Hypoxic preconditioning protects microvascular endothelial cells against hypoxia/reoxygenation injury by attenuating endoplasmic reticulum stress

Xu-Dong Wu • Zhen-Ying Zhang • Sheng Sun •Yu-Zhen Li • Xiao-Reng Wang • Xiu-Qin Zhu •Wei-Hong Li • Xiu-Hua Liu

Abstract

Endothelial cells (ECs) are directly exposed to hypoxia and contribute to injury during myocardial ischemia/ reperfusion. Hypoxic preconditioning (HPC) protects ECs against hypoxia injury. This study aimed to explore whether HPC attenuates hypoxia/reoxygenation (H/R) injury by suppressing excessive endoplasmic reticulum stress (ERS) in cultured microvascularECs(MVECs)from ratheart.MVECs injury was measured by lactate dehydrogenase (LDH) leakage, cytoskeleton destruction, and apoptosis. Expression of glucose regulating protein 78 (GRP78) and C/EBP homologous protein (CHOP), activation of caspase-12 (pro-apoptosis factors)andphosphorylationofp38mitogen-activatedprotein kinase (p38 MAPK) were detected by western blot analysis. HPC attenuated H/R-induced LDH leakage, cytoskeleton destruction, and cell apoptosis, as shown by flow cytometry, Bax/Bcl-2 ratio, caspase-3 activation and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling. HPCsuppressed H/R-inducedERS, asshown bya decrease in expression of GRP78 and CHOP, and caspase-12 activation. HPCenhancedp38MAPKphosphorylationbutdecreasedthat ofproteinkinaseR-likeERkinase(PERK,upstreamregulator of CHOP). SB202190 (an inhibitor of p38 MAPK) abolished HPC-induced cytoprotection, downregulation of GRP78 and CHOP, and activation of caspase-12, as well as PERK phosphorylation. HPC may protect MVECs against H/R injury by suppressing CHOP-dependent apoptosis through p38 MAPK mediated downregulation of PERK activation.

Keywords Hypoxic preconditioning Hypoxia/ reoxygenation Microvascular endothelial cells Endoplasmic reticulum stress

Introduction

Endothelial cells (ECs) are directly exposed to hypoxia and contribute to endothelial dysfunction-related diseases, such as coronary heart disease, atherosclerosis, and hypertrophy [1]. One strategy to prevent ECs injury could be to evoke endogenous cytoprotective mechanisms. In ischemic preconditioning (IPC), which can be simulated by hypoxic preconditioning (HPC) [2], brief ischemic episodes can attenuate subsequent sustained ischemia or ischemia/ reperfusion (I/R) injury, protect the heart against myocardial infarction and apoptosis, and attenuate coronary endothelium dysfunction [3]. However, the cytoprotective mechanism of IPC is incompletely understood; some evidence suggests that it prevents excessive endoplasmic reticulum stress (ERS) induced by I/R [4, 5].
The endoplasmic reticulum (ER) is a principal site for protein synthesis and folding, calcium storage, and signaling [6]. Alterations in the ER environment, such as perturbation of Ca2? homeostasis, elevated protein synthesis, deprivation of glucose, altered glycosylation, and the accumulation of misfolded proteins, cause ERS [7]. ERS induces the degradation of misfolding proteins and activates a highly conserved transcriptional program to increase ER capacity of protein folding and calcium storage [8]. ERS can be induced by the ER stress inducers thapsigargin (TG), which depletes Ca2? from ER, and tunicamycin (TM), which inhibits protein N-linked glycosylation. By coordinating the suppression of protein synthesis and upregulating ER-resident chaperones, such as glucose-regulated protein 78 (GRP78) and calreticulin, moderate ERS is often able to restore cellular homeostasis for a cytoprotective effect. However, when ERS is excessive, the ER-related apoptotic process is initiated with the induction of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) and activation of caspase-12.
Protein kinase R-like ER kinase (PERK) is an important ER transmembrane protein. The relocation of GRP78 from the luminal domain of PERK to misfolded proteins leads to PERK activation, which induces translation of activating transcription factor 4 (ATF4) and its target genes such as CHOP [9], thus contributing to apoptosis in I/R injury [10], atherosclerosis [11] and diabetes [12]. The ER is abundant in ECs and is critical for endothelial function and apoptosis control. Our previous study showed that HPC attenuated ER stress-related apoptosis induced by sustained hypoxia/ reoxygenation (H/R) in neonatal cardiomyocytes [13].
How HPC regulates ER stress is not fully characterized. Preconditioning-induced cytoprotection has been attributed, in part, to the change in activation of mitogen-activated protein kinases (MAPKs). MAPKs include extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, which are important in transducing extracellular signals into intracellular events [14]. JNK and p38 MAPK are regulated by extracellular stresses including oxidative stress, heat shock, and ultraviolet radiation. Delayed protection induced by adenosine A1 receptor agonist 2-chloro-N 6-cyclopentyladenosine (CCPA) was abolished by the p38 MAPK inhibitor SB203580 in the mouse heart, which suggests an essential role of p38 MAPK in cardioprotection [15]. TG-induced ER stress activated JNK before apoptosis in Jurkat T cells. The c-Jun-negative mutant prevented TG-induced apoptosis, which suggests that JNK also mediates ERS-induced apoptosis [16]. However, the role of p38 MAPK in regulation of H/R-induced ERS has not been investigated.
We hypothesized that HPC protects microvascular ECs (MVECs) against H/R injury by suppressing intense ERSinduced CHOP-dependent apoptosis, through p38 MAPK mediated down-regulating of PERK activation.

