Cleaved caspase-3 antibodies (cat. no. 9664) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Monoclonal actin (α-sarcomeric; α-sa) antibodies (cat. no. A2172), Dobutamine, Evans Blue dye and 2,3,5-triphenyl-tetrazolium (TTC) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The in situ cell death detection kit (cat. no. 11684795910) was purchased from Roche Diagnostics (Indianapolis, IN, USA). β-actin antibodies (cat. no. 66009-1-Ig), PEDF-R rabbit polyclonal antibodies (cat. no. 55190-1-AP) and RAC family small GTPase 1 (rac1) antibodies (cat. no. 24072-1-AP) were purchased from ProteinTech Group, Inc. (Chicago, IL, USA). The MitoSOX™ Red mitochondrial superoxide indicator was purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Hoechst 33342 and the Annexin V-allophycocyanin (APC)/propidium iodide (PI) Apoptosis Detection kit were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Malondialdehyde (MDA; cat. no. S0131), Dihydroethidium (DHE; cat. no. S0063), Dabco 4 and 6-diamidino-2-phenylindole (DAPI; cat. no. C1005) were purchased from Beyotime Institute of Biotechnology (Haimen, China). The TIANamp Genomic DNA kit was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). The NADPH oxidase activity assay kit was purchased from Shanghai Genmed Pharmaceutical Technology Co., Ltd. (Shanghai, China). The xanthine oxidase activity assay kit was purchased from Cayman Chemical Company (Ann Arbor, MI, USA).

Recombinant lentivirus (LV; Shanghai GeneChem Co., Ltd, Shanghai, China) was prepared as described previously (16). PEDF overexpression plasmid (Shanghai GeneChem Co., Ltd.) was successfully constructed and then packaged in 293T cells (American Type Culture Collection, Manassas, VA, USA). PEDF was cloned and ligated into the pGC287 plasmid using AgeI and BamHI sites. The sequences of the PEDF primers used were as follows: Forward, 5′-CGACCGGTGCCACCATGCAGACCCTGG-3′ and reverse, 5′-GGAATTCGGATCCTTAAGTGCTGCTGG-3′. The concentrated titer of virus suspension was 2×10¹² TU/l. Transient transfection of H9c2 cells with 20 μM small interfering (si)RNA targeting the PEDF-R genes (5′-GCGGCATTTCAGACAACTTGC-3′) was performed using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. A free-combination sequence of siPEDF-R was used as negative control (5′-ACGGTTATGCTCGAACTCGCA-3′), all the siRNA were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Recombinant rat PEDF (GenBank accession no. NM_177927) was synthesized by Cusabio Biotech, Co., Ltd. (Wuhan, China). In brief, 293T cells were transfected with the 20 μg recombinant vector pGEX 6P-1 (GE Healthcare, Chicago, IL, USA), containing glutathione S-transferase (GST)-tagged PEDF using Lipofectamine^® 3000 according to the manufacturer's protocols. GST-tagged PEDF proteins were purified by high-pressure liquid chromatography purification (>90% purity) and amino-terminal sequence determination (23,24). The resultant proteins were soluble in aqueous solutions.

Adult male Sprague-Dawley rats (8-10 weeks old, weighing 200-250 g, n=85) were purchased from the Experimental Animal Centre of Xuzhou Medical University (Xuzhou, China) and housed in a controlled environment (humidity, 50-60%). A total of 3 rats were housed per cage and were maintained at room temperature under a 12 h light/dark cycle; rats were provided free access to food and water. The experiments described in this manuscript conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Publication, 8th Edition, 2011, Bethesda, MD, USA) (25). All animal care and experimental protocols were approved by the Animal Care and Use Committee of Xuzhou Medical University (license no. SYXK 2002-0038, Jiangsu, China) and also followed the international guidelines (European Council Directive 2010/63/EU) on the ethical use of animals (26).

