Integrinβ3 Mediates the Protective Effect of Soluble Receptor for Advanced Glycation End-products During Myocardial Ischemia/Reperfusion

doi:10.21203/rs.3.rs-798612/v1

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Integrinβ3 Mediates the Protective Effect of Soluble Receptor for Advanced Glycation End-products During Myocardial Ischemia/Reperfusion

https://doi.org/10.21203/rs.3.rs-798612/v1

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Soluble receptor for advanced glycation end-product (sRAGE) was reported to protect myocardial ischemia/reperfusion (I/R) injuries via interacting with AGEs (the ligands of RAGE). Besides, sRAGE was also reported to interact with myocardial cell membranes to protect myocardial I/R injuries. However, the specific molecular of the interaction between sRAGE and myocardial cell membrane is not clearly defined. Integrins were the major adhesion receptors expressed on myocardial cells. The present study showed sRAGE affected the expression of integrinβ3 around integrinβ1 to β5 in I/R treated cardiomyocytes. Consequently, it was supposed that integrinβ3 might be related to the protective effects of sRAGE on myocardial ischemia-reperfusion injuries. The results in this study showed that the protective effects of sRAGE on cardiac function, cardiac infracting size and apoptosis in mice were cancelled by cilengitide (the inhibitor of integrinβ3). Myocardial apoptosis and autophagy decreased by sRAGE in I/R treated cardiomyocytes were showed to be increased by the inhibition of integrinβ3. Mechanistically, the inhibition of integrinβ3 decreased the phosphorylation of Akt and STAT3 but did not affect the phosphorylation of FAK, PTEN, and MAPKs (P38 MAPK, JNK, Erk) in OGD/R and sRAGE treated cardiomyocytes. In addition, the phosphorylation of STAT3 was significantly downregulated by the inhibition of Akt (LY294002,10μM) in OGD/R and sRAGE treated cardiomyocytes. The present study demonstrated integrinβ3 mediated the protective effects of sRAGE on myocardial I/R injuries through Akt-induced STAT3 signaling pathway thus may provide a novel molecular therapy strategy for ameliorating I/R injury.

Cardiac & Cardiovascular Systems

integrinβ3

myocardial ischemia/reperfusion

soluble receptor for advanced glycation end-products

STAT3

Cardiovascular diseases are the leading cause of death worldwide. Myocardial ischemia/reperfusion (I/R) injuries and aggravation of tissue injury after blood perfusion of the ischemic myocardium are considered risk factors for cardiac function impairment.[1]^,[2] I/R injury is characterized by the outbreak of reactive oxygen species (ROS),[3] mitochondrial dysfunctions, apoptosis, autophagy, and increased infarct area[3, 4] Previous studies have shown that soluble receptors for advanced glycation end-products (sRAGE) have a protective role in I/R injury by inhibiting myocardial apoptosis and autophagy.[5] sRAGE is produced by proteolytic cleavage or alternative RNA splicing in the extracellular area of the cell surface receptor.[6] It acts as a natural antagonist to RAGE signaling for isolating RAGE ligands, such as AGEs to inhibit RAGE-dependent cellular responses.[7] However, whether sRAGE depended on RAGE or other membrane receptors on cells to protect I/R myocardium remains unclear.

Integrins, which have been reported as a major adhesion receptor on the myocardium,[8] can promote mitochondrial function through STAT3 and lead to cell survival.[9] Previous studies have shown that RAGE can interact with integrins to mediate leukocyte adhesion.[10] Also, sRAGE might interact with integrins to induce the protective effects on cardiomyocytes during I/R injuries.

Integrinβ3 promotes cardiomyocyte proliferation and reduces hypoxia-induced apoptosis.[11] Yet, the role of integrinβ3 in sRAGE protecting cardiomyocytes from I/R injuries has not been reported so far. In this study, we investigated the relationship between integrinβ3 and sRAGE in the I/R mice model.

Animals

C57bL/6 adult male mice, 8 ~ 10 weeks old, were purchased from the Experimental Animal Laboratory, Capital Medical University. All the animals were housed in an environment with a temperature of 22 ± 1 ºC, relative humidity of 65 ± 5%, and a light/dark cycle of 12/12 hr, having free access to food and water. All animal studies (including the mice euthanasia procedure) were done in compliance with the regulations and guidelines of Capital Medical University institutional animal care and conducted according to the AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) and the IACUC (Institutional Animal Care and Use Committee) guidelines. The present study was approved by the Ethics Committee of Capital Medical University.

