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. 2018 Sep;34(5):424–431. doi: 10.6515/ACS.201809_34(5).20180708A

Melatonin Protects Against Hypoxia/Reoxygenation-Induced Dysfunction of Human Umbilical Vein Endothelial Cells Through Inhibiting Reactive Oxygen Species Generation

Shuo Zhao 1, Yueyuan Wang 1, Xiaoying Zhang 2, Lu Zheng 2, Bin Zhu 2, Shuang Yao 2, Ling Yang 3, Jun Du 1
PMCID: PMC6160513  PMID: 30271093

Abstract

Background

Hypoxia/reoxygenation (H/R) in human umbilical vein endothelial cells (HUVECs) induces oxidative stress and eventually leads to vascular injury.

Objective

The aim of this study was to examine the effect of melatonin on HUVECs injured by H/R and explore the underlying mechanisms.

Materials and Methods

A model of HUVECs under hypoxia/reoxygenation was established. Cell migration and adhesive ability was measured by wound healing and adhesion assays. Cell proliferation was measured by EdU assay. Production of reactive oxygen species (ROS) was evaluated by CM-H2DCFDA staining. Actin cytoskeleton rearrangement was examined by immunofluorescence. Western blotting analysis were used to analyze P38 and HSP27 phosphorylation levels.

Results

H/R inhibited HUVEC proliferation, cell migratory and adhesive capacities, whereas melatonin (1~100 μM) inhibited these effects in a dose-dependent manner. Melatonin alone did not affect HUVEC viability, however, it inhibited the increase in ROS production and cytoskeleton disruption elicited by H/R, and it dose-dependently prevented H/R-induced upregulation of P38 and HSP27 phosphorylation. In addition, the ROS scavenger N-acetyl-L-cysteine markedly inhibited increased phosphorylation levels of P38 and HSP27 under H/R.

Conclusions

Melatonin may have a potential clinical effect in trials of H/R-induced vascular injury through its antioxidant property.

Keywords: HSP27, HUVEC, Hypoxia/reoxygenation, Melatonin, P38

INTRODUCTION

Hypoxia/reoxygenation (H/R) injury is common in severe trauma and ischemic disease. Vascular endothelial dysfunction and structural damage are early pathological changes that occur in H/R, and they also play an important role in the development of injuries.1 Although novel strategies against H/R injury in endothelial cells have been developed, therapy failure remains a major challenge in the clinical setting. Therefore, determining an effective drug therapy for the treatment of vascular endothelial H/R injury is an important focus of research.

Among the compounds tested for cardiovascular disease, melatonin is of great interest because of its efficacy and lack of toxicity.2,3 Melatonin, an endogenous hormone involved in synchronization of the circadian rhythms of the human body, is essential for maintaining physiological functions including sleep timing, blood pressure regulation, seasonal reproduction, and many others. Previous studies have demonstrated that melatonin has significant therapeutic effects on myocardial ischemia-reperfusion injury, hypertension, atherosclerosis and other cardiovascular diseases.4-7 Of note, it has been reported that melatonin can protect against H/R-induced apoptosis of villous trophoblast.8 In addition, recent results from our laboratory showed that melatonin blocked ERK/Rac1 activation and subsequent HIF-1α upregulation in hypoxic human umbilical vein endothelial cells (HUVECs),9 however, its role in H/R-induced injury in HUVECs has not yet been reported.

In the present study, we examined the effects of H/R on proliferation, migration and cell adhesion in HUVECs. Using pharmacologic analysis, we further investigated whether melatonin exerted protective effects on H/R-induced vascular endothelial dysfunction, and explored the mechanisms involved in melatonin treatment.

MATERIALS AND METHODS

Cell culture

HUVECs were obtained from the Cell Biology Institute of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, high glucose) (Hyclone, Thermo Scientific, Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Hyclone, ThermoScientific, Waltham, MA, USA) and antibiotics (100 U/mL streptomycin and 100 μg/mL penicillin) (Invitrogen, USA) in a humidified incubator at 37 °C with 5% CO2. Cells were grown on coverslips for fluorescence staining and on plastic dishes for protein extraction.