Materials and methods

Chemicals

M199 medium (31100019) was from Gibco Co. (Carlsbad, CA, USA); endothelial cell growth supplement (ECGS, E0760), collagenase I (C0130), fluorescein isothiocyanate (FITC)-labeledalbumin(A7016), phalloidin-FITC (P-5282), tetramethyl ethylene diamine (TEMED, T-7024), phenylmethyl sulfonylfluoride (PMSF, P-7626), sodium dodecyl sulphate (SDS, L-3771), taurine, and SB202190 were from Sigma Chemical Co. (St. Louis, MO, USA). SB-203580 was from Research Biochemicals International (Natick, MA, USA). 29 Taq PCR MasterMix (KT201), 100 bp DNA Ladder (MD109), and agarose (RT101) were from Tiangen Biotech Co. (Beijing); rabbit anti-rat GRP78 (SPA-826) polyclonalantibodywas from StressgenCo.(San Diego,CA, USA); rabbit anti-human glyceraldehyde phosphate dehydrogenase (GAPDH, Sc-25778), rabbit anti-mouse CHOP (Sc-575), rabbit anti-human Bax (Sc-493) and Bcl-2 (Sc-783), goat anti-human PERK (Sc-9479), rabbit antihuman phospho-PERK (Sc-32577), phospho-specific antibody against p38 MAPK, p38 MAPK, monoclonal antibody against VE-cadherin, and enhanced chemiluminescence immunodetection kit (Sc-2048) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); peroxidase-conjugated AffiniPure goat anti-rabbit IgG (111-035-003), goat antimouseIgG(115-035-003)andrabbitanti-goatIgG(305-005003) were from Jackson ImmunoResearch Co. (West Grove, PA, USA); rabbit anti-mouse caspase-12 polyclonal antibody (3182-100) was from Biovision (CA, USA); TG (586005)wasfromCalbiochemCo.(Israel);immobilizedpH gradientbuffer(pH3–10),DestreakReagent,low-molecularweight calibration kit for SDS electrophoresis, and isoelectrofocusing Sample Appl Piece, Immobiline DryStrip (pH 3–10) were from Amersham Biosciences Co. (Buckinghamshire, UK); cocktail tablets were from Roche Co. (Basel, Switzerland).