Treatment and grouping

The recombinant adenovirus vector was purchased from Shandong Veizhen Biotechnology Co. LTD(China). Two weeks before the acute myocardial infarction/reperfusion operation, an adeno-associated virus with GFP or sRAGE (10¹¹v.g/kg) (Veizhen Co., Ltd. Shandong. China) was injected into mice through the tail vein. The mice were then randomly divided into five groups: (1) Sham group: animals received GFP adeno-associated virus injection without undergoing left anterior descending (LAD) ligation; (2) sRAGE group: sRAGE adeno-associated virus was injected intravenously (10¹²v.g./kg) without LAD ligation; (3) I/R group: GFP adeno-associated virus was injected intravenously and the LAD ligation (n = 8) was performed; (4) I/R + sRAGE group: sRAGE adeno-associated virus was intravenously injected, and a LAD ligation was performed; (5) I/R + sRAGE + CLG group: sRAGE adeno-associated virus was intravenously injected with cilengitide (75mg·kg^− 1)[22] (ApexBio technology LLC. Houston. USA) via intraperitoneal injection 2h before LAD ligation surgery.

I/R operation

The mice were subjected to I/R operation as previously described.[23] Briefly, the hearts of isoflurane-anesthetized mice were treated with 6 − 0 silk thread 30min after coronary artery occlusion and 24h reperfusion, after which the hearts were exposed through the fourth intercostal space.[5]

All animals were anesthetized by pentobarbital sodium (50mg·kg^− 1) and then euthanized by cervical dislocation. The hearts were collected and stored in the − 80°C refrigerator for further experiments.

Echocardiography

Twenty-four hours after reperfusion, mice were anesthetized with an intraperitoneal injection of tribromoethanol (100 mg/kg, T48402; Sigma-Aldrich, USA). An echocardiogram was then performed using the Vevo 2100 imaging system (Visual Sonics Inc., Toronto, Ontario, Canada) to measure Left ventricular ejection fraction (LVEF), left fractional ventricular shortening (LVFS), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV) as previously described.[24] Vevo LAB 3.3.1.was used to calculate the data.

2,3,5-triphenyl tetrazolium chloride (TTC) staining

Hearts were immediately removed after refilling with 1%TTC (Sigma-Aldrich St. Louis. MO. USA) solution. [25] Hearts were incubated in 1%TTC solution at 37°C for 3–5min and fixed in 4% (wv^− 1) paraformaldehyde for 5min. They were then cut vertically and photographed with a digital camera. Image J software (NIH) was used to quantify the area of infarction and normal sections. The data were expressed as the percentage of the infarction area and total area.

Immunofluorescence staining

Myocardial tissue was fixed with 10% buffered formalin and embedded in paraffin, after which it was cut into 4µm thick slices. The sections were incubated in 0.1% (vv^− 1) Triton X-100 for 8min and blocked with normal horse serum for 30min at room temperature. The sections were then incubated with cleaved-caspase3 antibody (1:600, 9664, Cell Signaling, Inc. Danvers, MA, USA) and α-actinin (1:600, A7811, Sigma-Aldrich, St Louis, MO, USA) at 4°C overnight,[26] and then with corresponding fluorescent secondary antibody for 1.5h at room temperature in dark. The sections were then stained with DAPI and observed by Olympus BX51 fluorescence microscope (Olympus America Inc., Center Valley, PA, USA). ImageJ software (NIH) was used to calculate the positive area.

TUNEL assay

TUNEL staining was performed in accordance with the instructions from TMR Red (Roche, Mannheim, Germany).[27] Tissues were paraffin-embedded and cut into 4µm thick slices. The myocardial cells were washed twice with PBS and fixed for 15min at room temperature. The slices or cells were incubated in 0.1% (VV-1) Triton X-100 for 8min and sealed with 3% (WV-1) bovine serum for 1h at room temperature. Each section was incubated with TUNEL conjugated dUTP at 37°C for 60min. After staining with α-actin and 4 ', 6-diamino-2-phenylindoles (DAPI), sections were stored in a refrigerator at -20°C. Images were obtained using an Olympus BX51 fluorescence microscope (Olympus America Inc., Center Valley, PA, USA) at 20× magnification. Image J (NIH) was used to calculate the number of TUNEL-positive cells and the total cells in each field.

Isolation and culture of the myocardium

Neonatal rat ventricular cardiomyocytes were isolated from the hearts of 1-to 2-day-old Sprague-Dawley rats with 0.25% trypsin as previously described.[28] Rats were purchased from Viton Lever Laboratory Animal Technology Co., LTD. (Beijing, China). Cell pellets were resuspended in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and cultured in an incubator of 5%CO₂ and 21%O₂ at 37°C for 1.5h to remove fibroblasts. Then, 1×10⁶ cells/well were cultured in DMEM/F12 medium containing 0.3g/l glutamine, 4.5g/l glucose, 10% FBS, and 1% penicillin/ streptomycin at an atmosphere of 5% CO₂ and 21%O₂ at 37°C for 48h. After the cardiomyocytes adhered to the wall, the subsequent operation for further experiments was carried out.