HUVEC treatment with H/R and melatonin or N-acetyl-L-cysteine (NAC)

An H/R model was established using the method described by Feng et al.10 In brief, for H/R, HUVECs were exposed to hypoxia (1% O2) using a continuous flow of a humidified mixture of 1% O2, 5% CO2, and 94% N2 at 37 °C for 2 h. The cells were then returned to culture in a normal incubator with 20% O2, 5% CO2, and 75% N2 at 37 °C for 4 h.

Melatonin (Sigma, St Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO), and the cells were treated with melatonin for the indicated doses. In experiments to determine the effects of the inhibitor NAC (Beyotime, China) on P38/HSP27 signaling inhibition, the cells were treated with NAC 1 mM for 30 min prior to H/R treatment.

MTT assay

Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously.11 Briefly, HUVECs were seeded at a density of 5 × 103 cells per well on a 96-well plate (Corning Inc., Corning, NY, USA) and treated with melatonin for the indicated times and doses. After culture, MTT was added and the plate was incubated in the dark for 4 h, followed by measurements at 490 nm using a microplate absorbance reader (Bio-Tek, Elx800, USA).

EdU staining

EdU staining was done according to the manufacturer’s instruction (Keygen Technology, Beijing, China). Briefly, cells were cultured on coverslips until reaching 70% confluence, and then EdU was added to the culture media for 2 h. After labeling, the cells were washed three times with PBS followed by formaldehyde fixation. The cells were then incubated once with glycine and washed with PBS containing 0.5% Triton X-100. After counterstained with DAPI, the cells were mounted and imaged using fluorescence microscopy.

Cell adhesion assay

The 96-well plates were coated with Matrigel (BD, Franklin Lakes, NJ, USA) at 37 ° C for 1 h, and then blocked with 1% BSA in PBS. The cells were detached from the dishes and plated in the wells for 1 h at 37 °C. The cells were then washed twice with PBS and the rate of attached cells was obtained using an MTT assay.

Wound healing assay

When the cells were 95~100% confluent, they were incubated overnight in DMEM and wounding was performed by scraping through the cell monolayer with a 10-μl pipette tip. Medium and nonadherent cells were removed, and the cells were washed twice with PBS, followed by the addition of new medium. The cells were then permitted to migrate into the area of clearing for 6 h. Wound closure was monitored by visual examination under a microscope (Carl Zeiss Meditec, Jena, Germany).

Actin cytoskeleton staining

Cells were fixed in ice-cold methanol for 20 min, permeabilized in 0.1% Triton X-100 and blocked in PBS containing 1% BSA for 1 h at room temperature. The cells were then incubated with FITC-conjugated phalloidin (0.1 μg/ml, Sigma, St. Louis, MO, USA) for 1 h at room temperature within a moist chamber. After washing with PBS, the samples were mounted with DAPI Fluoromount G (Southern Biotech, Birmingham, AL, USA). Images were acquired using an Olympus BX51 microscope coupled with an Olympus DP70 digital camera.

Western blotting analysis

Subconfluent cells were washed with PBS, and proteins were collected for 20 min on ice in RIPA buffer containing 1% protease inhibitor cocktail. The protein concentration was determined using a BCA protein assay reagent kit. The cellular proteins were then separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with primary antibodies overnight at 4 °C, and then incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The following antibodies were used: rabbit anti-P38 antibody, rabbit anti-P-P38 antibody, mouse anti-HSP27 antibody, rabbit anti-P-HSP27 antibody (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-β -actin antibody (Santa Cruz, CA, USA). Protein bands were detected by incubating with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotech, Santa Cruz, CA, USA) and visualized with ECL reagent (Millipore, Billerica, MA, USA). Digital images of immunoblots were obtained with Chemidoc XRS and analyzed using the image analysis program Quantity One (Bio-Rad, Hercules, CA).