The rat MI/R model was produced as described previously (27). Intramyocardial gene delivery was performed one week prior to the MI/R experiment in the rats. PEDF-LV (2×10⁷ TU) in 20 μl enhanced infection solution (pH 7.4, Shanghai GeneChem Co., Ltd.) was delivered with a 20-μl syringe and 25-gauge needle into the myocardium along the left-anterior descending coronary artery (LAD). Sham-operated animals underwent an identical surgical procedure without artery ligation. For the rat MI/R model, after 30 min of ischemia treatment, reperfusion was allowed for 24 h by releasing the ligatures. The animal models were randomly divided into five groups as follows: i) Sham group, surgical procedure without artery ligation (n=18); ii) Sham+P group, surgical procedure without artery ligation, PEDF-LV was transferred prior to surgery (n=18); iii) MI group, 0.5 h ischemia (n=6); iv) MI/R group, 0.5 h ischemia and 24 h reperfusion (n=18); v) MI/R+P group, 0.5 h ischemia and 24 h reperfusion, PEDF-LV was transferred prior to surgery (n=18).

Myocardial infarct size was measured by Evans Blue/TTC staining as previously described (28). Briefly, at the end of reperfusion or ischemia, the LAD was again occluded and 1 ml 3% Evans Blue dye was retrogradely injected into the ascending aorta to demarcate the ischemic area at risk (AAR). The heart was rapidly excised and frozen at −20°C prior to being cut into 1 mm thick sections perpendicular to the axis of the LAD. At this point, sections were immediately incubated in 1% TTC in phosphate buffer (pH 7.4) at 37°C for 15 min to discriminate infarcted tissue from viable myocardium. All sections were scanned from both sides using a color CCD camera (FV-10; Fujifilm Holdings Corporation, Tokyo, Japan), and in each slide, infarct area was compared with the AAR using digital planimetry software (Image-Pro Plus 6.0; Media Cybernetics, Inc., Rockville, MD, USA). Following correction with the weight of the sections, the myocardial infarct size was measured and expressed as a percentage of infarct size over total AAR.

Echocardiography was conducted under anesthesia with sodium pentobarbital (30 mg/kg, intraperitoneal; Sigma-Aldrich; Merck KGaA), as described previously (22). Two-dimensional-guided M-mode echocardiography was performed at rest and during dobutamine stress 24 h after reperfusion in order to determine left ventricular (LV) chamber volume at systole and diastole and contractile parameters, including left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV). Left ventricular ejection fraction (EF) and fractional shortening (LVFS) were calculated as follows: EF(%) = (EDV-ESV)/EDV×100. FS(%) = (LVEDD-LVESD)/LVEDD ×100. All measurements represent the mean of at least 3 consecutive cardiac cycles. Dobutamine (1 μg/g body weight; Sigma-Aldrich; Merck KGaA) was given intraperitoneally. Cardiac reserve was investigated 10 min after dobutamine injection.

The embryonic rat heart-derived H9c2 cell line was obtained from the American Type Culture Collection, and the cells were cultured in DMEM medium supplemented with 10% foetal bovine serum (both from Gibco; Thermo Fisher Scientific, Inc.) and 100 mg/ml penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. The medium was replaced every 2-3 days, and cells were subcultured or subjected to experimental procedures at 80-90% confluence.

To establish the H/R model, the culture medium was changed to serum-free low glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) and placed into a tri-gas incubator (Heal Force Bio-meditech Holdings, Ltd., Shanghai, China) that was purged with 94% N₂, 5% CO₂, and 1% O₂ for 4, 8 and 12 h respectively. Following hypoxia, reoxygenation was initiated by incubating in serum-free high glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂ for a further 0 or 2 h. H9C2 cells (~80-90% confluence) were treated with or without 10 nM PEDF 1 h prior to H/R. The following experimental groups were included: N group (control, normoxia), N+P group (PEDF, normoxia), H/R group (control, 8 h hypoxia and 2 h reoxygenation), H/R+P group (PEDF, H/R), H/R+PEDF+siPEDF-R group (PEDF+siPEDF-R, H/R), H/R+PEDF+vector group (PEDF+vector, H/R).

