Conditioned medium from adipose-derived stem cells attenuates ischemia/reperfusion-induced cardiac injury through the microRNA-221/222/PUMA/ETS-1 pathway

Abstract

Rationale: Cardiovascular diseases, such as myocardial infarction (MI), are the leading causes of death worldwide. Reperfusion therapy is the common standard treatment for MI. However, myocardial ischemia/reperfusion (I/R) causes cardiomyocyte injury, including apoptosis and fibrosis. We aimed to investigate the effects of conditioned medium from adipose-derived stem cells (ADSC-CM) on apoptosis and fibrosis in I/R-treated hearts and hypoxia/reoxygenation (H/R)-treated cardiomyocytes and the underlying mechanisms. Methods: ADSC-CM was collected from ADSCs. The effects of intramuscular injection of ADSC-CM on cardiac function, cardiac apoptosis, and fibrosis examined by echocardiography, Evans blue/TTC staining, TUNEL assay, and Masson's trichrome staining in I/R-treated mice. We also examined the effects of ADSC-CM on apoptosis and fibrosis in H/R-treated H9c2 cells by annexin V/PI flow cytometry, TUNEL assay, and immunocytochemistry. Results: ADSC-CM treatment significantly reduced heart damage and fibrosis of I/R-treated mice and H/R-treated cardiomyocytes. In addition, the expression of apoptosis-related proteins, such as p53 upregulated modulator of apoptosis (PUMA), p-p53 and B-cell lymphoma 2 (BCL2), as well as the fibrosis-related proteins ETS-1, fibronectin and collagen 3, were significantly reduced by ADSC-CM treatment. Moreover, we demonstrated that ADSC-CM contains a large amount of miR-221/222, which can target and regulate PUMA or ETS-1 protein levels. Furthermore, the knockdown of PUMA and ETS-1 decreased the induction of apoptosis and fibrosis, respectively. MiR-221/222 overexpression achieved similar results. We also observed that cardiac I/R markedly increased apoptosis and fibrosis in miR-221/222 knockout (KO) mice, while ADSC-CM decreased these effects. The increased phosphorylation of p38 and NF-κB not only mediated myocardial apoptosis through the PUMA/p53/BCL2 pathway but also regulated fibrosis through the ETS-1/fibronectin/collagen 3 pathway. Conclusions: Overall, our results show that ADSC-CM attenuates cardiac apoptosis and fibrosis by reducing PUMA and ETS-1 expression, respectively. The protective effect is mediated via the miR-221/222/p38/NF-κB pathway.

Keywords: ADSC-CM; Ischemia/reperfusion injury; apoptosis; fibrosis; miR-221/222.

Figure 1

ADSC-CM treatment reduced I/R-induced cardiac apoptosis and fibrosis. The left anterior descending coronary artery (LAD) of male C57/B6J mice was ligated for 30 min and then reperfused for 3 h (I/R 3 h) or 3 d (I/R 3 d). In ADSC-CM-treated animals, ADSC-CM (4 μg/mL) was injected into the anterior wall of the left ventricle. Sham-operated animals underwent the same procedure without occlusion of the LAD. (A) Representative echocardiogram from the control, I/R 3 d, and I/R 3 d+CM mice. Quantitative data of the fractional shortening (FS) and the ejection fraction (EF). (B) The infarct area was determined by TTC staining. The ischemic area showed pale and viable myocardium showed red. Infarct area was quantified as a percentage of total slice area. Infarct area and area at risk quantification by Evans blue and TTC double staining. Graphic representation of the infarct size expressed as percentage of infarct area over area at risk. (C)The morphology of cardiac sections was observed by HE staining (scale bar = 50 μm). (D) Measurement of intracellular ROS was performed by DHE staining (scale bar = 50 μm). (E) LDH levels in serum were measured by an LDH cytotoxicity detection kit. Myocardial apoptosis was determined and quantified by TUNEL assay (TUNEL-positive cells: brown; nuclei: blue; scale bar = 50 μm). (F) The presence of collagen deposition was evaluated by Masson's trichrome (MT) staining (scale bar =100 μm). (G) The expression of fibronectin and collagen 3 was examined by immunohistochemistry (scale bar = 50 μm). (H) The expression levels of p-p53, BCL2, fibronectin, and collagen 3 expression were measured by Western blotting. The data are expressed as the mean ± SEM (n = 3-5). *P < 0.05 vs. control, ^†P < 0.05 vs. the I/R 3 h group, ^‡P< 0.05 vs. the I/R 3 d group.

