The Bioactive Substance Secreted by MSC Retards Mouse Aortic Vascular Smooth Muscle Cells Calcification

Abstract

Background: Vascular calcification, which is associated with low-level chronic inflammation, is a complication that occurs during aging, atherosclerosis, chronic kidney disease, diabetes mellitus, and hyperlipaemia. In this study, we used conditioned media from mesenchymal stem cells (MSC-CM), a source of autologous cytokines, to test the hypothesis that MSC-CM inhibits vascular smooth muscle cell (VSMC) calcification by suppressing inflammation and apoptosis.

Methods: VSMCs were treated with β-glycerophosphate (β-GP) to induce calcification and MSC-CM was used as a treatment. Calcium deposition was evaluated using alizarin red and von Kossa staining after a 7-day induction period. Intracellular calcium contents were measured via the o-cresolphthalein complexone method, and alkaline phosphatase (ALP) activity was determined using the para-nitrophenyl phosphate method. The expressions of specific-osteogenic markers, inflammatory cytokines, and apoptosis-associated genes/proteins were examined by real-time polymerase chain reaction or western blotting.

Results: MSC-CM inhibited β-GP-induced calcium deposition in VSMCs and decreased intracellular calcium content and ALP activity. Additionally, MSC-CM suppressed the β-GP-induced increases in BMP2, Msx2, Runx2, and osteocalcin expression. Additionally, MSC-CM decreased the expression of TNF-α, IL-1β, and IL-6 in VSMC. MSC-CM also partly blocked β-GP-induced VSMC apoptosis, which was associated with an increase in the Bcl-2/Bax expression ratio and a decrease in caspase-3 expression.

Conclusion: Our study results suggest that MSC-CM can inhibit VSMC calcification. This suggests a potential novel clinical application for MSCs in the treatment of vascular calcification and associated diseases.

Figure 1

MSC-CM suppresses mineral deposition and ALP activity in VSMCs. VSMCs from different treatment groups were incubated for 7 days. Images of (a) alizarin red staining (magnification ×100) and (b) von Kossa staining (×100). The graphs depict the quantification of alizarin red staining (c), calcium content (d), and ALP activity (e) analyses.

Figure 2

MSC-CM inhibits the expression of osteogenesis-specific markers in VSMCs. (a) The mRNA expression levels of BMP-2, Msx2, Runx2, and osteocalcin were determined by RT-PCR after 3 days. (b) The protein expression levels of BMP-2 and Runx2 were measured by western blotting; β-actin was used as an endogenous control. (c) BMP-2 and Runx2 protein levels were quantified densitometrically. ^∗P < 0.05.

Figure 3

MSC-CM suppresses β-GP-induced inflammatory cytokine expression in VSMCs. (a–c) Expression levels of TNF-α, IL-1β, and IL-6 mRNA are shown. (d) TNF-α protein expression was determined by western blotting; β-actin was used as an endogenous control.

Figure 4

MSC-CM prevents β-GP-induced VSMC apoptosis. (a) A CCK8 assay was used to test VSMC viability (^∗P < 0.05). (b) The frequency of VMSC apoptosis was analysed using flow cytometry and calculated as quartile 2 (Q2) (FITC+/PI+) + Q3 (FITC+/PI−). (c) Apoptotic cells were determined among Hoechst-stained VSMCs (×200). (d) The relative mRNA expression (e) and protein expression of caspase-3 were determined by RT-PCR and western blotting, respectively. (f) The Bcl-2/Bax mRNA expression ratio was determined by RT-PCR. ^∗P < 0.05.