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Basic Science Aspects

Calreticulin Ameliorates Hypoxia/Reoxygenation-Induced Human Microvascular Endothelial Cell Injury By Inhibiting Autophagy

Wang, You∗,†; Tao, Tian-Qi; Song, Dan-Dan; Liu, Xiu-Hua

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doi: 10.1097/SHK.0000000000000905
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Abstract

INTRODUCTION

Ischemic heart disease, particularly coronary artery disease, is a leading cause of death worldwide (1). Restoration of blood flow (reperfusion) timely to the ischemic myocardium is the preferred strategy to rescue ischemic myocardium after myocardial infarction. However, the efficacy of the therapy is attenuated by ischemia/reperfusion injury, which induces additional damage to the myocardium, with the identification of myocardial stunning, arrhythmia, no-reflow, and so on.

The no-reflow phenomenon is a major contributor to final infarct size and an independent predictor of morbidity/mortality (2), which is characterized by damaged microvessels with intraluminal thrombosis and swollen endothelial cells. Microvascular endothelial cells (MECs) are affected directly by reperfusion (3). Therefore, determining the mechanisms underlying the protection of MECs from reperfusion injury is important for reducing ischemia/reperfusion injury.

Calreticulin (CRT) is a 46 kDa multifunctional protein predominantly located in endoplasmic reticulum (ER) and highly conserved in diverse species. ER-localized CRT is a Ca2+-binding protein, which is involved in ensuring proper conformation of proteins and glycoproteins, as well as in maintaining calcium homeostasis and regulating apoptosis. In recent years, CRT has been demonstrated to also exist in the surface of the cell membrane, cytoplasm, nucleus, and extracellular matrix, regulating cells proliferation, adhesion, migration, and apoptosis (4). It is also involved in pathological processes such as wound healing, immune response, neoplasia, cardiocerebrovascular diseases, and diabetes. Exogenous CRT shows a marked beneficial effect on ischemic diseases and chronic wounds healing in clinical practicing (5). Our previous studies confirmed that exogenous CRT alleviates microwave radiation-induced MECs injury (6, 7) and the mechanisms involved in attenuating ER stress (ERS)-induced apoptosis. Recently, autophagy has garnered considerable attention with regard to endothelial cell injury, which is a lysosome-dependent cellular degradation process that functions in nutrient recycling, energy generation, and the clearance of damaged proteins and organelles. Li et al. (8) reported that CRT expression on the surface of the cell membrane is associated with autophagy suppression. Autophagy was found to suppress CRT surface exposure (9, 10). However, it is not known whether exogenous CRT ameliorates hypoxia/reoxygenation (H/R)-induced MECs injury by inhibiting excessive autophagy. The present study focuses on the role of autophagy in CRT-mediated MECs protection to explore new strategies against H/R injury. We hypothesize that exogenous CRT alleviates H/R-induced MECs injury by suppressing mammalian target of rapamycin (mTOR)-mediated autophagy. We found that H/R induced autophagy through the mTOR pathway, and CRT suppressed rapamycin- and H/R-induced autophagosome formation and Beclin 1 expression in human MECs through the upregulating of mTOR phosphorylation, consequently attenuating H/R-induced human MECs injury.

MATERIALS AND METHODS

Cells, antibodies, and reagents

Human MECs were purchased from the Institutes of Biomedical Sciences, Fudan University, and cultured as described in an earlier study (7). Endothelial cell medium (ECM) was purchased from ScienCell (Carlsbad, Calif). Trypsin was purchased from Amresco (Solon, Ohio). Rapamycin, 3-methyladenine (3-MA), protease inhibitor, penicillin/streptomycin, and Triton X-100 were purchased from Sigma (St. Louis, Mo). Rabbit polyclonal antibodies against phosphorylated mTOR (p-mTOR), Beclin 1, and LC3 were purchased from Cell Signaling Technology (Danvers, Mass). Human CRT recombinant protein (10-288-22432F) was obtained from GenWay Biotech, Inc. (San Diego, Calif). Bovine serum albumin (BSA) and phosphatase inhibitor were purchased from Merck (Rahway, NJ). The Cell Counting Kit-8 (CCK-8) detection kit was from Kaiji Biological Engineering Company (Nanjing, China). Rabbit polyclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the enhanced chemiluminescence kit were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) was purchased from Epitomics (Burlingame, Calif).

Cell culture and protocol

Human MECs in the logarithmic phase were cultured in low-serum medium (ECM with 1% FCS and 1% penicillin/streptomycin) for 12 h and then divided randomly into groups as follows (each experiment was repeated thrice independently; n = 3).