Figure 2

ADSC-CM protected against myocardial I/R injury in mice through miR-221/222. (A) The levels of miR-221/222 were measured by RT-qPCR. (B) ADSC-CM contains a large amount of miR-221/222. (C) Nucleotide resolution of the predicted miR-221/222 binding sequence in PUMA and ETS-1. Luciferase activity in H9c2 cells cotransfected with PUMA-WT or PUMA-MUT, ETS-1-WT or ETS-1-MUT and miR-NC mimic or miR-221/222 mimics. (D). The expression of PUMA and ETS-1 in cardiac tissues was determined by immunohistochemistry (scale bar = 50 μm). (E) Western blot analysis of PUMA and ETS-1 expression. The data are expressed as the mean ± SEM (n = 3-5). ^*P < 0.05 vs. control, ^†P < 0.05 vs. the I/R 3 h group, ^‡P< 0.05 vs. the I/R 3 d group or KO control.

Figure 3

ADSC-CM attenuated hypoxia/reoxygenation (H/R)-induced apoptosis and fibrosis in H9c2 cells. Cardiomyocytes were pretreated with or without ADSC-CM for 4 h and then exposed to hypoxia for 24 h. The cells were then exposed to normoxia for another 12 h. (A) DCFH-DA staining was used to examine cytoplasmic H₂O₂ by fluorescence microscopy (scale bar = 50 μm). (B) Apoptosis and cell viability were evaluated by flow cytometry with annexin V/PI staining and MTT assays. (C) Apoptosis was examined by TUNEL assay (green: TUNEL-positive nuclei; blue: DAPI-positive nuclei; scale bar = 50 μm). (D) The expression levels of PUMA and ETS-1 were examined by immunofluorescent staining and Western blot (scale bar = 50 μm). (E) MiR-221/222 levels were measured by RT-qPCR. (F) The levels of apoptosis markers (p-p53 and BCL2) were examined by Western blot. (G) The levels of fibrosis markers (fibronectin and collagen 3) were examined by Western blot and immunofluorescent staining (scale bar = 50 μm). The data are expressed as the mean ± SEM (n = 3-4). ^*P < 0.05 vs. control, ^†P < 0.05 vs. H/R.