Adenovirus transfection and OGD/R model simulation in cardiomyocytes

The recombinant adenovirus vector was purchased from Hanheng Biotechnology Co Ltd (Shanghai, China). Myocardial cells were transfected by recombinant adenovirus expressing GFP or a sequence of the extracellular domain of human RAGE for 24h before stimulation with I/R injury. Cells were then treated with an “ischemia buffer” (pH 6.3), which included 118 NaCl, 24 NaHCO₃, 1.0 NaH₂PO₄, 2.5 CaCl₂·2H2O, 1.2 MgCl₂, 20 sodium lactate, 16 KCl, and 10 deoxyglucose (mM) for 2h in 1%O₂ and 5%CO₂ at 37°C (ischemia).[29] Subsequently, DMEM/F12 was added to the cells in an atmosphere of 5%CO₂ and 21%O₂ at 37°C (reperfusion). In the control groups, cells were incubated with DMEM/F12 in an atmosphere of 5%CO₂ and 21%O₂ at 37°C. In the I/R and sRAGE group, cilengitide (1µM)[30] was added to the medium 30 min before and during the ischemia and reperfusion.

Autophagic flux assessment

As mentioned before,[9] an adenovirus expressing mRFP-GFP-LC3 (Hanheng Biotechnology Co Ltd., Shanghai, China) was transfected into cultured cardiomyocytes for 24 hours to assess autophagic flux. Recombinant sRAGE protein (900µM)[31](Aidi Bo biological Ltd. Beijing. China) was then applied to the medium 30minutes before I/R stimulation. After reperfusion, cardiomyocytes were fixed with 4% (wv^− 1) paraformaldehyde for 15min, and the nucleus was stained by DAPI. Twenty images were collected using confocal fluorescence microscopy (TCS SP8 MP, Leica, Buffalo Groove, Illinois, USA) to observe LC3 spots in cells.

Real-time PCR

Real-time polymerase chain reaction (real-time PCR) was performed as previously described.[32] Approximately 1–2µg of total mRNA was reverse-transcribed to complementary DNA (cDNA) using Go Script TM reverse transcription system (S1000 Thermal Cycler, California, USA). Expression of integrins was analyzed via q-PCR using SYBR-Green PCR Master Mix (RR420A; TaKaRa Bio) with a 7500 Real-Time PCR System. The mouse primers were as follows: Integrinβ1: forward primer: 5′-GACCTGCCTTGGTGTCTGTGC-3′, reverse primer: 5′-AGCAACCACACCAGCTACAAT-3′; Integrinβ2: forward primer: 5′-TTCGGGTCCTTCGTGGACA-3′, reverse primer: 5′-ACTGGTTGGAGTTGTTGGTCA-3′; Integrinβ3: forward primer: 5′-ACCGGGGAAAGCTACATGAA-3′, reverse primer: 5′-TAGGTACAGTTCACCGCGTT-3′; Integrinβ4: forward primer: 5′-GAGGGTGTCATCACCATTGAA-3′, reverse primer: 5′-CCAGCGACTACACTATTGGAT-3′; Integrinβ5: forward primer: 5′-CCGGTTGGGATTTGGGTATT-3′, reverse primer: 5′-CTCAGGGGCATCTCGGTTC-3′; GAPDH: forward primer: 5′-CAAGTTCAACGGCACAGTCA-3′, reverse primer: 5′-CCCCATTTGAT GTTAGCGGG-3′.

Western blot

Cardiac proteins of the same amounts (40µg) were separated by SDS-PAGE (6–15%) and transferred to PVDF membranes. After being blocked by 5% skim milk at room temperature for 1 hour, the membranes were incubated in primary antibodies anti-phospho-STAT3 (1:1000; 9131; Cell Signaling Technology, MA, USA), anti-STAT3 (1:1000; 4904; Cell Signaling Technology), anti-phospho-FAK (1:1000; 3283; Cell Signaling Technology), anti-FAK (1:1000; 3285; Cell Signaling Technology), anti-integrinβ3 (1:1000; ab119992; Abcam), anti-P62 (1:1000; 23214; Cell Signaling Technology), anti-LC3A/B (1:1000; ARG55799; Arigo), anti-Beclin-1 (1:1000; 3738; Cell Signaling Technology), anti-cleaved-caspase3 (1:1000; 9664; Cell Signaling Technology), anti-phospho-Akt (1:1000; 9271; Cell Signaling Technology), anti-Akt (1:1000; 4691; Cell Signaling Technology), anti-phospho-p38 MAPK (1:1000; 4511; Cell Signaling Technology), anti-p38 MAPK (1:1000; 8690; Cell Signaling Technology), anti-phosphoErk1/2 (1:2000; 4370; Cell Signaling Technology), anti-Erk1/2 (1:1000; 4695; Cell Signaling Technology), anti- phospho-JNK(1:1000;9251; Cell Signaling Technology), anti-JNK (1:1000; 4668; Cell Signaling Technology) and anti-GAPDH (1:1000; 5174; Cell Signaling Technology) at 4°C overnight. The PVDF membranes were then balanced for 20min at room temperature and incubated with a horseradish peroxidase-conjugated secondary antibody anti-rabbit IgG (1:2000; 7074; Cell Signaling Technology) for 1 hour. Specific bands were exposed to ultrafiltration HRP substrate (WBKLS0500, Millipore, Billerica, MA, USA). Fluor Chem FC3 (Protein Simple, Wallingford, CT) was used to capture the images. The density was determined by Image J software (NIH). Loading differences were controlled by probing the blot with an antibody against GAPDH.