To evaluate the effect of CRT on cell viability in human MECs, cells were divided into 14 groups: 1) control group: human MECs remained in a 5% CO2 incubator at 37°C for 24 h and 30 min; 2)–4) different concentrations of rapamycin groups: to investigate the dose–effect of rapamycin on cell viability, human MECs were treated with rapamycin, an agonist of autophagy, at 50 nM (R50 nM), 100 nM (R100 nM), and 200 nM (R200 nM), respectively, for 24 h and 30 min; 5)–7) different concentrations of 3-MA groups: to investigate the dose–effect of 3-MA on cell viability, human MECs were treated with 3-MA, an antagonist of autophagy, at 2.5 mM (M2.5 mM), 5 mM (M5 mM), and 10 mM (M10 mM), respectively, for 24.5 h; 8) CRT group: cells were incubated with CRT (25 pg/mL) for 24 h and 30 min to investigate the effect of CRT on cell viability; 9) to 11) CRT and different concentrations of rapamycin groups: to investigate the effect of CRT on the autophagy induced by rapamycin at different concentrations, human MECs were treated with CRT (25 pg/mL) and rapamycin at 50 nM (CRT+R50 nM group), 100 nM (CRT+R100 nM group), and 200 nM (CRT+R200 nM group), respectively, for 24 h and 30 min; and 12)–14) CRT and different concentrations of 3-MA groups: to investigate the effect of CRT on the autophagy suppressed by 3-MA at different concentrations, human MECs were treated with CRT (25 pg/mL) and 3-MA at 2.5 mM (CRT+M2.5 mM group), 5 mM (CRT+M5 mM group), and 10 mM (CRT+M10 mM group), respectively, for 24 h and 30 min.

To evaluate the effect of CRT on cell viability in H/R-treated human MECs, cells were divided into 11 groups: 1) control group: cells were cultured in a 5% CO2 incubator at 37°C for 24 h and 30 min; 2) H/R group: human MECs were cultured in an incubator (Thermo Fisher Scientific, Waltham, Mass) filled with a gas mixture of 90% N2, 5% O2, and 5% CO2 for 8 h of hypoxia and then placed back in the normoxic CO2 incubator at 37°C for 16 h of reoxygenation; 3)–6) different concentrations of rapamycin+H/R groups: to investigate the effect of rapamycin on human MECs injury induced by H/R, human MECs were pretreated with rapamycin at 50 nM (R50 nM+H/R group), 100 nM (R100 nM+H/R group), and 200 nM (R200 nM+H/R group), respectively, for 30 min, and then treated the same way as group 2; 7) to 9) different concentrations of 3-MA groups: human MECs were pretreated with 3-MA at 2.5 mM (M2.5 mM+H/R group), 5 mM (M5 mM+H/R group), and 10 mM (M10 mM+H/R group), respectively, for 30 min, and then treated the same way group 2; 10) CRT+H/R group: cells were preincubated with CRT (25 pg/mL) for 30 min and then treated the same way as group 2; and 11) CRT+rapamycin+H/R group (CRT+R+H/R group): human MECs were pretreated with CRT (25 pg/mL) and rapamycin (100 nM) for 30 min and then treated the same way as group 2.

To evaluate the effect of CRT on autophagosome formation in human MECs, cells were divided into four groups: control group: cells were cultured in a 5% CO2 incubator at 37°C for 24 h and 30 min; CRT group: cells were incubated with CRT (25 pg/mL) for 24 h and 30 min; rapamycin group: cells were incubated with rapamycin (100 nM) for 24 h and 30 min; and CRT+rapamycin group: cells were incubated with CRT (25 pg/mL) and rapamycin (100 nM) for 24 h and 30 min.

To evaluate the effect of CRT on autophagy in H/R-treated human MECs, cells were divided into six groups: 1) control group: cells were cultured in a 5% CO2 incubator at 37°C for 24 h and 30 min; 2) H/R group: human MECs were cultured in an incubator filled with a gas mixture of 90% N2, 5% O2, and 5% CO2 for 8-h hypoxia and then placed back in the normoxic CO2 incubator at 37°C for 16-h reoxygenation; 3) CRT+H/R group: human MECs were preincubated with CRT (25 pg/mL) for 30 min and then treated the same way as group 2; 4) rapamycin+H/R group (R+H/R group): human MECs were preincubated with rapamycin (100 mM) for 30 min and then treated as group 2; 5) CRT+rapamycin+H/R group (CRT+R+H/R group): human MECs were preincubated with CRT (25 pg/mL) and rapamycin (100 mM) for 30 min and then treated the same way as group 2; 6) 3-MA+H/R group: cells were incubated with 3-MA (2.5 mM) for 30 min and then treated as group 2. After the treatment, human MECs were harvested for electron microscopy and western blot analysis.