Cell culture and experimental protocol

All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and approved by the local animal care and use committee. The culture and identification of myocardial MVECs from Sprague–Dawley rats were as described [17]. Briefly, male and female rats were killed by intraperitoneal injection of a lethal dose of pentobarbital (100 mg/kg body weight) and the left ventricles were fully minced and digested with 0.1 % collagenase I for 10 min at 37 C in a shaking water bath. 0.1 % trypsin was added for incubation for another 10 min at 37 C. The digested solution was filtered through 100 lm mesh filter, and the filtrate was collected and suspended in M199 medium containing 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 lg/mL streptomycin, 25 % fetal calf serum, 40 U/mL heparin, and 0.02 g/L ECGS. Then, the suspension was cultured in a humidified atmosphere with 5 % CO2 at 37 C.
The purity of MVECs was identified by staining with EC-specific marker VE-Cadherin (an EC-specific adhesion molecule and determinant of microvascular integrity in vivo; Fig. 1). MVECs were grown on collagen-coated coverslips and fixed in 3 % paraformaldehyde, permeabilized in 0.3 % Triton X-100, and blocked in 2 % bovine serum albumin. The cells were stained with rabbit antihuman antibody against VE-Cadherin (1:50), then goat anti-mouse Alexa 488 (Molecular Probes). Immunofluorescence images were viewed under a confocal laser scanning microscope. MVECs was also identified by microarray analysis as described [17].
TG was dissolved in DMSO and stored at -20 C. After 6–7 days, cells were plated at 5 9 104/cm2 (except for cytoskeleton and ER staining, for which the density was 1 9 104/cm2 to obtain individually plated cells) and allowed to rest overnight before the application of stress. The medium was replaced with serum-free M199 medium, and MVECs were incubated for an additional 24 h and divided randomly into the following groups for treatment: (1) control: culture normally with 95 % air and 5 % CO2; (2) H/R: under sterile conditions, cells on culture plates were placed into the hypoxia chamber (Anero-Pack series, Mitsubishi Gas Chemical Co.) for 3 h to induce hypoxia, then re-oxygenated with maintenance medium for 24 h to induce reoxygenation through p38 MAPK mediated downregulating of PERK activation; (3) HPC: incubation with 95 % N2–5 % CO2 for 20 min, then culture normally for 24 h to induce HPC. (4) HPC ? H/R: at 24 h after HPC, cells were exposed to lethal H/R treatment described for group (2); (5) SB202190: incubation with SB202190 (inhibitor of p38 MAPK, 5 lmol/L) for 10 min and cultured normally with 95 % air and 5 % CO2 for 24 h; (6) SB202190 ? HPC ? H/R (SB ? HPC ? H/R): preincubation with SB202190 (5 lmol/L) for 10 min before HPC and H/R treatment described previously.
To investigate the effects of HPC on ERS-induced injury, MVECs were divided into the following groups for treatment: (1) control: culture normally with 95 % air and 5 % CO2; (2) TG: incubation with 0.1, 0.2, 0.5, or 1 lmol/L TG (ER stress inducer) for 24 h; (3) HPC ? TG: culture with 0.5 lmol/L TG for 24 h after HPC treatment described above.
To demonstrate that HPC protects MVECs through attenuating excessive ERS, the MVECs are divided randomly into the following groups: (1) control, (2) TG: culture with 0.5 lmol/L TG for 24 h, (3) HPC ? TG group: culture with 0.5 lmol/L TG for 24 h after HPC treatment, (4) Taurine ? TG group: Culture with taurine (an ER stress inhibitor, 40 mM) for 1 h, followed by incubating with 0.5 lmol/L TG for 24 h, (5) HPC ? taurine ? TG group: Culture with taurine (40 mM) for 1 h, prior to HPC and TG treatment.
To further demonstrate that p38 MAPK was involved in HPC’s suppression of ERS-related injury induced by H/R, another structurally different inhibitor SB203580 was used in the experiment. MVECs were divided randomly into control, H/R, HPC, HPC ? H/R, SB203580 (incubation with SB203580, 5 lmol/L for 10 min), and SB203580 ? HPC ? H/R (incubation with SB203580, 5 lmol/L for 10 min, followed by HPC and H/R) groups. MVECs were analyzed for activation of caspase-3 and caspase-12 by Western blotting. Apoptosis were detected by Terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) as described below.