Apoptosis analysis by flow cytometry

Cell apoptosis levels were measured with an Annexin V-FITC apoptosis detection kit (Beyotime, shanghai, China). In brief, cells were seeded in a 25 cm2 dish and collected, resuspended in 100 μL of 1 × binding buffer containing 2.5 μL of FITC-conjugated Annexin-V and 1 μL of PI (100 μg/mL) and incubated for 15 min in the dark. The stained cells were then analyzed using flow cytometry.

Determination of reactive oxygen species (ROS) generation

2’,7’-dichlorofluorescein diacetate (CM-H2DCFDA) (Invitrogen Life Technologies, Carlsbad, CA, USA), an ROS-specific fluorescent probe, was used to determine the intracellular ROS levels. HUVECs were incubated with 5 μM CM-H2DCFDA for 15 min at 37 °C, and then washed twice with PBS. The cover slips were then mounted on glass slides. Images were obtained using an Olympus BX51 microscope coupled with an Olympus DP70 digital camera. Fluorescence intensity was quantified using Image J software.

Statistical analysis

Data were analyzed using ImageJ software, and statistical analyses were carried out using SPSS software version 15.0 (SPSS Inc., Chicago, IL). The Student’s t test was used to analyze differences between two groups. Statistical significance was considered when p < 0.05.

RESULTS

Effect of melatonin on cell viability of H/R-treated HUVECs

HUVECs were treated with different doses of melatonin, and cell viability was measured by MTT assay. As shown in Figure 1A, cell viability decreased after H/R treatment. Melatonin, at doses ranging from 1 to 100 μM, rescued cell viability. Treatment with melatonin alone had almost no significant effect on the viability and proliferation of HUVECs (Figure 1B&1E). We then assessed the proliferation rate of HUVECs under H/R. As shown in Figure 1C&1D, the decreased proliferation rate of H/R-treated HUVECs was attenuated by melatonin treatment. In addition, H/R produced a small increase in the number of apoptotic cells, and the upregulation of cell apoptosis was slightly reduced when the HUVECs were pretreated with melatonin (Figure 2).

Figure 1.

Figure 1

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Effect of melatonin on proliferation in H/R-treated HUVECs. (A) MTT assay showed that the decreased viability of H/R-treated HUVECs was attenuated by the addition of melatonin. (B&E) Melatonin had no effect on viability (B) or proliferation (E) when the HUVECs were cultured in normal condition (n = 10 for each group). (C&D) EdU staining showed that the decreased numbers of EdU+ in H/R was attenuated by melatonin pretreatment (n = 5 for each group). Scale bar, 10 μm. * p < 0.05, ** p < 0.01.

Figure 2.

Figure 2

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Effect of melatonin on apoptosis in H/R-treated HUVECs. HUVECs were pretreated with the indicated doses of melatonin and exposed to H/R conditions. Cell apoptosis was determined by flow cytometry. The experiments were repeated three times independently. * p < 0.05.

Effect of melatonin on adhesive and migratory abilities of H/R-treated HUVECs

HUVECs were pretreated with melatonin, and their adhesive and migratory abilities under control or H/R conditions were determined. As shown in Figure 3, H/R treatment produced a significant decrease in HUVEC adhesion (Figure 3A) and motility (Figure 3B&3C). Melatonin pretreatment abolished the effects induced by H/R, indicating that melatonin could rescue the HUVEC adhesive and migratory abilities impaired by H/R.

Figure 3.

Figure 3

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Effect of melatonin on adhesive and migratory abilities in H/R-treated HUVECs. Adhesive and wound-healing assays showed that melatonin dose-dependently attenuated H/R-induced the inhibition of HUVEC adhesive and migratory ability (n = 10 for each group). * p < 0.05, ** p < 0.01, *** p < 0.001.

Effect of melatonin on ROS production of H/R-treated HUVECs

ROS is the main initiator of cell injury response to H/R,12 so in the next step, we explored ROS production in H/R and melatonin + H/R-treated HUVECs. As shown in Figure 4, in the HUVECs, CM-H2DCFDA fluorescence, which indicated the intracellular ROS levels, was increased under H/R conditions, and dose-dependently decreased when melatonin was added to the culture.

Figure 4.