Statistical analysis

Data were expressed as the mean ± SEM. SPSS V25.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. A nonparametric test was used when data deviated from a normal distribution. Significance between three groups or more was determined by the parametric test of one-way ANOVA. The least significant difference test (LSD) was used to evaluate the significant difference between the two groups. A p < 0.05 was considered statistically significant.

sRAGE upregulated the expression of integrinβ3 after I/R treatment

To decide which subunit of integrins was influenced by I/R and sRAGE, integrin β1, β2, β3, β4, and β5 in cardiomyocytes were detected by real-time PCR. The results showed that I/R decreased relative integrinβ3 mRNA levels, which were reversed by sRAGE in cardiomyocytes (p < 0.05) (Fig. 1c); these data were consistent with Western blot results, which revealed that I/R decreased integrinβ3 protein expression, which was reversed by sRAGE treatment (p < 0.05) (Fig. 1f and 1g). However, I/R or sRAGE had no effects on the expression of other integrins (Fig. 1a, b, d, and e). These results suggested that integrinβ3 might be involved in the antagonistic effects of sRAGE on I/R injuries in cardiomyocytes.

Integrinβ3 inhibition reversed the antagonistic effects of sRAGE on cardiac I/R injury

To identify the effects of integrinβ3 on sRAGE and I/R treated hearts, cilengitide, an integrinβ3 inhibitor, was applied. The results showed that after integrinβ3 was inhibited, a cardiac function decreased: LVEF decreased from 40.8%±3.84% (I/R and sRAGE treated mice) to 30.48%±1.35% (sRAGE, I/R and cilengitide treated mice, n = 6–8, p < 0.05. Figure 2a, b); LVFS decreased from 25.8%±0.74% (I/R and sRAGE treated mice) to 15.34%±0.67% (sRAGE, I/R, and cilengitide treated mice) (n = 6–8, p < 0.01. Figure 2c); LVESV decreased from 29.19 ± 1.55% (I/R and sRAGE treated mice) to 23.7 ± 1.34% (sRAGE, I/R and cilengitide treated mice) (n = 6–8, p < 0.05. Figure 2d) and LVEDV decreased from 77.29 ± 0.20% (I/R and sRAGE treated mice) to 72.31 ± 0.06% (sRAGE, I/R, and cilengitide treated mice)(n = 6–8, p = 0.42. Figure 2e). In addition, results from TTC staining showed that the myocardial infarction size was enlarged from 19.98–29.92% after inhibition of integrinβ3 by cilengitide (n = 6–8, p < 0.05. Figure 2f, g). These results suggested that integrinβ3 mediated the antagonistic effects of sRAGE on cardiac function and myocardial infarction size of hearts during I/R injuries.

The inhibition of integrinβ3 abolished the antagonistic effects of sRAGE on myocardial apoptosis after I/R injury

The results from the TUNEL assay, which was used to determine myocardial apoptosis in the marginal zone of myocardial infarction, showed that cilengitide increased the ratio of TUNEL-positive cells from 0.0462 ± 1.23% (I/R and sRAGE treated mice) to 0.3140 ± 2.88% (sRAGE, I/R, and cilengitide treated mice) (n = 6–8, p < 0.05, Fig. 3a, c), which was remarkably decreased by sRAGE in I/R-treated hearts. In addition, cleaved-caspase3 immunofluorescent staining was used to detect the distribution of apoptotic cardiomyocytes. The results showed that after integrinβ3 was inhibited by cilengitide, the relative cleaved-caspase3 positive area was increased from 11.13%±0.83% (I/R and sRAGE treated mice) to 37.33%±0.88% (sRAGE, I/R, and cilengitide treated mice) (n = 6–8, p < 0.05, Fig. 3b, d). These results indicated that integrinβ3 mediates the antagonistic effects of sRAGE on myocardial apoptosis during I/R injuries.

The inhibition of integrinβ3 diminished the antagonistic effects of sRAGE on cardiomyocytes apoptosis after I/R injuries

Cardiomyocytes were subjected to hypoxia, and a TUNEL assay was used to determine the hypoxia-induced cardiomyocytes apoptosis. It was observed that after integrinβ3 was inhibited by cilengitide, the percentage of TUNEL-positive cells was significantly increased from 9.95%±0.01% (I/R and sRAGE treated cardiomyocytes) to 24.51%±0.02% (sRAGE, I/R, and cilengitide treated cardiomyocytes) (n = 6–10, p < 0.05, Fig. 4a, b). Meanwhile, Western blotting was performed to detect the expression of cleaved-caspase3. After integrinβ3 was inhibited by cilengitide, the expression of cleaved-caspase3 was significantly increased from 0.89 folds (I/R and sRAGE treated cardiomyocytes) to 1.76 folds (sRAGE, I/R, and cilengitide treated cardiomyocytes) (n = 3, p < 0.05, Fig. 4c, d). These results confirmed that integrinβ3 influenced the antagonistic effects of sRAGE on cardiomyocytes' apoptosis during hypoxia/reperfusion stimulation.