Detection of cell viability

Cells were seeded in 96-well plates (5 × 103 cells/100 μL in each well). After the treatment, cells were incubated with CCK-8 in a 5% CO2 incubator at 37°C for 3 h. Samples were measured using a microplate reader at 450 nm wavelength (Tecan Infinite f 200 Pro; Tecan Group Ltd., Männedorf, Switzerland). The absorbance value is proportional to the cell viability.

Immunofluorescence staining

Cells grown on coverslips (1 × 104 cells/cm2) were washed 3 times with phosphate-buffered saline (PBS) very gently, fixed with cold methanol (precooled at −20°C) for 5 min, and then fixed with 4% paraformaldehyde for 15 min at room temperature. Subsequently, cells were blocked in 10% donkey serum in PBS containing 1% BSA and 0.2% Triton X-100 for 50 min. We identified autophagosome by indirect immunofluorescence staining with anti-LC3 rabbit polyclonal antibody (1:100) overnight at 4°C and then with Texas red–conjugated donkey anti-rabbit secondary antibody (1:100) for 1 h at room temperature. The coverslips were mounted on glass slides with mounting medium and DAPI. Images were acquired using a confocal scanning microscope (Zeiss LSM-510 Meta, Jena, Germany). A 63 × oil immersion objective with a numerical aperture of 1.4 was used. Images were analyzed using Image J.

Transmission electron microscopy

After the treatment, culture medium was removed. Cells were washed with PBS (0.1 M, 4°C) and harvested with a cell scraper, and then centrifuged at 3,000 rpm for 30 s. The supernatant was discarded. Cells were fixed with 2.5% glutaraldehyde at 4°C for 2 h. Thereafter, the samples were subjected to acetone gradient dehydration, Epon812 embedding, semithin section optical positioning, and ultrathin sectioning. The sections were double-stained with uranyl acetate and lead citrate. Ultrastructure was examined using an H-7650 transmission electron microscope (10,000×; Hitachi7650 TEM, Tokyo, Japan).

Western blot analysis

The protein concentration of the cellular lysate was detected using the Bradford assay. Fractions were resolved on 8% (for p-mTOR), 10% (for Beclin 1 and GAPDH) or 15% (for LC3 and GAPDH) SDS-PAGE using 80 μg protein per lane. Following electrophoresis, proteins were electrophoretically transferred to nitrocellulose membranes and then blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) at room temperature for 1 h. Then membranes were probed with primary antibodies against p-mTOR, Beclin 1, LC3 and GAPDH (all 1:500 diluted), respectively, at 4°C overnight. The antibody-tagged membranes were incubated with a secondary antibody solution consisting of a 1:1,000 dilution of HRP-conjugated goat anti-rabbit IgG. An enhanced chemiluminescence detection system was used for immunoblot protein detection. Optical densities of the bands were analyzed using Image J software, and the densitometry results were normalized to those of GAPDH.

Statistical analysis

The SPSS v13.0 program (Chicago, Ill) was used for statistical analysis. Values are presented as mean ± SD. For multiple-group comparisons, one-way analysis of variance followed by Newman–Keuls post hoc analysis was performed. Pearson's bivariate correlation analysis was applied to determine the correlation between variables. P < 0.05 was considered to be statistically significant.

RESULTS

Effect of CRT on autophagy of human MECs

Effect of rapamycin and 3-MA on human MECs viability

Cell viability detected by the CCK-8 assay is shown in Figure 1. Upon cell exposure to different concentrations of rapamycin (50 nM, 100 nM, and 200 nM), the viability of human MECs decreased in a dose-dependent manner. Rapamycin, an inducer of autophagy, binds to the intercellular receptor FKBP12, and then predominantly inhibits mTOR complexes 1 (mTORC1) signaling, which inhibits autophagosome formation. Rapamycin at 50 nM, 100 nM, and 200 nM decreased human MECs viability by 21.7%, 22.4%, and 25.5%, respectively (P < 0.05 vs. control). However, 3-MA at different concentrations had different effects on cell viability. 3-MA inhibits autophagy by blocking autophagosome formation via the inhibition of type III Phosphatidylinositol 3-kinases. Compared with the control group, there was no significant difference between the M2.5 mM group and M5 mM group (P > 0.05). However, the cell viability in the M10 mM group decreased by 12% (P < 0.05 vs. control). These results indicated that the enhancement or excessive inhibition of autophagy resulted in a decrease in cell viability.