Figure 4

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Effect of melatonin on ROS production in H/R-treated HUVECs. Melatonin treatment dose-dependently reduced ROS production when the HUVECs were exposed to H/R (n = 5 for each group). Scale bar, 10 μm. * p < 0.05, ** p < 0.01.

Effect of melatonin on the cytoskeleton reorganization of H/R-treated HUVECs

As shown in Figure 5, parallel stress fibers of actin cytoskeleton collapsed under H/R conditions in the HUVECs, indicating an actin cytoskeleton disruption induced by H/R. The addition of melatonin also greatly prevented H/R-induced collapse of actin filaments.

Figure 5.

Figure 5

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Effect of melatonin on cytoskeleton reorganization in H/R-treated HUVECs. After pretreatment with the indicated doses of melatonin, HUVECs were exposed to H/R. Cells were then fixed and stained with FITC phalloidin to label filamentous actin. The actin cytoskeleton was visualized by fluorescence microscopy. Scale bar, 2 μm. The experiments were repeated three times independently.

Effect of melatonin on phosphorylation levels of P38 and HSP27 in H/R-treated HUVECs

First, we determined whether melatonin was capable of regulating the phosphorylation of P38 and HSP27 in H/R-treated HUVECs. As shown in Figure 6A&6B, a significant induction of phosphorylated P38 and HSP27 was observed after H/R treatment. Pretreatment with melatonin dose-dependently reversed this upregulation. In contrast, the levels of total P38 and HSP27 were constant at all dose points. Melatonin alone did not significantly change levels of p-P38 and p-HSP27 (Figure 6C&6D). Next, to determine the involvement of ROS in H/R-stimulated changes in P38/HSP27 phosphorylation, NAC, a specific scavenger for ROS, was applied. As shown in Figure 7, levels of phosphorylated P38 and HSP27 in the HUVECs under H/R conditions were greatly increased, and this was markedly blocked by pretreatment with NAC. These results confirmed the mechanism of ROS in modulating H/R-induced injury in HUVECs.

Figure 6.

Figure 6

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Effect of melatonin on P38 and HSP27 phosphorylation. (A&B) After pretreatment with the indicated doses of melatonin, HUVECs were exposed to H/R. P38 (A) and HSP27 (B) phosphorylation levels were determined by immunoblotting analysis. β-actin was used as a loading control. * p < 0.05, ** p < 0.01, *** p < 0.001. (C&D) HUVECs were exposed to the indicated doses of melatonin. P38 (C) and HSP27 (D) phosphorylation levels were also determined by immunoblotting analysis. The experiments were repeated three times independently.

Figure 7.

Figure 7

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Effect of ROS scavenger on P38 and HSP27 phosphorylation in H/R-treated HUVECs. After treatment with 1 mM of the ROS scavenger NAC for 30 min, HUVECs were treated with H/R and then analyzed by immunoblotting assay for P-P38 and P-HSP27 expressions. ** p < 0.01. The experiments were repeated three times independently.

DISCUSSION

Vascular endothelial cells play an important role in regulating vascular tension, anti-thrombosis, inhibiting proliferation of smooth muscle cells and inflammatory reactions of blood vessel walls. Endothelial dysfunction is the most common pathological basis of many cardiovascular diseases.13 Recent evidence highlights the role of H/R in inducing oxidative stress, eventually promoting vascular and myocardial injuries.14-16 The dysfunction of vascular endothelial cells induced by H/R may lead to thrombosis and atherosclerotic plaque formation, vasospasm, and further damage to parenchymal cells. Thus, protecting vascular endothelial cells is important to prevent H/R damage. HUVECs are high purity endothelial cells, therefore, they can be used to mimic changes in the structure and function of vascular endothelium during H/R injury.

In this study, we found that H/R exerted an inhibitory effect on HUVEC proliferation, migration, and adhesion. Different from other studies which focused on the proapoptotic effect of H/R,17,18 we found that H/R slightly promoted apoptosis in HUVECs. Melatonin itself had no cytotoxicity to HUVECs. Further, treatment with melatonin under H/R conditions could antagonize those roles, revealing that melatonin could reduce H/R-induced injury of vascular endothelial cells and protect the ability of endothelial cells to repair tissue.