Figure 4

MiR-221/222 was involved in the ADSC-CM-mediated reduction in H/R-induced apoptosis and fibrosis in H9c2 cells. (A) Western blot analysis of PUMA, p-p53, and BCL2 expression in cells transfected with miR-221/222 mimics. (B) PUMA expression was examined by immunofluorescent staining (scale bar = 50 μm). (C) Transfection with miR-221/222 mimics significantly decreased H/R-induced apoptosis, as examined by TUNEL assay and flow cytometry using annexin V/PI (scale bar = 50 μm). (D) Knockdown of PUMA in H9c2 cells exposed to H/R increased BCL2 expression and decreased p-p53, as shown by Western blot. (E) The number of apoptotic H9c2 cells after siPUMA transfection and H/R treatment was examined by TUNEL assay (scale bar = 50 μm). (F) Transfection of miR-221/222 mimics significantly reduced ETS-1, fibronectin and collagen 3 expression in H/R-treated H9c2 cells, as shown by Western blot. (G) ETS-1 expression was examined by immunofluorescent staining (scale bar = 50 μm). (H) Knockdown of ETS-1 in H9c2 cells exposed to H/R reduced fibronectin and collagen 3 expression, as shown by Western blot. (I) The overexpression of PUMA significantly decreased BCL2 expression and increased p-p53 expression, as shown by Western blot. (J) PUMA-overexpressing cells transfected with miR-221/222 mimics decreased apoptosis, as shown by TUNEL assay (scale bar = 50 μm). (K) ETS-1-overexpressing cells transfected with miR-221/222 mimics decreased fibronectin and collagen 3 expression, as shown by Western blot. (L) ADSC-CM-treated cells treated by H/R and transfected with miR-221/222 inhibitors increased PUMA and ETS-1 expression, as shown by Western blot. (M) ADSC-CM-treated cells exposed to H/R and transfected with miR-221/222 inhibitors increased the number of apoptotic cells, as shown by TUNEL assay (scale bar = 50 μm). (N, O) Western blot analysis showed that the miR-221/222 inhibitors increased the protein levels of p-p53, fibronectin and collagen 3. (P) ADSCs were transfected with miR-221/222 inhibitors and then the conditioned media were collected. MiR-221/222 levels were measured by RT-qPCR. (Q, R) MiR-221/222 inhibitors-ADSC-CM was used to examine the effects of miR-221/222 on the expression of PUMA, ETS-1, p-p53, BCL2, fibronectin, and collagen 3 in H/R-treated H9c2 cells. (S) MiR-221/222 inhibitors-ADSC-CM was used to examine the effects of miR-221/222 on cell apoptosis in H/R-treated H9c2 cells, as shown by TUNEL assay (scale bar = 50 μm). (T) MiR-221/222 inhibitors-ADSC-CM was used to examine the effects of miR-221/222 on the expression of fibronectin and collagen 3 by immunofluorescent staining (scale bar = 50 μm). The data are expressed as the mean ± SEM (n = 3-7). ^*P < 0.05 vs. control, ^†P < 0.05 vs. H/R, PUMA-OE, ETS-1-OE, ^‡P< 0.05 vs. H/R+scramble, H/R+miR221/222 mimics, H/R+ADSC-CM.

Figure 4

MiR-221/222 was involved in the ADSC-CM-mediated reduction in H/R-induced apoptosis and fibrosis in H9c2 cells. (A) Western blot analysis of PUMA, p-p53, and BCL2 expression in cells transfected with miR-221/222 mimics. (B) PUMA expression was examined by immunofluorescent staining (scale bar = 50 μm). (C) Transfection with miR-221/222 mimics significantly decreased H/R-induced apoptosis, as examined by TUNEL assay and flow cytometry using annexin V/PI (scale bar = 50 μm). (D) Knockdown of PUMA in H9c2 cells exposed to H/R increased BCL2 expression and decreased p-p53, as shown by Western blot. (E) The number of apoptotic H9c2 cells after siPUMA transfection and H/R treatment was examined by TUNEL assay (scale bar = 50 μm). (F) Transfection of miR-221/222 mimics significantly reduced ETS-1, fibronectin and collagen 3 expression in H/R-treated H9c2 cells, as shown by Western blot. (G) ETS-1 expression was examined by immunofluorescent staining (scale bar = 50 μm). (H) Knockdown of ETS-1 in H9c2 cells exposed to H/R reduced fibronectin and collagen 3 expression, as shown by Western blot. (I) The overexpression of PUMA significantly decreased BCL2 expression and increased p-p53 expression, as shown by Western blot. (J) PUMA-overexpressing cells transfected with miR-221/222 mimics decreased apoptosis, as shown by TUNEL assay (scale bar = 50 μm). (K) ETS-1-overexpressing cells transfected with miR-221/222 mimics decreased fibronectin and collagen 3 expression, as shown by Western blot. (L) ADSC-CM-treated cells treated by H/R and transfected with miR-221/222 inhibitors increased PUMA and ETS-1 expression, as shown by Western blot. (M) ADSC-CM-treated cells exposed to H/R and transfected with miR-221/222 inhibitors increased the number of apoptotic cells, as shown by TUNEL assay (scale bar = 50 μm). (N, O) Western blot analysis showed that the miR-221/222 inhibitors increased the protein levels of p-p53, fibronectin and collagen 3. (P) ADSCs were transfected with miR-221/222 inhibitors and then the conditioned media were collected. MiR-221/222 levels were measured by RT-qPCR. (Q, R) MiR-221/222 inhibitors-ADSC-CM was used to examine the effects of miR-221/222 on the expression of PUMA, ETS-1, p-p53, BCL2, fibronectin, and collagen 3 in H/R-treated H9c2 cells. (S) MiR-221/222 inhibitors-ADSC-CM was used to examine the effects of miR-221/222 on cell apoptosis in H/R-treated H9c2 cells, as shown by TUNEL assay (scale bar = 50 μm). (T) MiR-221/222 inhibitors-ADSC-CM was used to examine the effects of miR-221/222 on the expression of fibronectin and collagen 3 by immunofluorescent staining (scale bar = 50 μm). The data are expressed as the mean ± SEM (n = 3-7). ^*P < 0.05 vs. control, ^†P < 0.05 vs. H/R, PUMA-OE, ETS-1-OE, ^‡P< 0.05 vs. H/R+scramble, H/R+miR221/222 mimics, H/R+ADSC-CM.