Fig. 1
Fig. 1:
Effect of autophagy on the viability of human MECs.

Effect of CRT on the viability of human MECs exposed to rapamycin or 3-MA

Human MECs were incubated with CRT and different concentrations of rapamycin or 3-MA. Cell viability detected by the CCK-8 assay is shown in Figure 2. There was no significant difference between the control and CRT group (P > 0.05). CRT restored cell viability in rapamycin-treated samples at low and medium dose (50 nM and 100 nM), as shown by a 16.8% increase in the CRT+R50 nM group (P < 0.05 vs. R50 nM) and a 16.9% increase in CRT+R100 nM group (P < 0.05 vs. R100 nM), respectively. However, CRT did not restore cell viability in rapamycin-treated samples at a high dose of 200 nM, as evidenced by no significant difference between R200 nM and CRT+R200 nM groups (P > 0.05). These findings indicate that CRT protected human MECs from damage induced by rapamycin at low and medium dose.

Fig. 2
Fig. 2:
Effect of CRT on the viability of human MECs exposed to different concentrations of rapamycin or 3-MA.

As described above, the cell viability was not influenced by 3-MA at low or medium doses, but decreased with high-dose 3-MA. Interestingly, the cell viability was further decreased upon coincubation with CRT and 3-MA. Compared with 3-MA treatment alone (M2.5 mM, M5 mM, and M10 mM groups), the cell viability in the CRT+M2.5 mM, CRT+M5 mM, and CRT+M10 mM groups decreased by 10.2%, 16.5%, and 26.8% (P < 0.05), respectively. The results showed that as autophagy was inhibited, the protective effect of CRT on human MECs was abolished, indicating that moderate autophagy was necessary for CRT to attenuate cell injury.

Effect of CRT on autophagosome formation in human MECs

The above results showed that CRT treatment improved the viability of cell exposed to low- and medium-dose rapamycin (an autophagic inducer), and aggravated the decrease in cell viability induced by the autophagic inhibitor 3-MA, indicating that autophagy was involved in the protection of CRT in human MECs. To further investigate this, we detected the formation of autophagosome in human MECs by immunofluorescence staining of LC3 (Fig. 3), which is distributed evenly throughout the cytoplasm in normal cells and transforms from the cytoplasmic form (LC3-I) to a membrane-associated form (LC3-II) serving as the protein marker associated with autophagosome when autophagy is induced (11). Therefore red fluorescence intensity is proportionate to the number of autophagosome in cells (12). Fluorescence microscope images showed diffused and weak LC3 punctate dots in the control group. No significant difference was observed between the control group and CRT group (P > 0.05), indicating that CRT had no influence on autophagy in normal cells. Although in human MECs exposed to rapamycin (100 nM), abundant red LC3 punctate dots accompanied by aggregated puncta in the cytoplasm were observed. The mean fluorescence intensity in the rapamycin group increased by 73.7% (P < 0.05 vs. control), indicating that rapamycin markedly induced autophagy. Conversely, CRT significantly suppressed autophagosome formation induced by rapamycin, as evidenced by a 22.7% decrease in CRT+rapamycin group in the mean fluorescence intensity of the CRT+rapamycin group (P < 0.05 vs. the rapamycin group). The results suggested that CRT suppressed rapamycin-induced autophagy.

Fig. 3
Fig. 3:
Effect of CRT on rapamycin-induced autophagosome formation in human MECs.

Effect of CRT on H/R-induced autophagy in human MECs

To investigate whether CRT attenuates the H/R-induced injury by inhibiting autophagy, we used an H/R model of human MECs, mimicking ischemia/reperfusion injury in vivo, to study the effect of H/R and autophagy regulation on human MECs injury and the role of CRT on H/R-induced cell injury.