Under normal circumstances, vascular endothelial cells generate low amounts of ROS to maintain normal cellular redox homeostasis. After H/R treatment, large amounts of ROS are rapidly generated in endothelial cells leading to injury.19-22 High levels of ROS have been shown to promote apoptosis of vascular endothelial cells via the p38 MAPK pathway.23 Inhibition of ROS has also been reported to be closely related to the amelioration of high glucose-induced vascular inflammatory processes.24 Consistently, we found that H/R upregulated ROS production in HUVECs, and this may have promoted the processing of HUVEC injury.25

Besides its function as a synchronizer of the biological clock, melatonin is recognized as a powerful free-radical scavenger and wide-spectrum antioxidant.26,27 With regards to the mechanism, the high concentration of melatonin in mitochondria likely helps it to exert protective effects against oxidative stress.28 Melatonin has been reported to not only directly scavenge ROS, but also to lead to an upregulation of antioxidant enzymes and a downregulation of pro-oxidant enzymes as well as a reduction in toxic cytokine synthesis.29 In addition, melatonin has been shown to ameliorate smoke-induced vascular injury.30 It has also been reported to induce the activation of phase-2 antioxidative enzymes to protect human epidermal keratinocytes from ultraviolet radiation-mediated oxidative stress.31 In this study, we treated HUVECs with melatonin, and the results showed that melatonin significantly decreased ROS production of HUVECs under H/R conditions, suggesting that melatonin may exert an anti-injury effect on H/R-treated HUVECs by blocking ROS generation.

The accumulation of ROS has been shown to act as a messenger to activate the p38 MAPK pathway.32-34 HSP27 is thought to act as a p38 MAPK downstream effecter, and has been reported to regulate actin rearrangement when bound to actin filaments.35 These filaments comprise the cellular cytoskeletal architecture and support many important cellular processes, including cell motility, proliferation and maintenance of cell junctions.36-38 Our previous studies also showed that, in renal proximal tubule cells, ATP depletion reduced cell adhesive ability via activating the p38 MAPK/HSP27/actin cytoskeleton signaling pathway.39 Recent studies have shown that HSP27 accumulates in ischemia/reperfusion neurons.40 Melatonin has also been shown to reverse morphine tolerance by inhibiting HSP27 expression.41 In this study, we examined whether ROS was a potential activator of the p38 MAPK/HSP27 signaling pathway in our system. Our results showed that H/R induced a dose-dependent increase in P38 and HSP27 phosphorylation and the collapse of the actin cytoskeleton, and that melatonin ameliorated the upregulation and reconstruction of the disrupted actin cytoskeleton in HUVECs. Similarly, blocking ROS by NAC also significantly prevented H/R-induced P38 and HSP27 phosphorylation. Therefore, it may be reasonable to think that the generation of ROS upon treatment with H/R increased the activation of the p38 MAPK/HSP27/actin cytoskeleton pathway in HUVECs, which otherwise may be the main target for melatonin treatment to prevent injury induced by H/R.

CONCLUSIONS

In summary, melatonin inhibited endothelial cellular oxidative stress damage and showed a protective effect on vascular endothelium. Our results suggest that therapy with melatonin may have a potential clinical effect in H/R-induced vascular injury.

Acknowledgments

This work was supported by Foundation of The Key Projects of Changzhou Municipal Committee of Health and Family Planning (ZD201407), the National Natural Science Foundation of China (No. 81773107).

COMPETING INTERESTS

The authors declared that they have no competing interests.