Myocardial samples from the rat left ventricle were embedded in optimum cutting temperature compound tissue medium (Sakura Finetek Europe B. V., Flemingweg, The Netherlands), snap-frozen on dry ice and stored at −80°C. Cardiomyocyte apoptosis in vivo was determined by double-labeling TUNEL immunofluorescence staining, which was performed with an In Situ Cell Death Detection kit according to the manufacturer's protocol. Cardiomyocytes were identified using monoclonal actin (α-sa) antibodies. Specimens were blocked with 5% bovine serum albumin (Vicmed Life Sciences, Xuzhou, China) for 30 min at room temperature prior to incubation with primary antibody. Specimens were incubated with anti-α-sa (1:300) overnight at 4°C. Specimens were then washed three times in PBS and incubated with the Goat anti-Mouse immunoglobulin G (IgG; H+L) Cross-Adsorbed secondary antibody (Alexa Fluor^®488/green; 1:200; A11001; Thermo Fisher Scientific, Inc.) under light-protected conditions for 1 h at room temperature. DAPI staining conducted at room temperature for 15 min was used to count the total number of nuclei. Cardiomyocyte nuclei with a relatively large diameter are located within myofibers (16). In addition, 2×10⁴ H9c2 cells with Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) were seeded into each of a 48-well plate. Following the H/R treatments, H9c2 cells were treated with the In Situ Cell Death Detection kit according to the manufacturer's protocol. H9c2 cells were counterstained with DAPI for 30 min at room temperature. The cardiomyocytes and H9c2 cells were observed under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). The percentage of apoptotic cells was calculated as the ratio of the number of TUNEL-positive cells to the total number of cells, which were counted in three different random fields of view.

For western blot analysis, the cells were solubilized in lysis buffer (100 mmol/l Tris-HCl, 4% SDS, 20% glycerine, 200 mmol/l dithiothreitol and protease inhibitors; pH 6.8). Total cellular protein was denatured by boiling for 10 min with an equal volume of 2X Tris-glycine SDS buffer. Protein concentration from the supernatant was determined using a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). A total of 50 ng protein per lane was separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). Following blocking with 5% non-fat milk/PBS-T for 3 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C. Cleaved caspase-3, PEDF-R rabbit polyclonal and rac1 antibodies were used at a dilution of 1:1,000; β-actin antibody was used at a dilution of 1:5,000. Then, fluorescence-labeled secondary antibodies was added for 1 h at 37°C: Anti-rabbit IgG H+L DyLight™ 800 4X PEG (1:30,000; cat. no. 5151) and anti-mouse IgG (H+L DyLight™ 680, 1:15,000; cat. no. 5470) (both from Cell Signaling Technology, Inc.) and membranes were scanned by the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). ImageJ software (v1.50; NIH, Bethesda, MD, USA) was used for quantification.

The Annexin V-APC/PI Apoptosis Detection kit was used for the following assay. H9c2 cardiomyocytes (1×10⁶ per group) were collected, washed twice with PBS, resuspended with 500 μl 1X binding buffer, treated with 5 μl Annexin V-APC and 5 μl PI, and placed in the dark at room temperature for 5-15 min. Then, the cells were analyzed using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

DHE was used to stain for intracellular ROS, as previously described (29). DHE, an oxidant-sensitive probe, is widely used for the detection of ROS. Harvested cells (2×10⁴ per well) were incubated with 10 μM DHE for 30 min at 37°C, according to the manufacturer's protocol, then washed with DMEM without FBS three times. Fluorescence was observed under a fluorescence microscope. Fluorescence was calculated by viewing in four randomly selected fields for each group; Image-Pro Plus software (v6.0; Media Cybernetics, Inc.) was used for quantification.