Effect of autophagy on the viability of human MECs exposed to H/R

The viability of human MECs exposed to H/R measured by the CCK-8 assay revealed that H/R induced cells injury (Fig. 4). Compared with the control group, the cell viability in the H/R group decreased by 17% (P < 0.05). To investigate the effect of autophagy on human MECs viability after H/R, human MECs were preincubated with rapamycin (50 nM, 100 nM, and 200 nM) or 3-MA (2.5 mM, 5 mM, and 10 mM) for 30 min before H/R. We found that rapamycin pretreatment aggravated H/R-induced injury. Compared with the H/R group, the cell viability in the R50 nM+HR, R100 nM+HR and R200 nM+HR groups decreased by 11.4%, 14.9%, and 17.9%, (P < 0.05), respectively, indicating that rapamycin induced autophagy to aggravate H/R-induced injury. Moreover, 3-MA at a low dose (2.5 mM) alleviated HR-induced injury, as shown by an 8.2% increase in cell viability compared with the H/R group (P < 0.05). 3-MA at a moderate dose (5 mM) had no effect on the viability of H/R-treated human MECs (P > 0.05 vs. H/R). However, 3-MA at 10 mM aggravated H/R-induced human MECs injury, as evidenced by a 26.4% decrease in cell viability compared with the H/R group (P < 0.05), indicating that moderate inhibition of autophagy attenuated H/R-induced injury, whereas excessive inhibition of autophagy by 3-MA aggravated it.

Fig. 4
Fig. 4:
Effect of autophagy on the cell viability of human MECs exposed to H/R.

Effect of CRT on the viability of human MECs exposed to H/R

On the basis of the above results, rapamycin at 100 nM enhanced autophagy and its effect on autophagy was abolished by CRT. 3-MA at 2.5 mM inhibited autophagy and protected human MECs against H/R-induced injury. We chose rapamycin at 100 nM and 3-MA at 2.5 mM to investigate the role of autophagy in CRT-mediated protection against H/R injury (Fig. 5). Compared with the H/R group, the cell viability in the CRT+H/R and 3-MA+H/R groups was 12.3% and 16.7% higher (P < 0.05), respectively, indicating that exogenous CRT had the same protective effect on H/R-treated human MECs injury as 3-MA through moderate inhibition of autophagy. CRT pretreatment suppressed rapamycin-mediated cell viability decline, resulting in a 12% increase in cell viability in the CRT+rapamycin+H/R group (P < 0.05 vs. rapamycin+H/R group). However, compared with the CRT+H/R group, the cell viability in the CRT+rapamycin+H/R group decreased by 19.5% (P < 0.05), indicating that rapamycin-induced autophagy reduced the protection of CRT on H/R-treated human MECs. In summary, CRT partially inhibited H/R-induced cell death in human MECs.

Fig. 5
Fig. 5:
Effect of CRT on the cell viability of human MECs exposed to rapamycin or 3-MA under H/R conditions.

Effect of CRT on autophagy in human MECs exposed to H/R

The effect of CRT on the ultrastructure of human MECs exposed to H/R is shown in Figure 6. Transmission electron microscopy (TEM) analyses revealed that human MECs in the control group displayed typical endothelial features, visible as oval cells with a large central nucleus and intact mitochondria and ER. Human MECs in the H/R group showed obvious ultrastructural lesions, including cell swelling, appearance of numerous vesicles, ER dilation, mitochondrial swelling, and vacuolization, together with an increased number of autophagosomes. Compared with the H/R group, pretreatment with CRT significantly alleviated ultrastructural lesions and reduced autophagosome formation, as did 3-MA. Rapamycin aggravated ultrastructural lesions, as shown by nuclear envelope destruction and karyorrhexis as well as the lesions observed in the H/R group, indicating that excessive autophagy aggravated H/R-induced injury. Compared with the rapamycin+H/R group, ultrastructural lesions in the human MECs of the CRT+rapamycin+H/R group were repaired, evident as nuclear envelop integrity and decreased autophagosome number, suggesting that CRT reversed the unfavorable effect of rapamycin. The results suggested that CRT alleviated H/R-induced human MECs injury by inhibiting autophagy.

Fig. 6
Fig. 6:
Effect of CRT on autophagosome formation and ultrastructure in HR-treated human MECs assessed by transmission electron microscopy (10,000×).