REFERENCES

  • 1.Ferdinandy P, Hausenloy DJ, Heusch G, et al. Interaction of risk factors, comorbidities, and comedications with ischemia/reperfusion injury and cardioprotection by preconditioning, postconditioning, and remote conditioning. Pharmacol Rev. 2014;66:1142–1174. doi: 10.1124/pr.113.008300. [DOI] [PubMed] [Google Scholar]
  • 2.Pandi-Perumal SR, BaHammam AS, Ojike NI, et al. Melatonin and human cardiovascular disease. J Cardiovasc Pharmacol Ther. 2016;[E-pub ahead of print] doi: 10.1177/1074248416660622. [DOI] [PubMed] [Google Scholar]
  • 3.Sun H, Gusdon AM, Qu S. Effects of melatonin on cardiovascular diseases: progress in the past year. Current Opinion in Lipidology. 2016;27:408–413. doi: 10.1097/MOL.0000000000000314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Ekelof SV, Halladin NL, Jensen SE, et al. Effects of intracoronary melatonin on ischemia-reperfusion injury in ST-elevation myocardial infarction. Heart Vessels. 2016;31:88–95. doi: 10.1007/s00380-014-0589-1. [DOI] [PubMed] [Google Scholar]
  • 5.Simko F, Baka T, Paulis L, et al. Elevated heart rate and nondipping heart rate as potential targets for melatonin: a review. J Pineal Res. 2016;61:127–137. doi: 10.1111/jpi.12348. [DOI] [PubMed] [Google Scholar]
  • 6.Favero G, Rodella LF, Reiter RJ, et al. Melatonin and its athero-protective effects: a review. Mol Cell Endocrinol. 2014;382:926–937. doi: 10.1016/j.mce.2013.11.016. [DOI] [PubMed] [Google Scholar]
  • 7.Cimen B, Uz A, Cetin I, et al. Melatonin supplementation ameliorates energy charge and oxidative stress induced by acute exercise in rat heart tissue. Acta Cardiol Sin. 2017;33:530–538. doi: 10.6515/ACS20170331A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Lanoix D, Lacasse AA, Reiter RJ, et al. Melatonin: the watchdog of villous trophoblast homeostasis against hypoxia/reoxygenation-induced oxidative stress and apoptosis. Mol Cell Endocrinol. 2013;381:35–45. doi: 10.1016/j.mce.2013.07.010. [DOI] [PubMed] [Google Scholar]
  • 9.Yang L, Zheng J, Xu R, et al. Melatonin suppresses hypoxia-induced migration of huvecs via inhibition of erk/rac1 activation. Int J Mol Sci. 2014;15:14102–14121. doi: 10.3390/ijms150814102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Feng Y, Hu L, Xu Q, et al. Cytoprotective role of alpha-1 antitrypsin in vascular endothelial cell under hypoxia/reoxygenation condition. J Cardiovasc Pharmacol. 2015;66:96–107. doi: 10.1097/FJC.0000000000000250. [DOI] [PubMed] [Google Scholar]
  • 11.Deng W, Zhang Y, Gu L, et al. Heat shock protein 27 downstream of p38-pi3k/akt signaling antagonizes melatonin-induced apoptosis of sgc-7901 gastric cancer cells. Cancer Cell Int. 2016;16:5. doi: 10.1186/s12935-016-0283-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Yu D, Li M, Tian Y, et al. Luteolin inhibits ros-activated mapk pathway in myocardial ischemia/reperfusion injury. Life Sci. 2015;122:15–25. doi: 10.1016/j.lfs.2014.11.014. [DOI] [PubMed] [Google Scholar]
  • 13.Luchetti F, Crinelli R, Cesarini E, et al. Endothelial cells, endoplasmic reticulum stress and oxysterols. Redox Biol. 2017;13:581–587. doi: 10.1016/j.redox.2017.07.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Zhu M, Ding J, Jiang H, et al. Propofol ameliorates endothelial inflammation induced by hypoxia/reoxygenation in human umbilical vein endothelial cells: role of phosphatase a2. Vascul Pharmacol. 2015;73:149–157. doi: 10.1016/j.vph.2015.06.002. [DOI] [PubMed] [Google Scholar]