The inhibition of integrinβ3 abolished the antagonistic effects of sRAGE on autophagy in myocardial cells after I/R injury

After cardiomyocytes were infected with mRFP-GFP-LC3 adenovirus for at least 24 hours, autophagy flow was detected. Yellow and red dots represented autophagosomes and autolysosomes, respectively.[12] Image J (NIH) was used to analyze the number of spots. The results revealed that after integrinβ3 was inhibited by cilengitide, the number of autophagosomes increased from 16.5 ± 0.76% (I/R and sRAGE treated cardiomyocytes) to 26.33 ± 1.52% (sRAGE, I/R and cilengitide treated cardiomyocytes) (n = 6, p < 0.05, Fig. 5a, b) and the number of autolysosomes were not remarkably changed by the inhibition of integrinβ3 in I/R and sRAGE treated cardiomyocytes (n = 6–10, Fig. 5a, c). Besides, Western blot was performed to detect the expression of autophagy relative proteins. These results showed that after integrinβ3 was abolished by cilengitide, autophagy relative proteins such as LC3-II were activated from 1.19 folds (I/R and sRAGE treated cardiomyocytes) to 1.47 folds (sRAGE, I/R and cilengitide treated cardiomyocytes) (n = 5–6, p < 0.05, Fig. 5d, f) and Beclin-1 was activated from 1.11 folds (I/R and sRAGE treated cardiomyocytes) to 1.37 folds (sRAGE, I/R, and cilengitide treated cardiomyocytes) (n = 3–4, p = 0.2, Fig. 5d, g). Meanwhile, after integrinβ3 was inhibited by cilengitide, the expression of p62 was downregulated from 1.62 folds (I/R and sRAGE treated cardiomyocytes) to 1.14 folds (sRAGE, I/R, and cilengitide treated cardiomyocytes) (n = 4–7, p < 0.05, Fig. 5d, e). Briefly, this data suggested that integrinβ3 mediated the reductive effect of sRAGE on excessive autophagy in I/R-treated cardiomyocytes.

AKT and STAT3 signalings are downstream molecules of integrinβ3 induced by sRAGE

To demonstrate the intracellular signaling of integrinβ3 induced by sRAGE in I/R treated cardiomyocytes, the potential molecules were detected after integrinβ3 was inhibited by cilengitide. Briefly, the expression of integrinβ3 was showed to be decreased from 1.75 folds (I/R and sRAGE treated cardiomyocytes) to 0.48 folds (sRAGE, I/R, and cilengitide treated cardiomyocytes) (n = 6–8, p < 0.05, Fig. 6a, b) in OGD/R treated cardiomyocytes after integrinβ3 was inhibited by cilengitide, suggesting that cilengitide inhibited the expression of integrinβ3 in cardiomyocytes during I/R inducement. In addition, phosphorylated STAT3 was downregulated from 3.7 folds (I/R and sRAGE treated cardiomyocytes) to 0.73 folds (sRAGE, I/R, and cilengitide treated cardiomyocytes) (n = 5–6, p < 0.05, Fig. 6a, d), which had been proved to be increased from 0.47 folds (I/R treated cardiomyocytes) to 3.7 folds (I/R and sRAGE treated cardiomyocytes) (n = 5–6, p < 0.05, Fig. 6a, d) by sRAGE in I/R treated cardiomyocytes. However, no changes in the expression of phosphorylated FAK, phosphorylated PTEN, and MAPKs (JNK, Erk, P38) were observed among the groups in cardiomyocytes (n = 3–6, p > 0.05, Fig. 6a, c, e to i). Additionally, the expression of phosphorylated Akt was decreased from 0,67 folds (I/R and sRAGE treated cardiomyocytes) to 0.42 folds (sRAGE, I/R, and cilengitide treated cardiomyocytes) (n = 4, p < 0.05, Fig. 6e, j). To sum up, these results suggested that FAK, PTEN, and MAPKs were not the downstream pathway of sRAGE-integrinβ3 in cardiomyocytes during I/R injury, while STAT3 and Akt might be involved in the effects of integrinβ3 mediated function of sRAGE during cardiac I/R injuries.

To further explore the interaction between Akt and STAT3, LY294002 (an inhibitor of Akt) was adopted in I/R and sRAGE treated cardiomyocytes. The results showed that the phosphorylated STAT3 was downregulated after Akt was inhibited in I/R and sRAGE treated cardiomyocytes (n = 4, p < 0.05, Fig. 6k to m). These results suggested that Akt induced the activation of STAT3 signaling in I/R and sRAGE treated cardiomyocytes.