LC3 immunofluorescence staining is shown in Figure 7. Human MECs exposed to H/R exhibited abundant red LC3 puncta in the cytoplasm and fluorescence intensity in the H/R group increased by 80.7% (P < 0.05) compared with the control group. To determine the pathophysiological role of autophagy in HR-mediated cell injury, rapamycin (100 nM) and 3-MA (2.5 mM) were separately applied to regulate the autophagy of human MECs exposed to H/R. The results showed that 3-MA pretreatment inhibited the HR-induced increase in punctate dots, showing a 20.7% decrease in fluorescence intensity in the 3-MA+H/R group (P < 0.05 vs. H/R group). However, rapamycin pretreatment markedly increased the number of LC3 punctate dots, showing a 46.7% increase in fluorescence intensity in the rapamycin+H/R group (P < 0.05 vs. H/R group). Notably, CRT pretreatment suppressed H/R-induced or rapamycin+H/R-induced autophagy, resulting in a 22.3% decrease in fluorescence intensity in the CRT+H/R group (P < 0.05 vs. H/R group) and a 37.6% decrease in fluorescence intensity in the CRT+R+H/R group (P < 0.05 vs. R+H/R group). However, compared with the CRT+HR group, the fluorescence intensity in the CRT+R+H/R group increased by 17.9% (P < 0.05), indicating that rapamycin weakened the protective effect of CRT on H/R-induced human MECs injury. In conclusion, CRT inhibited autophagy in human MECs exposed to H/R.

Fig. 7
Fig. 7:
Effect of CRT on autophagosome formation in HR-treated human MECs assessed by immunofluorescence stainning.

Effect of CRT on autophagy signaling

Effect of CRT on mTOR phosphorylation in human MECs exposed to H/R

The original term “mTOR” was named as such to distinguish the “mammalian” target of rapamycin from its yeast counterpart, a major suppressor of autophagy (13). The active (phosphorylated) form of mTOR is a negative regulator of autophagy (14). The effect of CRT on phospho-mTOR (p-mTOR) was detected by western blotting. As shown in Figure 8, the decrease in p-mTOR in the H/R group was significant (48.3% decrease, P < 0.05) compared with the control group, indicating that H/R enhanced autophagy by downregulating p-mTOR. Compared with the H/R group, rapamycin decreased the level of p-mTOR by 15.9% (P < 0.05), whereas 3-MA increased the level of p-mTOR by 24.5% (P < 0.05). These results revealed that rapamycin and 3-MA modulated autophagy in H/R-treated human MECs by regulating mTOR phosphorylation. Compared with H/R alone, CRT pretreatment before H/R increased the p-mTOR level by 17.2% (P < 0.05). In addition, CRT abolished rapamycin-induced downregulation of p-mTOR, as evidenced by a 25.1% increase in the CRT+rapamycin+H/R group compared with the rapamycin+H/R group (P < 0.05), suggesting that CRT inhibited H/R-induced and rapamycin+H/R-induced autophagy via the mTOR pathway.

Fig. 8
Fig. 8:
Effect of CRT on p-mTOR in H/R-treated human MECs assessed by western blot assay.

Effect of CRT on the expression of Beclin 1 in human MECs exposed to H/R

Beclin 1, the mammalian ortholog of yeast Atg6, has a pivotal role in autophagy induction. Increased expression of Beclin 1 induces autophagy (15). The effect of CRT on Beclin 1 expression was detected by western blot analysis (Fig. 9). Under H/R conditions, Beclin 1 protein level was increased by 35.8% compared with the control group (P < 0.05), indicating that H/R induced autophagy. Rapamycin pretreatment followed by H/R treatment resulted in a 19.4% increase in Beclin 1 expression as compared with H/R alone (P < 0.05), whereas 3-MA pretreatment decreased Beclin 1 expression by 18.8% (P < 0.05 vs. H/R), suggesting that rapamycin aggravated H/R-induced autophagy, whereas 3-MA inhibited H/R-induced autophagy. Remarkably, CRT pretreatment followed by H/R treatment decreased Beclin 1 expression by 23.1% compared with the H/R group (P < 0.05). Moreover, CRT pretreatment also inhibited rapamycin+H/R-induced excessive autophagy, as shown by a 5.6% decrease in the CRT+rapamycin+H/R group compared with the rapamycin+H/R group (P < 0.05). These data provided further evidence that CRT alleviated H/R-induced autophagy in human MECs by downregulating Beclin 1.

Fig. 9
Fig. 9:
Effect of CRT on Beclin 1 expression in H/R-treated human MECs.

