AT1R-Mediated Apoptosis of Bone Marrow Mesenchymal Stem Cells Is Associated with mtROS Production and mtDNA Reduction

Abstract

Angiotensin II (Ang II) is used as an inducer for the differentiation of mesenchymal stem cells (MSCs). Whether the commonly used doses of Ang II for MSC differentiation affect cell apoptosis has not been elucidated. In this study, we investigated the effect of Ang II on the apoptosis of bone marrow MSCs (BMMSCs), and its relations to the activation of Ang II receptor-1- (AT1R-) signaling, mitochondrial ROS (mtROS) generation, and mitochondrial DNA (mtDNA) leakage. AT1R expression in BMMSCs was identified by immunostaining and Western-blotting assays. BMMSC viability was measured by MTT assay following exposure to 1 nM~1 mM Ang II for 12 hours. Cell apoptosis, mtROS, and mtDNA levels were detected by FAM-FLICA® Poly Caspase, MitoSOX™ superoxide, and PicoGreen staining, respectively. The expressions of Bcl2 and Bax were measured by Western-blotting assays. Next, we used losartan to block AT1R-signaling and subsequently measured apoptosis, mtROS, and mtDNA levels, again. The maximum viability of BMMSCs was in response to 100 nM Ang II, after that it began to decrease with the increase of Ang II doses, indicating that Ang II (≧1 μM) may cause apoptosis of BMMSCs. As expected, 1 μM and 10 μM Ang II both caused BMMSC apoptosis. Furthermore, 1 μM and 10 μM Ang II could also induce mtROS generation and cause a marked mtDNA leakage. The application of losartan markedly inhibited Ang II-induced mtROS production, mtDNA leakage, and BMMSC apoptosis. In conclusion, the activation of AT1R-signaling stimulates apoptosis of BMMSCs, which is associated mtROS production and mtDNA reduction.