  • 15.Zhang Q, Shang M, Zhang M, et al. Microvesicles derived from hypoxia/reoxygenation-treated human umbilical vein endothelial cells promote apoptosis and oxidative stress in h9c2 cardiomyocytes. BMC Cell Biol. 2016;17:25. doi: 10.1186/s12860-016-0100-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Chang ST, Chu CM, Yang TY, et al. Optimal duration of coronary ligation and reperfusion for reperfusion injury study in a rat model. Acta Cardiol Sin. 2016;32:491–497. doi: 10.6515/ACS20150824B. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Li HW, Meng Y, Xie Q, et al. Mir-98 protects endothelial cells against hypoxia/reoxygenation induced-apoptosis by targeting caspase-3. Biochem Biophys Res Commun. 2015;467:595–601. doi: 10.1016/j.bbrc.2015.09.058. [DOI] [PubMed] [Google Scholar]
  • 18.Kim YS, Kim JS, Kwon JS, et al. Bay 11-7082, a nuclear factor-kappab inhibitor, reduces inflammation and apoptosis in a rat cardiac ischemia-reperfusion injury model. Int Heart J. 2010;51:348–353. doi: 10.1536/ihj.51.348. [DOI] [PubMed] [Google Scholar]
  • 19.Yu G, Bolon M, Laird DW, et al. Hypoxia and reoxygenation-induced oxidant production increase in microvascular endothelial cells depends on connexin 40. Free Radic Biol Med. 2010;49:1008–1013. doi: 10.1016/j.freeradbiomed.2010.06.005. [DOI] [PubMed] [Google Scholar]
  • 20.Zhang YQ, Hu SY, Chen YD, et al. Hepatocyte growth factor inhibits hypoxia/reoxygenation-induced activation of xanthine oxidase in endothelial cells through the jak2 signaling pathway. Int J Mol Med. 2016;38:1055–1062. doi: 10.3892/ijmm.2016.2708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Michiels C, Arnould T, Houbion A, et al. Human umbilical vein endothelial cells submitted to hypoxia-reoxygenation in vitro: implication of free radicals, xanthine oxidase, and energy deficiency. J Cell Physiol. 1992;153:53–61. doi: 10.1002/jcp.1041530109. [DOI] [PubMed] [Google Scholar]
  • 22.Martin SF, Chatterjee S, Parinandi N, et al. Rac1 inhibition protects against hypoxia/reoxygenation-induced lipid peroxidation in human vascular endothelial cells. Vascul Pharmacol. 2005;43:148–156. doi: 10.1016/j.vph.2005.05.002. [DOI] [PubMed] [Google Scholar]
  • 23.Cheng R, Li C, Wei L, et al. The artemisinin derivative artesunate inhibits corneal neovascularization by inducing ros-dependent apoptosis in vascular endothelial cells. Invest Ophthalmol Vis Sci. 2013;54:3400–3409. doi: 10.1167/iovs.12-11068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Jayakumar T, Chang CC, Lin SL, et al. Brazilin ameliorates high glucose-induced vascular inflammation via inhibiting ros and cams production in human umbilical vein endothelial cells. Biomed Res Int. 2014;2014:403703. doi: 10.1155/2014/403703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Jiang J, Chen DY, Liu ZT, et al. Effect of n-perfluorooctane on hypoxia/reoxygenation injury in human umbilical vein endothelial cells. Acta Cardiol Sin. 2016;32:716–722. doi: 10.6515/ACS20151228D. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Patino P, Parada E, Farre-Alins V, et al. Melatonin protects against oxygen and glucose deprivation by decreasing extracellular glutamate and nox-derived ros in rat hippocampal slices. Neurotoxicology. 2016;57:61–68. doi: 10.1016/j.neuro.2016.09.002. [DOI] [PubMed] [Google Scholar]
  • 27.du Plessis SS, Hagenaar K, Lampiao F. The in vitro effects of melatonin on human sperm function and its scavenging activities on no and ros. Andrologia. 2010;42:112–116. doi: 10.1111/j.1439-0272.2009.00964.x. [DOI] [PubMed] [Google Scholar]