Effect of CRT on the conversion of LC3-I to LC3-II

LC3, a recognized marker of autophagy, is converted from the unconjugated form LC3-I to the conjugated form LC3-II, when autophagy is activated. Therefore, the LC3-II/LC3-I ratio serves as an indicator of autophagy (11). The ratio of LC3-II/LC3-I was measured by western blot analysis (Fig. 10). H/R promoted the conversion of LC3-I to LC3-II, showing that the LC3-II/LC3-I ratio in the H/R group increased by 35.8% compared with the control group (P < 0.05). Rapamycin aggravated autophagy in H/R-treated human MECs, showing that the LC3-II/LC3-I ratio in the rapamycin+H/R group increased by 12.9% (P < 0.05 vs. H/R group), whereas 3-MA alleviated autophagy in H/R-treated human MECs, resulting in a 13.6% decrease in the LC3-II/LC3-I ratio compared with the H/R group (P < 0.05). Notably, CRT pretreatment attenuated H/R-induced autophagy in human MECs, as evidenced by a 25.8% decrease in the LC3-II/LC3-I ratio in the CRT+H/R group (P < 0.05 vs. H/R group), suggesting that CRT might inhibit autophagy induced by H/R. CRT also reversed the effect of rapamycin on autophagy in H/R-treated human MECs, showing that the LC3-II/LC3-I ratio in the CRT+rapamycin+H/R group decreased by 29% compared with the rapamycin+H/R group (P < 0.05). Taken together, CRT inhibited H/R-induced autophagy in human MECs.

Fig. 10
Fig. 10:
Effect of CRT on the LC3-II/LC3-I ratio in H/R-treated human MECs.

DISCUSSION

Cardiovascular disease is a leading cause of death worldwide, with coronary artery disease accounting for the majority of deaths (1). The preferred treatment is the timely restoration of blood flow of the infarct-related artery to limit the myocardial infarct size. However, reperfusion injury reduces the benefit of blood flow restoration (16). Thus far, little attention has been paid to MECs, which are affected by reperfusion earlier than cardiomyocytes, this might be attributable to their structural and histological characteristics (3). Therefore, it is important to explore pharmacological agents that protect MECs against ischemia/reperfusion injury. Autophagy is an evolutionarily conserved basal cellular process mediating cytoprotection (17), whereas excessively accelerated or inhibited autophagy leads to cellular death (18). Thus, it would be a novel strategy to attenuate H/R-induced MECs injury by regulating autophagy. The present study demonstrated that CRT pretreatment significantly suppressed H/R-induced human MECs injury as evidenced by restored cell viability, alleviation of cell swelling, ER dilatation, and mitochondrial vacuolization. CRT preincubation significantly restored cell viability in rapamycin-treated samples at moderate and low doses, but aggravated the decrease in cell viability induced by the autophagy inhibitor 3-MA at a high dose. Our results also showed that CRT upregulated mTOR phosphorylation, downregulated Beclin 1 expression, and inhibited the conversion of LC3-I to LC3-II. Taken together, our data indicated that CRT protected cells against H/R injury by suppressing excessive autophagy by the mTOR/Beclin 1 pathway.

Autophagy is activated as a response to ischemia/reperfusion injury. Sybers et al. (19), found that cardiomyocyte autophagy was accelerated by the transient deprivation of oxygen and glucose, followed by oxygen and glucose resupply, in a model of fetal mouse heart organ culture. In the isolated cardiomyocytes subjected to H/R, autophagosomes also increased (20). However, whether autophagy plays a beneficial or unfavorable role in ischemia/reperfusion injury remains unclear. Several studies have suggested that autophagy has a favorable effect on ischemia/reperfusion injury (21, 22). Conversely, Aki et al. (23) found that in glucose-free cardiomyocytes, autophagy was enhanced, accompanied by increased cell death, whereas 3-MA inhibited autophagy and cell death. This inconsistency in findings might be ascribed to different experimental designs and limitations of the research methods. Our study showed that H/R resulted in decreased cell viability and increased autophagy. Pretreatment with rapamycin, an autophagy inducer, further enhanced H/R-induced autophagy and aggravated cell injury, whereas 3-MA at a low dose suppressed autophagy and subsequently alleviated H/R-induced MECs injury, suggesting that moderate inhibition of autophagy attenuated H/R-induced MECs injury. These findings were consistent with the results of Liu (24) and Xiao (25).

Growing evidence has demonstrated that exogenous CRT is a promising therapy in wound healing, cancer, and cardiocerebrovascular diseases (5), and enhanced CRT at the surface of tumor cells is related with inhibited autophagy (8, 9). Whether exogenous CRT protects MECs from H/R injury by inhibiting excessive autophagy remains to be determined. Our previous studies have demonstrated that exogenous CRT pretreatment protects human MECs against microwave radiation-induced injury (6, 7). In the present study, a model of H/R-induced human MECs injury was used to mimic ischemia/reperfusion injury in vivo. We found that CRT did not impact cell viability or autophagosome formation in normal endothelial cells, whereas CRT preincubation, before H/R, restored cell viability and inhibited autophagosome formation, indicating that CRT attenuated MECs injury and autophagy under H/R conditions. We further investigated the effect of CRT on autophagy induced by rapamycin and found that CRT antagonized rapamycin-aggravated cell viability and accelerated autophagosome formation under H/R conditions, suggesting that CRT attenuated H/R-induced endothelial cell injury by inhibiting excessive autophagy.

