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Inhibitory role of reactive oxygen species in the differentiation of multipotent vascular stem cells into vascular smooth muscle cells in rats: a novel aspect of traditional culture of rat aortic smooth muscle cells

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Abstract

Proliferative or synthetic vascular smooth muscle cells (VSMCs) are widely accepted to be mainly derived from the dedifferentiation or phenotypic modulation of mature contractile VSMCs, i.e., a phenotype switch from a normally quiescent and contractile type into a proliferative or synthetic form. However, this theory has been challenged by recent evidence that synthetic VSMCs predominantly originate instead from media-derived multipotent vascular stem cells (MVSCs). To test these hypotheses further, we re-examine whether the conventional rat aortic SMC (RASMC) culture involves the VSMC differentiation of MVSCs or the dedifferentiation of mature VSMCs and the potential mechanism for controlling the synthetic phenotype of RASMCs. We enzymatically isolated RASMCs and cultured the cells in both a regular growth medium (RGM) and a stem cell growth medium (SCGM). Regardless of culture conditions, only a small portion of freshly isolated RASMCs attaches, survives and grows slowly during the first 7 days of primary culture, while expressing both SMC- and MVSC-specific markers. RGM-cultured cells undergo a process of synthetic SMC differentiation, whereas SCGM-cultured cells can be differentiated into not only synthetic SMCs but also other somatic cells. Notably, compared with the RGM-cultured differentiated RASMCs, the SCGM-cultured undifferentiated cells exhibit the phenotype of MVSCs and generate greater amounts of reactive oxygen species (ROS) that act as a negative regulator of differentiation into synthetic VSMCs. Knockdown of phospholipase A2, group 7 (Pla2g7) suppresses ROS formation in the MVSCs while enhancing SMC differentiation of MVSCs. These results suggest that cultured synthetic VSMCs can be derived from the SMC differentiation of MVSCs with ROS as a negative regulator.

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Acknowledgements

This work was supported by the National Institute of Health (KO1 grant 5KO1DK080884-05), the Shandong University National Qianren Scholar Fund, the Taishan Scholar Fund and the National Natural Science Foundation of China (grant no. 81370267).

Conflicts of Interest

The authors declare that they have no conflicts of interest with biotech companies, financial foundations, or any other third parties and that no competing interests exist. The funders of this project had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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    1. Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan, 250012, Peopleʼs Republic of China

      Haibo Song, Hui Wang, Weiwei Wu, Lei Qi, Lei Shao, Fang Wang, Xing Li Wang, Dongqi Tang & Taixing Cui

    2. Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC, 29208, USA

      Yimu Lai, Desiree Leach, Bryan Mathis, Joseph S. Janicki, Dongqi Tang & Taixing Cui

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    1. Haibo Song
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    Haibo Song and Hui Wang contributed equally to this work.

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    Figure S1

    Flow cytometry analysis of CD29, CD90 and CD44H expression in RASMCs cultured in RGM over time. The results are representative of three independent experiments (P passage) (GIF 67 kb)

    High resolution image (TIFF 3984 kb)

    Figure S2

    Adipogenic, chondrogenic and osteogenic differentiation of RASMCs cultured in SCGM. P3 RASMCs grown in SCGM were subjected to adipogenic, chondrogenic and osteogenic differentiation by defined factors as indicated. a Adipogenesis. Oil red staining and qPCR analysis of PPARγ2, LPL and C/EBPα. n=4, *P<0.05 vs. control SCGM. b Chondrogenic differentiation. Alcian blue staining and qPCR analysis of aggrecan and OPN. n=4, *P<0.05 vs. control SCGM. c Osteogenic differentiation. Alizarin red staining and qPCR analysis of Runx2 and Alp. n=4, *P<0.05 vs. control SCGM (GIF 215 kb)

    High resolution image (TIFF 13254 kb)

    Figure S3

    Adipocyte, chondrocyte, or osteocyte differentiation potential of RASMCs cultured in SCGM over time. a Experimental flow chart. RASMCs growth in SCGM at various passages were subjected to adipocyte, chondrocyte, or osteocyte differentiation by defined factors as indicated. b Adipogenesis. qPCR analysis of PPARγ2, LPL and C/EBPα. n=4, *P<0.05 vs. control SCGM. c Chondrogenic differentiation. qPCR analysis of aggrecan and OPN. n=4, *P<0.05 vs. control SCGM. d Osteogenic differentiation. qPCR analysis of Runx2 and Alp. n=4, *P<0.05 vs. control SCGM (GIF 58 kb)

    High resolution image (TIFF 3400 kb)

    Figure S4

    Effect of NEDD8 knockdown on SMC differentiation of RASMCs cultured in SCGM. a Experimental flow chart. P2 RASMCs transfected with control (Ctr) interfering RNA (RNAi) and NEDD8 RNAi were cultured in SCGM, RGM, or RGM with PDGF-BB (50 ng/ml) for 7 days and then subjected to the analysis as indicated. b qPCR analysis of NEDD8 gene mRNA expression in P2 RASMCs grown in SCGM, RGM, or RGM with PDGF-BB (50 ng/ml) for 7 days. n=4, *P<0.05 vs. SCGM. c Representative results of NEDD8 protein expression in P2 RASMCs transfected with Ctr siRNA and NEDD8 siRNA and cultured in SCGM for 24 h. d qPCR analysis of SMC gene mRNA expression. n=4, *P<0.05 vs. SCGM. e Western blot analysis of SMC gene protein expression. The results are representative of four separate experiments (GIF 80 kb)

    High resolution image (TIFF 4718 kb)

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    Song, H., Wang, H., Wu, W. et al. Inhibitory role of reactive oxygen species in the differentiation of multipotent vascular stem cells into vascular smooth muscle cells in rats: a novel aspect of traditional culture of rat aortic smooth muscle cells. Cell Tissue Res 362, 97–113 (2015). https://doi.org/10.1007/s00441-015-2193-9

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