  • 28.Reiter RJ, Rosales-Corral S, Tan DX, et al. Melatonin as a mitochondria-targeted antioxidant: one of evolution’s best ideas. Cell Mol Life Sci. 2017;74:3863–3881. doi: 10.1007/s00018-017-2609-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Reiter RJ, Mayo JC, Tan DX, et al. Melatonin as an antioxidant: under promises but over delivers. J Pineal Res. 2016;61:253–278. doi: 10.1111/jpi.12360. [DOI] [PubMed] [Google Scholar]
  • 30.Wang Z, Ni L, Wang J, et al. The protective effect of melatonin on smoke-induced vascular injury in rats and humans: a randomized controlled trial. J Pineal Res. 2016;60:217–227. doi: 10.1111/jpi.12305. [DOI] [PubMed] [Google Scholar]
  • 31.Kleszczynski K, Zillikens D, Fischer TW. Melatonin enhances mitochondrial atp synthesis, reduces reactive oxygen species formation, and mediates translocation of the nuclear erythroid 2-related factor 2 resulting in activation of phase-2 antioxidant enzymes (gamma-gcs, ho-1, nqo1) in ultraviolet radiation-treated normal human epidermal keratinocytes (nhek). J Pineal Res. 2016;61:187–197. doi: 10.1111/jpi.12338. [DOI] [PubMed] [Google Scholar]
  • 32.Olavarria VH, Recabarren P, Fredericksen F, et al. Isav infection promotes apoptosis of shk-1 cells through a ros/p38 mapk/bad signaling pathway. Mol Immunol. 2015;64:1–8. doi: 10.1016/j.molimm.2014.10.016. [DOI] [PubMed] [Google Scholar]
  • 33.Yin Q, Lu H, Bai Y, et al. A metabolite of danshen formulae attenuates cardiac fibrosis induced by isoprenaline, via a nox2/ros/p38 pathway. Br J Pharmacol. 2015;172:5573–5585. doi: 10.1111/bph.13133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Wang G, Cui J, Guo Y, et al. Cyclosporin a protects h9c2 cells against chemical hypoxia-induced injury via inhibition of mapk signaling pathway. Int Heart J. 2016;57:483–489. doi: 10.1536/ihj.16-091. [DOI] [PubMed] [Google Scholar]
  • 35.Mounier N, Arrigo AP. Actin cytoskeleton and small heat shock proteins: how do they interact? Cell Stress Chaperones. 2002;7:167–176. doi: 10.1379/1466-1268(2002)007<0167:acashs>2.0.co;2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Gerthoffer WT, Gunst SJ. Invited review: focal adhesion and small heat shock proteins in the regulation of actin remodeling and contractility in smooth muscle. J Appl Physiol (1985) 2001;91:963–972. doi: 10.1152/jappl.2001.91.2.963. [DOI] [PubMed] [Google Scholar]
  • 37.Gefen A, Weihs D. Mechanical cytoprotection: a review of cytoskeleton-protection approaches for cells. J Biomech. 2016;49:1321–1329. doi: 10.1016/j.jbiomech.2015.10.030. [DOI] [PubMed] [Google Scholar]
  • 38.Provenzano PP, Keely PJ. Mechanical signaling through the cytoskeleton regulates cell proliferation by coordinated focal adhesion and rho gtpase signaling. J Cell Sci. 2011;124:1195–1205. doi: 10.1242/jcs.067009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Du J, Zhang L, Yang Y, et al. Atp depletion-induced actin rearrangement reduces cell adhesion via p38 mapk-hsp27 signaling in renal proximal tubule cells. Cell Physiol Biochem. 2010;25:501–510. doi: 10.1159/000303055. [DOI] [PubMed] [Google Scholar]
  • 40.Mohammad-Gharibani P, Modi J, Menzie J, et al. Mode of action of s-methyl-n, n-diethylthiocarbamate sulfoxide (detc-meso) as a novel therapy for stroke in a rat model. Mol Neurobiol. 2014;50:655–672. doi: 10.1007/s12035-014-8658-0. [DOI] [PubMed] [Google Scholar]
  • 41.Lin SH, Huang YN, Kao JH, et al. Melatonin reverses morphine tolerance by inhibiting microglia activation and hsp27 expression. Life Sci. 2016;152:38–43. doi: 10.1016/j.lfs.2016.03.032. [DOI] [PubMed] [Google Scholar]

Articles from Acta Cardiologica Sinica are provided here courtesy of Taiwan Society of Cardiology

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