Adipose-derived stem cell-released osteoprotegerin protects cardiomyocytes from reactive oxygen species-induced cell death

Abstract

Background: The paracrine effect is likely the major mechanism of the adipose-derived stem cell (ASC)-mediated cardioprotective effect. However, the exact composition and nature of ASC-released paracrine factors remain elusive. In the present study, we examined the effect of osteoprotegerin (OPG), a stem cell-released decoy receptor for death ligand, on the survival of cardiomyocytes exposed to oxidative stress.

Methods: The production of OPG from ASCs under oxidative stress was determined by ELISA and immunohistochemistry. The effects of OPG and the OPG-containing conditioned media of ASCs on the survival of cardiomyocytes were determined using a cell viability assay.

Results: Hydrogen peroxide (H₂O₂) significantly increased OPG production from ASCs in vitro, and OPG production from the ASCs transplanted into the ischemia-reperfusion-injured heart was also observed. OPG significantly attenuated cardiomyocyte death in vitro. OPG-containing conditioned media of ASCs also significantly protected cardiomyocytes. Delivery of siRNA specific to OPG significantly decreased the OPG production of ASCs, and also offset the protective effect of the conditioned media of ASCs.

Conclusions: Our study strongly suggests that OPG is one of the prosurvival factors released from ASCs that may contribute to the ASC-mediated cardioprotection and calls for further studies to elucidate detailed underlying mechanisms.

Keywords: Cardiomyocyte survival; Osteoprotegerin; Oxidative stress; Stem cell.

Fig. 1

Oxidative stress induced OPG secretion from ACSs. (a) Amount of OPG secreted from ASCs exposed to H₂O₂ (100 μM) determined using an ELISA kit specific to human OPG. Conditioned media of ASCs exposed to H₂O₂ up to 48 hours were used for the analysis. TGF-β1 (5 ng/ml) was included as a soluble factor commonly present in damaged heart. Inset: viability of ASCs treated with H₂O₂ (100 μM) or TGF-β1 (5 ng/ml) for 48 hours. *p < 0.05 compared to untreated control. (b) Expression of OPG in ischemia–reperfusion (I/R)-injured rat heart examined by immunofluorescence staining. I/R-injured rat heart was collected 1, 3, and 7 days after the injury without ASC transplantation, and damaged heart was stained with antibodies specific to CD90 (red), a stem cell marker, and OPG (green). Nucleus was stained with DAPI (blue). (c) Expression of OPG in I/R-injured heart with ASC transplantation (1 × 10⁶ cells/head). Two-dimensional immunofluorescence images were converted to 2.5D topological view images for clear presentation of staining intensity among groups. ASC adipose-derived stem cell, OPG osteoprotegerin, TGF-β1 transforming growth factor beta 1 (Color figure online)

Fig. 2

OPG protected cardiomyocytes from ROS-induced cell death. (a) Effect of OPG on H₂O₂-induced cell death of H9c2 examined using a cell counting kit. H9c2 cells were treated with 100 μM of H₂O₂ for 24 hours with or without OPG (1.5 and 3 ng/ml) treatment. *p < 0.05, #p < 0.05 compared to H₂O₂-treated group. (b) Expression of proapoptotic receptor TRAIL-R2 and activated (cleaved) caspase 8 in H9c2 cardiomyocytes exposed to H₂O₂ (100 μM) for 24 hours with or without OPG treatment (1.5 and 3 ng/ml) examined by western blot analysis. (c) Effect of ASC conditioned media with or without H₂O₂ conditioning on the viability of H9c2 cardiomyocytes exposed to H₂O₂. Normal conditioned media of ASC (NorCM) prepared by culturing ASCs for 48 hours and conditioned media with H₂O₂ conditioning (H₂O₂CM) prepared by culturing ASCs in the presence of 100 μM of H₂O₂ for 48 hours. H9c2 cells were cultured in NorCM or H₂O₂CM with or without addition of 100 μM of H₂O₂ for 24 hours. *p < 0.05, #p < 0.05 compared to H₂O₂-treated group. (d) Effect of ASC conditioned media with or without H₂O₂ conditioning on the viability of primary cardiomyocytes exposed to H₂O₂. The same experimental procedure used for H9c2 cardiomyocytes was applied to primary cardiomyocytes. *p < 0.05, #p < 0.05 compared to H₂O₂-treated group. OPG osteoprotegerin, TRAIL-R2 tumor necrosis factor-related apoptosis-inducing ligand receptor 2

Fig. 3

Downregulation of OPG offset protective effect of ASC conditioned media. (a) OPG-siRNA-mediated downregulation of OPG in H₂O₂-treated ASCs. ASCs were transfected with 20 nM of OPG-specific siRNA for 24 hours, and then exposed to 100 μM of H₂O₂ for an additional 48 hours. mRNA expression of OPG normalized by 18 s rRNA expression. *p < 0.05, #p < 0.05 compared to H₂O₂-treated group. (b) Effect of OPG-specific siRNA on the OPG production from ASCs exposed to H₂O₂. OPG-siRNA (20 nM)-transfected ASCs were cultured in the presence of 100 μM of H₂O₂ for 48 hours, and the amount of OPG in ASC culture media was determined using an OPG-specific ELISA kit. *p < 0.05 compared to untreated control, #p < 0.05 compared to H₂O₂-treated group. (c) Viability of OPG-siRNA-transfected ASCs exposed to 100 μM of H₂O₂. *p < 0.05 compared to H₂O₂-treated group. (d) Effect of three different types of H₂O₂CMs on H₂O₂-induced cell death of H9c2 cells. *p < 0.05. H₂O₂CM ASC conditioned media prepared by culturing ASCs with 100 μM of H₂O₂ for 48 hours, H₂O₂CM/Reagent conditioned media obtained using transfection reagent-treated ASCs, H₂O₂CM/siRNA conditioned media obtained using OPG-specific siRNA-transfected ASCs, OPG osteoprotegerin