The present study demonstrated that integrinβ3 mediated the protective effects of sRAGE on myocardial I/R injuries through Akt-induced STAT3 signaling pathway, which might be used as a potential molecular therapy strategy for ameliorating I/R injury in hearts.

Integrins are widely expressed on cell membranes, including cardiomyocytes.[13] The integrin family contains many subunits, including integrin β1, 2, 3, 4, and 5.[14] This study showed that integrinβ3 was markedly decreased by I/R, which was reversed by sRAGE in cardiomyocytes (Fig. 1). Previous studies have revealed that the expression of integrinβ3 was upregulated by hypoxia inducement in H9C2 cells (cell line derived from embryonic BD1X rat heart tissue).[15] However, the results in this study revealed that oxygen and glucose deprivation/reperfusion reduced integrinβ3 expression, which was reversed by sRAGE. Due to the previous results that sRAGE protected hearts from ischemia/reperfusion injuries, it was supposed that integrinβ3 might be related to the protective effects of sRAGE on I/R cardiomyocytes.

sRAGE was reported to protect cardiac function and the heart infarction size induced by I/R.[16] The same as the previous reports, the results in this study showed that sRAGE protected cardiac function and the infarction size in I/R treated hearts. However, the protective effects of sRAGE were reversed after integrinβ3 was inhibited by cilengitide (Fig. 2), which suggested that integrinβ3 might mediated the antagonistic effects of sRAGE on I/R injuries in hearts. In addition, sRAGE was also reported to inhibit apoptosis induced by I/R in hearts besides the cardiac function and the heart infarction size.[5] Did integrinβ3 mediate the effects of sRAGE on apoptosis in the hearts as well?

Then,myocardial apoptosis in mice was examined by TUNEL assay and cleaved-caspase3 immunofluorescent staining. The results showed that myocardial apoptosis was decreased by sRAGE in I/R treated mice, which was increased after integrinβ3 was inhibited in I/R and sRAGE treated hearts (Fig. 3). These results suggested that integrinβ3 mediated the suppressive effects of sRAGE on apoptosis during I/R injuries in mice hearts. Then the question arising was, did sRAGE interact with integrinβ3 on myocardial cells directly?

Consequently, cardiomyocytes were isolated and treated with sRAGE and cilengitide (the inhibitor of integrinβ3). The results showed that myocardial apoptosis induced by I/R and inhibited by sRAGE were reversed by the inhibition of integrinβ3 in I/R and sRAGE treated cardiomyocytes (Fig. 4), which confirmed that sRAGE interacted with integrinβ3 on myocardial cells directly to inhibit apoptosis in I/R treated cardiomyocytes. In addition, previous studies have reported that the inhibition of excessive autophagy resisted apoptosis induced by I/R in cardiomyocytes,[5] which contributed to the inhibiting effects on myocardial ischemia-reperfusion injuries.[17] Then, did the effects of sRAGE on apoptosis which mediated by integrinβ3 in cardiomyocytes also mediated via autophagy?

Therefore, autophagosomes and autolysosomes were detected by mRFP-GFP-LC3 virus transfection in cardiomyocytes. The results showed that the number of autophagosomes was increased, the expression of LC3-II and Beclin-1 were upregulated and P62 was downregulated after integrinβ3 was inhibited in I/R and sRAGE treated cardiomyocytes (Fig. 5). All the results suggested that integrinβ3 mediated the antagonistic effects of sRAGE on excessive autophagy in myocardial cells. The present study verified the assumption that integrinβ3 mediated the suppressive effects of sRAGE on excessive autophagy in I/R myocardium. Then, what was the intracellular signaling pathway of integrinβ3 to mediate the antagonistic effects of sRAGE on ischemia/reperfusion injuries in cardiomyocytes?

Integrin-FAK signaling was reported to enhance mitochondrial function[9] and decrease cell death[18] via STAT3 signaling. In this study, phosphorylation of STAT3 but not FAK was decreased after integrinβ3 was inhibited by cilengitide in I/R and sRAGE treated cardiomyocytes (Fig. 6a to d). Mechanistically, Akt signaling[19], MAPKs[20], JNK signaling[21] and PTEN[11] were reported to be affected by the activation of integrin in non-small cell lung cancer cells, human osteoarthritis chondrocytes, vascular endothelial cells, and cardiomyocytes, respectively. Nevertheless, the results in this study showed that the phosphorylation of PTEN, p38 MAPK,JNK, and Erk1/2 were not affected by the inhibitor of integrinβ3 in I/R and sRAGE treated cardiomyocytes (Fig. 6e to i). Interestingly, the phosphorylation of Akt was significantly upregulated after sRAGE administration in I/R treated cardiomyocytes, while it was downregulated after integrinβ3 was inhibited (Fig. 5b, f). Meanwhile, the phosphorylation of STAT3 was downregulated by the inhibitor of Akt (LY294002) in I/R and sRAGE treated cardiomyocytes, which suggested that STAT3 was the downstream molecule of Akt pathway. Based on these findings, the present study concluded that integrinβ3 mediated the protective effects of sRAGE during I/R in cardiomyocytes via Akt and STAT3 signaling pathways.

