H₂O₂ also caused -catenin to dissociate fromthe endothelial cytoskeleton, which was not prevented by PP1. Finally,we determined that PP1 did not prevent cadherin internalization. Thesedata suggest that oxidants like H₂O₂ produce biological effects through protein phosphotyrosine modifications bydecreasing total cellular phosphatase activity combined with increasedsrc kinase activity, resulting in increased endothelial solute permeability.

     

New Role of JAK2/STAT3 Signaling in Endothelial Cell Oxidative Stress Injury and Protective Effect of Melatonin

Previous studies have shown that the JAK2/STAT3 signaling pathway plays a regulatory role in cellular oxidative stress injury (OSI). In this study, we explored the role of the JAK2/STAT3 signaling pathway in hydrogen peroxide (H₂O₂)-induced OSI and the protective effect of melatonin against (H₂O₂)-induced injury in human umbilical vein endothelial cells (HUVECs). AG490 (a specific inhibitor of the JAK2/STAT3 signaling pathway) and JAK2 siRNA were used to manipulate JAK2/STAT3 activity, and the results showed that AG490 and JAK2 siRNA inhibited OSI and the levels of p-JAK2 and p-STAT3. HUVECs were then subjected to H₂O₂ in the absence or presence of melatonin, the main secretory product of the pineal gland. Melatonin conferred a protective effect against H₂O₂, which was evidenced by improvements in cell viability, adhesive ability and migratory ability, decreases in the apoptotic index and reactive oxygen species (ROS) production and several biochemical parameters in HUVECs. Immunofluorescence and Western blotting showed that H₂O₂ treatment increased the levels of p-JAK2, p-STAT3, Cytochrome c, Bax and Caspase3 and decreased the levels of Bcl2, whereas melatonin treatment partially reversed these effects. We, for the first time, demonstrate that the inhibition of the JAK2/STAT3 signaling pathway results in a protective effect against endothelial OSI. The protective effects of melatonin against OSI, at least partially, depend upon JAK2/STAT3 inhibition.