To assess the levels of mtROS, MitoSOX™, a fluorochrome specific to anion superoxide produced in the inner mitochondrial compartment (Invitrogen; Thermo Fisher Scientific, Inc.) was used. H9c2 cells (1×10⁴ per well) were seeded into each well of a 48-well plate, underwent their respective treatments as aforementioned, and subsequently loaded with 200 μl MitoSOX™ (5 mM stock in ethanol dissolved in Hank's Balanced Salt Solution to a working solution of 5 μM) for 10 min at 37°C. After washing three times with PBS, nuclei were counter-stained with Hoechst 33342 for 15 min at 37°C. Following three washes with PBS, the sample was observed by a fluorescence microscope (Olympus IX73; Olympus Corporation). Fluorescence was calculated by viewing in five randomly selected fields for each group. Image-Pro Plus software (v6.0; Media Cybernetics, Inc.) was used for quantification.

Total DNA was isolated from cells using the TIANamp Genomic DNA kit, according to the manufacturer's protocol. As mitochondrial DNA (mtDNA) is a primary target of ROS and a reflection of ETC, mtDNA copy number was detected by qPCR using SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR was conducted with a final volume of 20 μl containing 10 μl 2× SYBR-Green PCR Master Mix, 0.1 μM of each primer and 100 μg genomic DNA. The mixture was subjected to qPCR amplification for 95°C for 10 min, 45 cycles (95°C for 10 sec, 60°C for 10 sec, 72°C for 20 sec), 1 cycle (95°C for 1 min, 65°C for 1 min and 97°C with continuous) and then cooled to 40°C for 30 sec using a Roche Light Cycler 480 (Roche Diagnostics). Gene expression was normalized to that of 18s RNA. Gene expression was quantified by using the 2^−∆∆Cq method (30). The following primers, synthesized by GenScript (Piscataway, NJ, USA) were used: D-loop forward, 5′-TGGTTCATCGTCCATACGTT-3′ and reverse, 5′-TGACGGCTATGTTGAGGAAG-3′; 18sRNA forward, 5′-CATTCGAACGTCTGCCCTATC-3′ and reverse, 5′-CCTGCTGCCTTCCTTGGA-3′.

MDA levels, XO activity and NOX activity were measured using the respective detection kits, according to the manufacturers' protocol.

The results are expressed as the mean ± standard error of the mean. All statistical analyses were conducted with SPSS 19.0 for Windows (IBM Corp., Armonk, NY, USA). The results were analyzed using unpaired Student's t-test or repeated-measures one-way analysis of variance followed by Duncan's new multiple range method or Fisher's least significant difference test. P<0.05 was considered to indicate a statistically significant difference.

First, the protective effects of PEDF were verified in a MI/R rat model. As presented in Fig. 1A and B, an irregular small myocardial infarct area was observed in 3 of 6 hearts in the MI group, and infarcted myocar-dium was not observed in the rest of the MI group. However, the MI/R group had a significantly increased myocardial infarct size compared with the MI group. In addition, PEDF treatment resulted in a reduced myocardial infarct size compared with the MI/R group. Next, TUNEL staining was used to analyze the anti-apoptotic effect of PEDF in the I/R myocardium. There was a marked increase in cardiomyocyte apoptosis in MI/R hearts compared with the sham group, and treatment with PEDF reduced this effect (Fig. 1C and D). There was no significant difference between the Sham group and Sham+P group.

Cardiac function and cardiac functional reserve were measured using transthoracic M-mode echocardiography prior to and following dobutamine (1 μg/g) injection. The values of ejection fraction and fractional shortening, which are signs of myocardial function, were significantly increased in PEDF-treated rats compared with untreated MI/R hearts (Fig. 1E and F). The Δ ejection fraction and Δ fractional shortening to dobutamine infusions were increased in hearts transfected with PEDF compared with MI/R rats in vivo, which revealed that PEDF may increase cardiac functional reserve (Fig. 1G and H). Together, these results indicated that PEDF has a protective effect against I/R damage in rat hearts.