Lactate dehydrogenase (LDH) activity and apoptosis rate analysis

LDH activity in medium was measured spectrophotometrically by use of an LDH assay kit (Jiancheng Bioengineering Inst., Nanjing, China). For apoptosis analysis, cells were collected and stained by use of the Annexin V-FITC Apoptosis Detection Kit (KeyGEN Bioengineering Inst., Nanjing, China), and apoptosis was analyzed by use of the BD FACScalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

Transwell permeability assay

MVECs were plated on 6.5 mm Costar polyester transwells with pore size 3 lm (Corning Life Sciences, Wilkes Barre, PA, USA) coated with 1 % glutin; the permeability assay of MVECs was as described previously [18]. Briefly, at the end of the experiment, culture medium in the top well was replaced with 100 lL FITC–labeled albumin (250 lg/mL) for 4 h at 37 C. Duplicate 20 and 200 lL medium from the top and bottom wells, respectively, was put into wells of a 96-well plate for determination of fluorescence by a spectrophotometer (Viktor 1420, Wallac, PerkinElmer, Waltham, MA, USA). Percentage permeability was calculated as follows: % permeability = *fluorescencebottom/

ER and cytoskeleton staining

MVECs were plated on glass-bottomed culture dishes (Mattek Co., Ashland, MA, USA) and ER staining of freshly viable MVECs followed the kit protocol (GMS10041.2, Genmed, Arlington, MA, USA). Cytoskeleton staining was as described [19]. Briefly, MVECs fixed with 2.5 % glutaraldehyde were incubated with phalloidinFITC for 1 h at room temperature. After 3 washings with ice-cold phosphate buffered saline, stained MVECs were mounted in mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and viewed under a confocal laser scanning microscope (FV1000, Olympus, Tokyo, Japan).

TUNEL staining

MVECs were grown on collagen-coated coverslips and fixed with 2.5 % glutaraldehyde. Tunel assay involved use of the In Situ Cell Death Detection Kit (Promega G3250, Madison, WI, USA). The staining was observed under a laser confocal microscope (Olympus, Japan). At least 10 randomly chosen fields with 400 DAPI-positive cells were scored, and the number of TUNEL-positive cells was presented as a percentage of positive nuclei/total nuclei counted 9 100 %.

2-D gel electrophoresis (2-DE) and mass spectrometry

Protein was extracted from MVECs as described [17]. Protein concentration was detected by Bradford assay [20], and total protein was kept in liquid nitrogen until use. 2-DE involved the immobilized pH gradient technique as described [21]. In total, 1 mg protein was applied to 18 cm immobilized pH gradient strips (pH range, 3–10) and separated by isoelectric focusing electrophoresis at 20 C in a Multiphor II apparatus (Amersham Pharmacia Biotech, Switzerland), then 12.5 % SDS-PAGE was performed. After Coomassie brilliant blue staining and computer analysis, the protein spots expressed differently between control and TG treatments underwent mass spectrometry analysis, then a database search with the Mascot software (Matrix Science, Boston, MA) for peptide mapping.

RT-PCR

Total RNA was isolated from MVECs by use of the RNAsimple Total RNA kit, and reverse transcription involved the QuantScript RT kit (TianGen, Beijing, China). cDNA was stored at -20 C. PCR products were separated on 1.5 % agarose gel, and the expression of Bax and Bcl-2 was evaluated by use of Image-Pro Plus (Media Cybernetics, Silver Spring, MD, USA). For quantification, the mRNA expression of the genes was normalized to that of the housekeeping gene GAPDH, calibrated to 1.0.

Western blot analysis

Protein was extracted from MVECs as described [17]. Equal amounts of protein (60 lg/lane) underwent SDSPAGE (10 % resolving gel), then transfer to a nitrocellulose membrane for immunoblot analysis. After incubation with blocking buffer (10 % nonfat dry milk) for 4–6 h, blots were incubated with the antibodies anti-GRP78 (1:1,000), anti-caspase-3 (1:200), anti-caspase-12 (1:200), anti-Bax (1:200), anti-Bcl-2 (1:200), or phospho-specific antibody against p38 MAPK, p38 MAPK, and anti-GAPDH (1:500) overnight at 4 C. After incubation for 1 h with secondary antibody (1:1,000), the reaction was visualized by use of an enhanced chemiluminescence kit. The expression of proteins was quantified by use of Image-Pro Plus. The levels of analyzed proteins were normalized to that of GAPDH, calibrated to 1.0.

Statistical analysis

Results are presented as mean ± SD. Statistical differences among groups were assessed by one-way ANOVA with Student–Newman–Keuls post-test. P\0.05 was considered statistically significant.