Clinically, up to 40% of patients with acute myocardial infarction maintain inadequate myocardial microvasculature reperfusion despite successful reopening of the infarct-related artery through primary percutaneous coronary intervention. This phenomenon, termed as “no-reflow,” is closely related with ischemia–reperfusion injury. No-reflow is caused by impaired functionality of the coronary microcirculation involved in endothelial dysfunction, intravascular inflammation, and endothelial swelling (2). Various drugs, such as adenosine, verapamil, statin, and thromboxane-A2 receptor antagonists, are applied to prevent no-reflow by antiplatelet therapy, vasodilation, and attenuation of endothelial injury. We found that CRT alleviated H/R-induced MEC injury; therefore, CRT could protect myocardial microvasculature against ischemia–reperfusion injury. Our previous research suggested that H/R induced endogenous CRT expression, and that both hypoxic preconditioning (26) and hypoxic postconditioning (27) upregulate CRT expression and protect the myocardium from H/R injury. Exogenous CRT has been reported to increase cell-surface CRT (28) and accelerate MEC proliferation (29). We have already demonstrated that exogenous CRT enhances the cell-surface CRT expression of MECs (6) and inhibits microwave-induced MEC injury through the integrin-focal adhesion kinase pathway. Through in vivo experiments, we found that exogenous CRT alleviated auricular and muscular microcirculation injury induced by microwave irradiation in mice (30). Nanney et al. (29) reported that topically applied CRT enhances cutaneous wound healing in animal models. These studies suggested that CRT has promising potential for therapeutic applications in humans.

Autophagy is mediated by a series of autophagy-related genes (Atg). Myocardial ischemia/reperfusion injury has been found to be alleviated by activating the mTOR pathway to inhibit autophagy (24, 25). Matsui et al. (22) reported that both autophagy and myocardial injury are significantly attenuated in beclin 1(+/−) mice exposed to ischemia/reperfusion. LC3 is a marker protein localized at the surface of an autophagosome, and the conversion of LC3-I (free form) to LC3-II (phosphatidylethanolamine-conjugated form) is a critical step in autophagosome formation; therefore, the ratio of LC3-II (phosphatidylethanolamine-conjugated form) to LC3-I (free form) is often used to evaluate autophagic activity. We found that H/R treatment downregulated mTOR phosphorylation, upregulated Beclin 1 expression, and increased the LC3-II/LC3-I ratio in MECs, consistent with previous studies (24, 25, 31). In an earlier study, we demonstrated that exogenous CRT inhibited ERS, a condition caused by excess accumulation of unfolded/misfolded proteins in the ER lumen and intended to restore homeostasis in the ER by attenuating protein translation, upregulating ER-resident chaperone proteins, and initiating apoptosis if the stress is prolonged, in endothelial cells. Yorimitsu et al. (32) showed that ERS triggers autophagy. Moreover, ERS induces the release of Ca2+ and activates the Ca2+/calmodulin-dependent kinase kinase-beta/AMPK (33) and PKC pathways (34), thereby inhibiting mTOR and enhancing autophagy. Thus, exogenous CRT likely inhibits autophagy in H/R-treated endothelial cells by suppressing ERS. Given the complex function of CRT, the underlying mechanisms of its inhibitory effect on autophagy in H/R-treated endothelial cells remain to be clarified. Our present study found that CRT led to increased mTOR phosphorylation, downregulated Beclin 1 expression, and decreased the LC3-II/LC3-I ratio. Furthermore, in H/R-treated human MECs, CRT abolished the rapamycin-induced decrease in mTOR phosphorylation, upregulated Beclin 1 expression, and increased the LC3-II/LC3-I ratio, indicating that CRT protected MECs from H/R injury by inhibiting excessive autophagy via the mTOR pathway

In conclusion, we demonstrated, for the first time, that CRT pretreatment protected MECs from H/R injury by inhibiting excessive autophagy by mTOR-mediated Beclin 1 downregulation and inhibiting the conversion of LC3-I to LC3-II.

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Keywords:

Calreticulin; hypoxia/reoxygenation; mammalian target of rapamycin; microvascular endothelial cell

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