Angiotensin II and Angiotensin‐(1‐7) Modulate Mitochondrial Respiration in Mouse Mesenchymal Stromal Cells

Abstract

Mesenchymal stromal cells (MSCs) constitute bone marrow niches that support homing and quiescence of hematopoietic stem cells via paracrine functions. MSCs have the potential of differentiating into a variety of cell types and therefore have therapeutic potential in regenerative medicine. Mitochondria modulates cellular functions including paracrine functions by which MSCs modulate quiescence and differentiation of stem cells. Previous studies have provided evidence for the expression of renin-angiotensin system in MSCs. The current study evaluated the effects of Angiotensin II (Ang II) and Angiotensin-(1-7) (Ang-(1-7)) on mitochondrial respiration in mouse bone marrow MSCs. Study was carried out in cultured MSCs, passage eight or nine, and mitochondrial respiration was evaluated by using Seahorse (XFp). Expression of ACE, ACE2, AT1R, AT2R and Mas receptor were evaluated by real-time PCR and western blotting. Oxygen consumption rate (OCR) was determined by carrying out mito-stress test. For measuring OCR in real-time, the mito-stress test was carried out, which involved use of different pharmacological agents that modulate mitochondrial electron transport chain such as oligomycin (2µm), FCCP (0.5µm), rotenone/ antimycin A (0.5µm) to determine ATP-linked respiration, maximal respiration, non-mitochondrial respiration, respectively, after recording basal respiration. PCR detected mRNA transcripts of ACE, AT1R, AT2R, and Mas, but not ACE2. Mito-stress test showed dose-dependent (0.1nm, 1nm, 10nm) increase in basal, ATP-linked, maximal, and nonmitochondrial respiration by Ang-(1-7) and effects at 10nm concentration were significant (P<0.05, n=5) compared to the untreated cells (Figure 1). All these changes were blocked by A-779 (1µm). All of the mitochondrial respiratory parameters were significantly (P<0.05, n=5) decreased when only A-779 was present compared to untreated cells. Ang II (100nm) did not alter OCR in MSCs in mito-stress test (Figure 1). Proton leak was not changed by any treatments. Intriguingly, treatment with PD123391 (1µm) significantly increased basal respiration and maximal respiration compared to untreated cells (P<0.05). These effects were not observed when cells were treated with Ang II along with PD123391. Along similar lines, losartan (10µm) significantly increased the basal respiration compared to untreated MSCs (P<0.05), and this change was not observed in the presence of Ang II. This preliminary study shows that mitochondrial oxygen consumption in MSCs is differentially modulated by angiotensin peptides. Further investigations are needed to address the significance of mitochondrial modulation in the regenerative functions of MSCs by angiotensins.