Results

H/R and TG caused MVECs injury

MVECs viability in all groups was [95 % before treatment. Cellular viability detected by trypan blue assay, apoptosis by Annexin V-FITC detection, and LDH activity with treatment is in Table 1. Compared with control treatment, H/R decreased viability by 24 % (P\0.05), and increased apoptosis by 5.34 % (P\0.05). LDH activity was greater with H/R than control treatment (1226.21 ± 39.72 vs. 876.22 ± 11.21, P\0.05).
The ERS inductor TG dose-dependently induced significant cell injury in MVECs (Supplementary Figure 1). Compared with control treatment, 0.1 lmol/L TG increased LDH activity (710.17 vs. 456.30 U/L, P\0.05), and cell apoptosis was elevated significantly with 0.2 lmol/L TG and further increased with increasing TG concentration. Compared with controls, 0.5 lmol/L TG produced significant injury in MVECs, as shown by increased LDH activity (878.70 vs. 456.30 U/L, P\0.05) and cell apoptosis rate (26.7 vs. 12.8 %, P\0.05) (Supplementary Figure 2). Moreover, as compared with 0.5 lmol/L TG, 1 lmol/L TG produced serious MVECs injury, as shown by increased LDH activity (1138.15 vs. 878.70 U/L, P\0.05) and cell apoptosis (37.8 vs. 25.7 %, P\0.05). Because cell apoptosis was increased by 37.8 % with 1 lmol/L TG, we used 0.5 lmol/L TG for HPC experiments.

HPC protected MVECs against H/R injury

HPC significantly attenuated H/R injury as compared with H/Ralone.MVECsshowedan11 %increaseinsurvivalwith lethalH/RwithHPC,ascomparedwithH/Ralone(P\0.05; Table 1). Apoptosis rate was decreased with HPC ? H/R than H/R alone (9.12 ± 0.48 vs. 12.62 ± 1.21 %, P\0.05) and LDH release was decreased (968.11 ± 32.91 vs. 1226.21 ± 39.72, P\0.05). SB202190 (an inhibitor of p38 MAPK) had no effect on apoptosis rate, survival, or LDH release from MVECs in control group (P[0.05). However, compared with HPC alone, SB202190 pretreatment abolished the cytoprotection induced by HPC, as shown by decreased survival rate by 8 % and reduced LDH activity (968.11 ± 32.91 vs. 1116.28 ± 41.26 U/L, P\0.05), and increased cell apoptosis, by 2.2 % (11.32 ± 0.81 vs. 9.12 ± 0.48 %, P\0.05) (Table 1).
MVECs were analyzed for caspase-3 activation by detecting proteolytic cleavage (Fig. 2). H/R increased 17 kD active caspase-3 subunit and HPC inhibited the H/R-induced activity. However, with SB203580 treatment, the inhibitory effect of HPC on caspase-3 activation was attenuated.
We used TUNEL staining to quantify the number of MVECs undergoing apoptosis (Fig. 3). The fraction of TUNEL-positive MVECs with negative control was low as compared with H/R treatment (9.2 ± 2.1 vs. 69.1 ± 5.2 %, P\0.05). HPC significantly attenuated the H/R-induced TUNEL-positive MVECs with HPC. MVECs in HPC group showed decrease in TUNEL-positive cells with lethal H/R as compared with H/R alone (42.6 ± 6.5 vs. 69.1 ± 5.2 %; P\0.05). SB203580 had no effect on apoptosis rate, as shown by TUNEL-positive MVECs (P[0.05). However, compared with HPC ? H/R, SB203580 pretreatment abolished the cytoprotection induced by HPC, as shown increase in TUNEL-positive MVECs (60.9 ± 4.9 vs.
Transwell permeability assay was used to access the effects of HPC on cell permeability induced by H/R. Cell permeability was increased significantly in MVECs subjected to H/R as compared with controls, with percentage permeability elevated 1.2-fold (P\0.05; Table 1). HPC significantly attenuated the H/R-induced increase in permeability by 48.81 % as compared with H/R alone (P\0.05). SB202190 alone had no notable effects on cell permeability (P[0.05 compared with controls). SB202190 pretreatment abolished the HPC-induced protection of cell permeability, as shown by percentage permeability increased 74.58 % as compared with HPC alone (P\0.05).