Figure 5

ADSC-CM reduced apoptosis and fibrosis in H/R-treated H9c2 cells through the p-p38/NFκB p65 pathway. (A) Cardiomyocytes were pretreated with or without ADSC-CM for 4 h and then exposed to hypoxia for 24 h. The cells were then exposed to normoxia for another 12 h. The phosphorylation levels of p38 and NFκB p65 were examined by Western blot. (B, C) Cells were pretreated with or without SB203580 (10 μM, p38 inhibitor) for 1 h and then induced with H/R. The expression levels of PUMA, p-p53, BCL2, ETS-1, fibronectin, and collagen 3 were examined by Western blot. SB203580 reduced the expression of PUMA, p-p53, ETS-1, fibronectin, and collagen 3, while increasing BCL2 expression in H/R-treated H9c2 cells. (D, E) Cells were pretreated with or without Bay 11-7082 (2.5 μM, NFκB inhibitor) for 1 h and then induced with H/R. Bay 11-7082 reduced the expression of PUMA and p-p53 and increased the expression of BCL2. Bay 11-7082 also significantly reduced H/R-induced ETS-1, fibronectin, and collagen 3 expression in H9c2 cells. (F) The transfection of miR-221/222 mimics significantly ameliorated H/R-reduced p38 and p65 phosphorylation, as shown by Western blot. (G) The p38 inhibitor reduced the phosphorylation of NFκB, while the NF-κB inhibitor did not affect p38 phosphorylation, as shown by Western blot. (H, I) Treatment with the p38 inhibitor or with the NFκB inhibitor decreased apoptosis and fibrosis, as shown by TUNEL assay and immunofluorescent staining, respectively. (scale bar = 50 μm). (J) I/R significantly increased p-p38 and p-p65 compared to those of the control mice, as shown by Western blot, while ADSC-CM reversed these effects in vivo. The data are expressed as the mean ± SEM (n = 3-7). ^*P < 0.05 vs. control, ^†P < 0.05 vs. H/R, I/R, ^‡P< 0.05 vs. H/R+scramble, H/R+miR221/222 mimics.