In summary, the present study demonstrated that integrinβ3 mediated the suppressive effects of sRAGE on I/R-induced apoptosis and autophagy via the Akt and STAT3 signaling pathway in myocardial cells, which contributed to the antagonistic effects of sRAGE on cardiac function and the infarct size in hearts during I/R injuries.

Ethics approval and Consent to participate

All animal studies (including the mice euthanasia procedure) were done in compliance with the regulations and guidelines of Capital Medical University institutional animal care and conducted according to the AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) and the IACUC (Institutional Animal Care and Use Committee) guidelines. The study was reported in accordance with ARRIVE guidelines. This study did not involve human participants.

Consent for publication

This work did not involve the identification images or other personal or clinical details of participants. The authors of this paper consent for publication.

Availability of data and materials

The data that support the findings of this study are not openly available and are available from the first author or the corresponding author upon reasonable request.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 81370313,81570321,81870265) and the China Young and Middle-aged Clinical Research Foundation (Grant No. 2017CCA-VG045).

Conflict of interest and authors' contributions

All authors contributed to the study conception and design. Material preparation was performed by X.G. and J.Z., data collection and analysis were performed by X.H., J.C. and L.C.. The first draft of the manuscript was written by X.H. and X.G.. X.Z., H.W. and F.D. commented on previous versions of the manuscript. C.G. provided the funding acquisition and supervised the study. All authors read and approved the final manuscript. The authors declare no potential conflict of interest.