HPC protected MVECs against TG-induced ERS injury

In TG-treated MVECs, HPC significantly attenuated LDH leakage and cell apoptosis (Supplementary Figure 2A, B), and the apoptosis rate did not differ from that in controls.
Cell permeability was increased significantly in TG-treated MVECs, with percentage permeability elevated 92.5 % of that in controls (P\0.05). HPC significantly blocked the TG-induced increase in permeability, with a 13.9 % decrease as compared with TG alone (P\0.05; Supplementary Figure 2C).
Staining for F-actin in TG-treated MVECs showed disarrangement or dissolved cytoplasm, with obvious vacuoles. HPC prevented the ERS-induced destruction in cytoskeleton, and F-actin staining showed proper arrangement with no vacuoles, similar to that in controls (Supplementary Figure 2D).

ER staining

ER staining in freshly viable MVECs revealed the structure severely destroyed with TG treatment. The fluorescence density of ER showed a non-uniform distribution, accompanied by vacuoles. HPC inhibited the ER injury, as shown by ER fluorescence localized uniformly around the nucleus without any vacuoles and similar to controls (Supplementary Figure 3).

Protein expression profiles in MVECs

The 2-DE expression map contained 530 ± 26 protein spots with TG treatment, as determined by use of Image Master 2D Elite. Seven proteins were expressed differentially in responseto0.5 lmol/LTG,with6proteinsupregulatedand1 protein downregulated as compared with controls (Table 2). Among the identified proteins, GRP78 and protein disulfide isomerase associated 3 (PDIA3), markers of ERS, were significantly upregulated in TG-treated MVECs (Fig. 4).
The 2-DE expression map contained 589 ± 36 protein spots with HPC treatment (Fig. 5a, b). Ten proteins were expressed differentially in response to HPC treatment, with 8 proteins downregulated and 1 protein disappeared, and 1 protein can only be detected in HPC-treated MVECs as compared with controls (Table 3). Among the identified proteins, the expression of markers of ER stress GRP78, PDIA3, and reticulocalbin-1 precursor, as well as calumenin, was decreased by 2.6-, 4.3-, 2.7-, and 2.7-fold, respectively, with HPC treatment as compared with controls (P\0.05; Fig. 5c, d).

Western blot analysis

GRP78 protein expression was significantly upregulated in MVECs subjected to H/R with a 191.6 % increase, comparing with controls (P\0.05; Fig. 6). HPC significantly attenuated the H/R-induced upregulation of GRP78 expression with a 37.18 % decrease (P\0.05) as compared with H/R alone. SB202190 treatment abolished the HPC-induced downregulation of GRP78 expression, as shown by increased GRP78 expression by 39 % as compared with HPC ? H/R (P\0.05).

HPC attenuated H/R-induced caspase-12 activation

Activity of the pro-caspase form of caspase-12 (55 kDa) was similar with all treatment (P[0.05; Fig. 7a, c). The activity of the 35-kDa proteolytic fragment of caspase, caspase-12, was increased by 174.1 % with H/R as compared with controls (P\0.05). HPC reduced the activation of caspase-12 by 46.3 % as compared with H/R alone (P\0.05).SB202190 pretreatment abolished the HPC reduced activation of caspase-12, as shown by an increase in activity of 64.9 % as compared with HPC ? H/R (P\0.05). SB203580 (another p38 MAPK inhibitor) also suppressed the down-regulation of caspase-12 activation induced by HPC (P[0.05; Supplementary Figure 4). HPC-downregulated CHOP expression
The level of CHOP was increased two-fold with H/R as compared with controls (P\0.05; Fig. 7b, d). HPC reduced CHOP expression by 43.3 % as compared with H/R (P\0.05). SB202190 pretreatment abolished the HPC reduced CHOP expression, as shown by an increase in level of 99.0 % as compared with HPC ? H/R (P\0.05).

HPC reduced TG-induced Bax/Bcl-2 ratio

The mRNA and protein ratio of Bax/Bcl-2 was increased by 181.5 and 107.5 %, respectively, in TG-treated MVECs as compared with controls (P\0.05; Fig. 8). HPC reduced the mRNA and protein ratio of Bax to Bcl-2 by 76.1 and 40.4 %, respectively, as compared with TG alone (P\0.05).