Figure 6

ADSC-CM ameliorated myocardial I/R injury in miR-221/222 KO mice. The LAD of miR-221/222 KO mice was occluded for 30 min and then reperfused for 3 h or 3 d. In ADSC-CM-treated animals, ADSC-CM (4 μg/mL) was injected into the anterior wall of the left ventricle. (A) ADSC-CM significantly inhibited I/R-increased LDH levels. (B, C) The levels of apoptosis and fibrosis were examined by TUNEL assay and Masson's trichrome (MT) staining, respectively (scale bar = 50 μm; 100 μm). (D) The protein levels of PUMA and ETS-1 were evaluated by Western blot analysis and immunohistochemistry (scale bar = 100 μm). (E) The expression levels of p-p53, BCL2, fibronectin and collagen 3 were examined by Western blot and immunohistochemistry (scale bar = 50 μm). (F) The effects of the in vivo transfection of miR-221/222 mimics on apoptosis and fibrosis were examined by TUNEL assay and Masson's trichrome staining, respectively, in normal mice and miR-221/222 KO mice. (scale bar = 50 μm). (G) The effect of the in vivo transfection of miR-221/222 mimics treatment on the expression of PUMA, p-p53, BCL2, ETS-1, fibronectin and collagen 3 was measured by Western blot. (H) The effects of the in vivo transfection of miR-221/222 mimics treatment on the expression of PUMA and ETS-1 were examined by immunohistochemistry (scale bar = 50 μm). (I) The effects of the in vivo transfection of miR-221/222 inhibitors on apoptosis and fibrosis were examined by TUNEL assay and Masson's trichrome staining, respectively (scale bar = 50 μm). (J, K) The expression of PUMA and ETS-1 in WT mice transfected with miR-221/222 inhibitors was evaluated by Western blot and immunohistochemistry. The data are expressed as the mean ± SEM (n = 3-5). ^*P < 0.05 vs. control, ^†P < 0.05 vs. the I/R 3 h or KO control group, ^‡P< 0.05 vs. the I/R 3 d or KO I/R group.

Figure 6

ADSC-CM ameliorated myocardial I/R injury in miR-221/222 KO mice. The LAD of miR-221/222 KO mice was occluded for 30 min and then reperfused for 3 h or 3 d. In ADSC-CM-treated animals, ADSC-CM (4 μg/mL) was injected into the anterior wall of the left ventricle. (A) ADSC-CM significantly inhibited I/R-increased LDH levels. (B, C) The levels of apoptosis and fibrosis were examined by TUNEL assay and Masson's trichrome (MT) staining, respectively (scale bar = 50 μm; 100 μm). (D) The protein levels of PUMA and ETS-1 were evaluated by Western blot analysis and immunohistochemistry (scale bar = 100 μm). (E) The expression levels of p-p53, BCL2, fibronectin and collagen 3 were examined by Western blot and immunohistochemistry (scale bar = 50 μm). (F) The effects of the in vivo transfection of miR-221/222 mimics on apoptosis and fibrosis were examined by TUNEL assay and Masson's trichrome staining, respectively, in normal mice and miR-221/222 KO mice. (scale bar = 50 μm). (G) The effect of the in vivo transfection of miR-221/222 mimics treatment on the expression of PUMA, p-p53, BCL2, ETS-1, fibronectin and collagen 3 was measured by Western blot. (H) The effects of the in vivo transfection of miR-221/222 mimics treatment on the expression of PUMA and ETS-1 were examined by immunohistochemistry (scale bar = 50 μm). (I) The effects of the in vivo transfection of miR-221/222 inhibitors on apoptosis and fibrosis were examined by TUNEL assay and Masson's trichrome staining, respectively (scale bar = 50 μm). (J, K) The expression of PUMA and ETS-1 in WT mice transfected with miR-221/222 inhibitors was evaluated by Western blot and immunohistochemistry. The data are expressed as the mean ± SEM (n = 3-5). ^*P < 0.05 vs. control, ^†P < 0.05 vs. the I/R 3 h or KO control group, ^‡P< 0.05 vs. the I/R 3 d or KO I/R group.