To investigate whether PEDF suppresses H9c2 cell apoptosis under H/R conditions, the level of the cleaved caspase-3 following H/R was dected in H9c2 cells at different time points using western blotting. Onset of hypoxia for up to 8 h resulted in an increase in cleaved caspase-3 protein expression, and cleaved caspase-3 expression was also significantly increased in the hypoxia (8 h)/reoxygenation (2 h) group in comparison with the hypoxia (8 h) group (Fig. 2A and B). Treatment with PEDF could significantly reduce the level of the cleaved caspase-3 compared with the H/R group (Fig. 2C and D). To further confirm the observation that PEDF treatment inhibited H9c2 cell apoptosis under H/R condition, flow cytometric detection of early apoptosis was performed. The results demonstrated similar trends (Fig. 2E and F). These observations indicated that PEDF prevented H/R-induced H9c2 cell apoptosis.

Next, to investigate whether PEDF-R is involved in the PEDF-mediated repression of cell injury, H9c2 cells were treated with PEDF under normoxic and H/R conditions, and RNA interference assays were used to silence PEDF-R. PEDF-R siRNA significantly reduced PEDF-R expression levels under normoxic conditions in H9c2 cells (Fig. 3). As presented in Fig. 4A and B, PEDF-R siRNA prevented the PEDF-induced reduction of cleaved caspase-3 protein expression under H/R conditions in H9c2 cells. This was not observed in the PEDF+vector group. TUNEL staining was also used to identify apoptotic H9c2 cells, and the results revealed the critical involvement of PEDF-R in the effect of PEDF in H/R injury (Fig. 4C and D). These results suggested that the anti-apoptotic effect of PEDF is dependent on PEDF-R.

The ROS burst is a key factor of myocardial ischemia/reperfusion injury. Therefore, whether PEDF inhibited the H/R-induced burst of ROS via PEDF-R was investigated. The changes in ROS levels in H9c2 cells are presented in Fig. 5A and B. Compared with the normal group, ROS levels were significantly increased in the H/R groups. Nonetheless, PEDF markedly decreased the level of ROS induced by H/R, while this effect was reversed by PEDF-R siRNA. Intracellular MDA levels were measured to further confirm the antioxidative function of PEDF. In the H/R group, MDA content was increased compared with the normal group. PEDF dampened the MDA increase, but PEDF-R siRNA attenuated this effect (Fig. 5C). These results suggested that PEDF has antioxidant activities in response to H/R injury in H9c2 cells, and these activities are dependent on PEDF-R.

To study the detailed mechanisms underlying the antioxidative effect of PEDF, which protects cardiomyocytes against H/R-induced apoptosis, our group developed a novel hypothesis: That PEDF attenuates H/R-induced oxidative stress via PEDF-R through inhibition of ROS generation. The main source of ROS is the mitochondria, which produce ROS primarily through single electron transport to molecular oxygen in the ETC (11). In addition, mtDNA is the primary target of the ROS, and a reflection of ETC function (31). Thus, mtROS level and the mtDNA copy number were measured to investigate mitochondrial-derived ROS synthesis. Under H/R conditions, mtROS levels were increased while the mtDNA copy number was decreased. This effect was reversed by PEDF treatment, but PEDF-R siRNA inhibited this effect (Fig. 6). The results demonstrated that PEDF attenuates the H/R-induced burst of ROS by reducing mtROS generation via PEDF-R.

NOX and XO are the main sources of ROS formation in the cytoplasm (11), thus, examination of XO and NOX activity permits the evaluation of cytoplasmic ROS production. XO and NOX activity was increased in H/R and H9c2 cells and, while PEDF reduced thid activity, PEDF-R siRNA reversed this effect (Fig. 7A and B). In addition, the expression of rac1, which has an important effect on NOX activation (32), was detected. Rac1 protein expression was altered in a similar manner to NOX activity (Fig. 7C and D). These observations suggested that PEDF attenuates the H/R-induced ROS burst by reducing cytoplasmic ROS generation via PEDF-R.