Taurine pretreatment reduced HPC-induced MVECs protection

MVECs showed an increase in survival and decrease in LDH leakage in HPC and taurine group, as compared with TG alone (P\0.05), respectively. Pre-incubation of MVECs with taurine prior to HPC partially abolished the protection induced by HPC (Supplementary Table 1). p38 MAPK are involved in HPC suppressed ERS signaling

p38 MAPK activity

The phosphorylation level of p38 MAPK was increased by 32.8 % with H/R as compared with controls (P\0.05; Fig. 9a, c). HPC further increased the activation of p38 MAPK by 67.1 % as compared with H/R alone (P\0.05). Pretreatment with the inhibitor SB202190 abolished the activation of p38 MAPK by 65.5 % as compared with HPC ? H/R (P\0.05).

Phosphorylation and expression of PERK

The phosphorylation level of PERK was increased by 147.6 % with H/R as compared with controls (P\0.05; Fig. 9b, d). HPC suppressed the phosphorylation of PERK by 50.49 % as compared with H/R alone (P\0.05). Pretreatment with the p38 MAPK inhibitor SB202190 reduced the phosphorylation of PERK by 60.41 % as compared with HPC ? H/R (P\0.05).

Discussion

Myocardial MVECs are susceptible to hypoxia injury, which influences the pathogenesis, development, and prognosis of coronary heart disease [1]. Evoking endogenous cytoprotective mechanisms is one of the strategies to prevent MVECs injury induced by ischemia or hypoxia. IPC is a potent endogenous protective mechanism capable of protecting the heart against myocardial infarction and reduces coronary endothelium dysfunction [3]. In present study, we showed that HPC protected MVECs against injury induced by H/R or the ERS inducer TG, with a marked reduction in LDH leakage and cell apoptosis. HPC also prevented cytoskeletal damage and cell permeability increase in ERS-treated MVECs.
The mechanism by which HPC protects ECs against H/R injury has been obscure; some evidence suggests that preventing an increase in cytosolic calcium level and synthesis of proteins is involved [10]. Previous investigation on cardiomyocytes showed that HPC protects cardiomyocytes against apoptosis following reoxygenation through reducing peroxynitrite (ONOO-) formation [22]. The ER, abundant in MVECs, is a principal site for protein synthesis and folding, calcium storage, and signaling [6]. Hypoxia, ischemia, calcium overload, oxidative stress, and ONOO- formation cause ER stress [7]. Preventing I/Rinduced excessive ERS in the myocardium and brain are protective mechanisms of HPC. Elevated Ca2? level in cytoplasm activates Ca2?-dependent calpain, which results in systolic protein degeneration and cytoskeletal structure deterioration [23]. We revealed that HPC significantly inhibited the damaged ER and cytoskeleton and prevented the elevation in cell permeability in TG-treated MVECs, which suggests that the prevention of ERS by HPC is involved in the protection against cytoskeleton destruction. HPC attenuated TG-induced MVEC injury, as shown by decreased LDH leakage and cytoskeleton damage, which suggests that HPC protected MVECs against H/R injury by suppressing excessive ERS.
ERS usually exhibits upregulation of ERS-related molecules such as GRP78 [24] and PDIA3 involved in protein quantity control, which is upregulated by ischemia or oxidative stress [25]. Our 2-DE and mass spectrometry analysis revealed that TG treatment upregulated the expression of GRP78andPDIA3inMVECs,andHPC-downregulatedthat of GRP78, PDIA3, and reticulocalbin-1 precursor, and calumenin (markers of ERS). The alteration in GRP78 protein level was confirmed by western blot analysis in TG- and HPC-treated MVECs, so HPC inhibited H/R-induced excessiveERSinMVECs.ComparingwithTG-treatedcells, survival of MVECs increased while LDH leakage decreased in HPC and taurine group, respectively. This result indicated that HPC and taurine (an ER stress inhibitor) attenuated TGinduced MVECs injury. However, pre-incubation of MVECs with taurine prior to HPC partially reduced the protection induced by HPC, which means that HPC protects MVECs partly through attenuating excessive ER stress.
