Impact of Enhanced Production of Endogenous Heme Oxygenase-1 by Pitavastatin on Survival and Functional Activities of Bone Marrow-derived Mesenchymal Stem Cells

Abstract

Although mesenchymal stem cells (MSCs) have a therapeutic potential for the repair of tissue injuries, their poor viability in damaged tissue limits their effectiveness. Statins can induce an increased production of heme oxygenase-1 (HO-1), which may prevent this detrimental effect in MSCs. We investigated the protective effect of statin-induced overexpression of HO-1 by examining changes in gene expression and function in MSCs after pitavastatin treatment. The relative expression of the HO-1 and endothelial nitric oxide synthase genes in MSCs was significantly increased after treatment with pitavastatin (MSCs). Immunocytological analysis showed that MSCs also stained with phospho-Akt. After exposure to oxidative stress, MSCs showed increased resistance to induced cell death compared with control MSCs. Under serum starvation conditions, MSCs treated with 1 μM pitavastatin showed enhanced cell proliferation and a marked increase in vascular endothelial growth factor production compared with control MSCs. Interestingly, MSCs showed enhanced tube formation under both normoxia and hypoxia. These results demonstrate that pitavastatin can enhance endogenous HO-1 expression in MSCs, which may protect the cells into the environment of oxidative stress with partial activation of endothelial nitric oxide synthase and Akt phosphorylation.

FIGURE 1

Effect of pitavastatin treatment on HO-1 and eNOS mRNA levels in MSCs. HO-1 (A) and eNOS (B) mRNA levels increased in a dose-dependent manner after pitavastatin treatment. Data are shown as mean ± SD (n = 6). ∗P < 0.05 versus control.

FIGURE 2

Expression of HO-1 (A) and phospho-Akt (B) in MSCs (^PitaMSCs) with or without pitavastatin treatment. Immunostaining (A and B) and Western blotting (C and D) analyses with duplicate fashion showed that both HO-1 and Akt phosphorylation were enhanced by pitavastatin treatment. The reason why we detected at least 2 bands of phospho-Akt by Western blotting using anti-phospho-Akt (Ser473) antibody might be that Akt was phosphorylated at several sites including Thr308, Tyr315, and Tyr326.

FIGURE 3

Effects of pitavastatin-induced increase in HO-1 expression on cell viability during oxidative stress. Cell viability (vertical axis) was evaluated in cells exposed to H₂O₂ (400 µM) for 2 hours. Data are mean ± SD (n = 8). ∗P < 0.05 versus control.

FIGURE 4

Effect of pitavastatin on MSC proliferation. Cultured MSCs were treated with a range of pitavastatin concentrations (0, 0.1, 0.5, and 1 µM) in serum-free medium or medium containing 10% FBS. Data are mean ± SD (n = 6). ∗P < 0.05 versus control (FBS⁻), †P < 0.05 versus control (FBS⁺).

FIGURE 5

A, VEGF production and (B) expression of the hypoxia-inducible factor (HIF)-1 gene in MSCs before and after pitavastatin treatment. Changes in VEGF concentrations (A, vertical axis) in culture media from MSCs and ^PitaMSCs under normoxia or hypoxia. Real-time RT-PCR analysis of HIF-1α mRNA levels (B) in MSCs or ^PitaMSCs that were cultured for 24 hours under normoxia or hypoxia. Data are mean ± SD (n = 6). ∗P < 0.05 versus normoxia (pitava⁻), ΨP < 0.05 versus normoxia (pitava⁺), †P < 0.05 versus hypoxia (pitava⁻).

FIGURE 6

Effect of pitavastatin treatment on capillary tube formation in cultured MSCs. A, Representative photographs of MSCs cultured on Matrigel after incubation under normoxia (upper) or hypoxia (lower). Culture of MSCs was performed without serum (left), with serum (center), and with serum-free plus pitavastatin (1 µM, right) (n = 4 for each group). B, Quantitative analysis of tube formation. Data are mean ± SD (n = 4) for each group. ΨP < 0.05 versus normoxia (FBS⁻, pitava⁻), ∗P < 0.05 versus normoxia (FBS⁺, pitava⁻), ∬P < 0.05 versus hypoxia (FBS⁻, pitava⁻), †P < 0.05 versus hypoxia (FBS⁺, pitava⁻).