Binder, A. et al. Myocardial protection from ischemia-reperfusion injury post coronary revascularization. Expert Review of Cardiovascular Therapy, 13 (9), 1045–1057 (2015).
Chen, M., Li, X., Yang, H., Tang, J. & Zhou, S. Hype or hope: Vagus nerve stimulation against acute myocardial ischemia-reperfusion injury. Trends Cardiovasc Med, 30 (8), 481–488 (2020).
Cadenas, S. ROS and redox signaling in myocardial ischemia-reperfusion injury and cardioprotection. Free Radic Biol Med, 117, 76–89 (2018).
Chang, J. C. et al. Intermittent Hypoxia Prevents Myocardial Mitochondrial Ca(2+) Overload and Cell Death during Ischemia/Reperfusion: The Role of Reactive Oxygen Species.Cells2019, 8(6).
Dang, M. et al. Soluble receptor for advance glycation end-products inhibits ischemia/reperfusion-induced myocardial autophagy via the STAT3 pathway. Free Radic Biol Med, 130, 107–119 (2019).
Kumar, V., Sulaj, A., Fleming, T. & Nawroth, P. P. Purification and Characterization of the Soluble form of the Receptor for Advanced Glycation End-Products (sRAGE): A Novel Fast, Economical and Convenient Method. Exp Clin Endocrinol Diabetes, 126 (3), 141–147 (2018).
Egana-Gorrono, L. et al. Receptor for Advanced Glycation End Products (RAGE) and Mechanisms and Therapeutic Opportunities in Diabetes and Cardiovascular Disease: Insights From Human Subjects and Animal Models. Front Cardiovasc Med, 7, 37 (2020).
Chen, C., Manso, A. M. & Ross, R. S. Talin and Kindlin as Integrin-Activating Proteins: Focus on the Heart. Pediatr Cardiol, 40 (7), 1401–1409 (2019).
Visavadiya, N. P. et al. Integrin-FAK signaling rapidly and potently promotes mitochondrial function through STAT3. Cell Commun Signal, 14 (1), 32 (2016).
Frommhold, D. K. A. et al. RAGE and ICAM-1 cooperate in mediating leukocyte recruitment during acute inflammation in vivo.. Blood 2010 116(5):841–849.
Wei, L. et al. Integrin beta3 promotes cardiomyocyte proliferation and attenuates hypoxia-induced apoptosis via regulating the PTEN/Akt/mTOR and ERK1/2 pathways. Int J Biol Sci, 16 (4), 644–654 (2020).
Wang, Y. et al. Metformin promotes autophagy in ischemia/reperfusion myocardium via cytoplasmic AMPKα1 and nuclear AMPKα2 pathways. Life Sci, 225, 64–71 (2019).
Israeli-Rosenberg, S., Manso, A. M., Okada, H. & Ross, R. S. Integrins and integrin-associated proteins in the cardiac myocyte. Circ Res, 114 (3), 572–586 (2014).
Civitarese, R. A., Kapus, A., McCulloch, C. A. & Connelly, K. A. Role of integrins in mediating cardiac fibroblast-cardiomyocyte cross talk: a dynamic relationship in cardiac biology and pathophysiology. Basic Res Cardiol, 112 (1), 6 (2017).
Su, Y., Tian, H., Wei, L., Fu, G. & Sun, T. Integrin beta3 inhibits hypoxia-induced apoptosis in cardiomyocytes. Acta Biochim Biophys Sin (Shanghai), 50 (7), 658–665 (2018).
Jiang, X. et al. A soluble receptor for advanced glycation end-products inhibits myocardial apoptosis induced by ischemia/reperfusion via the JAK2/STAT3 pathway., 20 (8), 1033–1047 (2015).
Wu, S. et al. Trimetazidine protects against myocardial ischemia/reperfusion injury by inhibiting excessive autophagy. J Mol Med (Berl), 96 (8), 791–806 (2018).
Visavadiya, N. P. et al. Correction to: Integrin-FAK signaling rapidly and potently promotes mitochondrial function through STAT3. Cell Commun Signal, 18 (1), 64 (2020).
Yang, Y. et al. Integrin alpha5 promotes migration and invasion through the FAK/STAT3/AKT signaling pathway in icotinib-resistant non-small cell lung cancer cells. Oncol Lett, 22 (1), 556 (2021).
Lohberger, B. et al. Mechanical exposure and diacerein treatment modulates integrin-FAK-MAPKs mechanotransduction in human osteoarthritis chondrocytes. Cell Signal, 56, 23–30 (2019).
Siu, M. K. Y. et al. PDK1 promotes ovarian cancer metastasis by modulating tumor-mesothelial adhesion, invasion, and angiogenesis via alpha5beta1 integrin and JNK/IL-8 signaling. Oncogenesis, 9 (2), 24 (2020).
Wilisch-Neumann, A. et al. The integrin inhibitor cilengitide affects meningioma cell motility and invasion. Clin Cancer Res, 19 (19), 5402–5412 (2013).
Gao, E. et al. A novel and efficient model of coronary artery ligation and myocardial infarction in the mouse. Circ Res, 107 (12), 1445–1453 (2010).
Kong, P. et al. Opposing Actions of Fibroblast and Cardiomyocyte Smad3 Signaling in the Infarcted Myocardium., 137 (7), 707–724 (2018).
Wang, F. et al. Artemisinin suppresses myocardial ischemia-reperfusion injury via NLRP3 inflammasome mechanism. Mol Cell Biochem, 474 (1–2), 171–180 (2020).
Tu, L. et al. Astragaloside IV protects heart from ischemia and reperfusion injury via energy regulation mechanisms. Microcirculation (New York, NY: 1994) 2013, 20(8):736–747.
Su, Q. et al. Inhibition of lncRNA TUG1 upregulates miR-142-3p to ameliorate myocardial injury during ischemia and reperfusion via targeting HMGB1- and Rac1-induced autophagy. J Mol Cell Cardiol, 133, 12–25 (2019).
Song, J. Q., Teng, X., Cai, Y., Tang, C. S. & Qi, Y. F. Activation of Akt/GSK-3beta signaling pathway is involved in intermedin(1–53) protection against myocardial apoptosis induced by ischemia/reperfusion., 14 (11), 1299–1307 (2009).
Dang, M. et al. Interferon-γ mediates the protective effects of soluble receptor for advanced glycation end-product in myocardial ischemia/reperfusion. Lab Invest, 99 (3), 358–370 (2019).
Kanazawa, H., Imoto, K., Okada, M. & Yamawaki, H. Canstatin inhibits hypoxia-induced apoptosis through activation of integrin/focal adhesion kinase/Akt signaling pathway in H9c2 cardiomyoblasts. PLoS One, 12 (2), e0173051 (2017).
Cao, X. et al. Soluble receptor for advanced glycation end-products promotes angiogenesis through activation of STAT3 in myocardial ischemia/reperfusion injury., 25 (5–6), 341–353 (2020).
Dang, M. Q. et al. Gene expression profile in the early stage of angiotensin II-induced cardiac remodeling: a time series microarray study in a mouse model. Cell Physiol Biochem, 35 (2), 467–476 (2015).

No competing interests reported.

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Integrinβ3 Mediates the Protective Effect of Soluble Receptor for Advanced Glycation End-products During Myocardial Ischemia/Reperfusion

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Abstract

Figures

Introduction

Materials And Methods

Animals

Treatment and grouping

I/R operation

Echocardiography

2,3,5-triphenyl tetrazolium chloride (TTC) staining

Immunofluorescence staining

TUNEL assay

Isolation and culture of the myocardium

Adenovirus transfection and OGD/R model simulation in cardiomyocytes

Autophagic flux assessment

Real-time PCR

Western blot

Statistical analysis

Results

sRAGE upregulated the expression of integrinβ3 after I/R treatment

Integrinβ3 inhibition reversed the antagonistic effects of sRAGE on cardiac I/R injury

AKT and STAT3 signalings are downstream molecules of integrinβ3 induced by sRAGE

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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