The unfolded protein response (UPR) is one of the most important responses in ERS and is involved in regulating H/R-related apoptosis and protein quality control. The initial aim of the UPR is to reestablish the ER function and begins with activation of 3 UPR sensors at the ER: PERK, inositol-requiring protein 1 (IRE1), and ATF6 [26]. PERK activation leads to transduction of apoptotic signals or transcription of genes encoding molecular chaperones and proteins involved in the ER-associated degradation pathway. PERK-mediated apoptotic signaling upregulates ATF4 and consequent expression of CHOP, also named growth arrest and DNA damage inducible gene 153 (GADD153), thus leading to apoptosis. CHOP can directly regulate the target genes in the nucleus and increase sensitivity to cell apoptosis [27]. CHOP-/- cells are resistant to ERS-mediated apoptosis [28], and overexpression of CHOP can lead to cell cycle arrest and/or apoptosis [29]. Many studies demonstrate that overexpression of CHOP leads to decreased Bcl-2 protein level and translocation of Bax protein from the cytoplasm to the mitochondria [30]. We revealed that HPC significantly inhibited the H/Rinduced upregulation of PERK phosphorylation and CHOP expression, as well as Bax/Bcl-2 ratio, in ERS-treated MVECs, so HPC protected MVECs against excessive ERSrelated apoptosis by preventing the PERK–CHOP-mediated apoptotic signaling pathway. Besides PERK-CHOP pathway, ER stress triggers a specific caspase cascade in apoptosis, in which ER-localized caspase-12 specifically cleaves and activates procaspase-9 and the activated caspase-9 catalyzes cleavage of procaspase-3 [31]. Our data revealed that HPC suppressed the activation of caspase-12 and caspase-3 in MVECs subjected to H/R, which means that the ER stress-specific caspase 12 was also involved in HPC’s anti-apoptotic effect in MVECs.
.The signals by which HPC inhibits H/R-induced excessive ERS are not fully characterized. IPC-induced cardioprotection has been attributed, in part, to MAPKs, including ERK, JNK, and p38 MAPK. MAPKs are important regulatory proteins that transduce various extracellular signals into intracellular events [32]. p38 MAPK and MAPKAP kinase-2 are strongly activated by ischemia in the perfused rat heart [33]. Direct activation of p38 MAPK by anisomycin mimicked IPC in hearts and myocytes [15] against hypoxic insults. Hung and colleagues showed that GRP78 and calreticulin attenuates H2O2-induced cell injury in LLC-PK1 renal epithelial cells by potentiating ERK activation and decreasing JNK activation [34]. We revealed greater p38 MAPK phosphorylation with HPC in MVECs with H/R, which was positively associated with PERK activation. Inhibition of p38 MAPK with SB202190 abolished the HPC-downregulated PERK activation, CHOP expression and caspase-12 activation, and the cytoprotective effect of HPC, as shown by decreased survival rate, increased apoptosis rate and LDH leakage. Another p38 MAPK inhibitor SB203580 also abolished the HPC-downregulated activation of caspase12, caspase-3, and the anti-apoptotic effect as shown by increased TUNEL-positive MVECs. Our data suggests that p38 MAPK might be involved in H/R-induced apoptosis in MVECs by downregulating ER stress-specific PERK phosphorylation, CHOP expression, and caspase-12 activation.
Our findings demonstrate that suppression of excessive ERS by HPC acts via the p38 MAPK pathway to modulate MVECs injury in response to H/R. With SB202190 preincubation before HPC and H/R, p38 MAPK activity was decreased significantly as compared with HPC ? H/R alone, although the level was higher than that of the control. Thus, HPC-induced p38 MAPK activation is complicated. As a protein kinase, p38 MAPK activity depends on phosphorylation as well as dephosphorylation. We showed that SB202190 inhibited HPC-induced p38 MAPK phosphorylation and HPC might downregulate dephosphorylation of p38 MAPK, which increased p38 MAPK activity with inhibitor pretreatment as compared with the control.
In summary, we have demonstrated that inhibition of excessive ERS attenuates H/R-induced apoptosis in MVECs by downregulating PERK phosphorylation, CHOP expression, and caspase-12 activation, which is mediated by the p38 MAPK pathway during HPC. Inhibition of p38 MAPK with SB202190 abolished the downregulation of PERK phosphorylation, CHOP expression, caspase-12 activation, and cytoprotective effect of HPC. The p38 MAPK signaling pathway is a critical upstream effecter of the suppression of excessive ERS and protection afforded by HPC against H/R injury in MVECs.

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