Rationale Our previous studies have developed an efficiency method for producing a large number of smooth muscle cells (SMCs) from embryonic stem (ES) cells. However, little is known about the underlying mechanism.

Methodology and results Nuclear proteins were harvested and isolated from undifferentiated and differentiating ES cells at different time points, and subjected to proteomics analysis. Notably, the majority of upregulated nuclear proteins during SMC differentiation were involved in chromatin remodelling, cellular morphogenesis, cell proliferation, DNA replication, protein synthesis, mRNA transport and RNA processing processes. We further focused on chromobox protein homologue 3 (Cbx3) owing to its involvement in the regulation of gene-specific expression. Knockdown of Cbx3 in the differentiating ES cells resulted in downregulation of smooth muscle differentiation markers, while enforced expression of this gene enhanced SMC differentiation in a dose-dependent manner. Our data also suggested that Cbx3 mediates SMC differentiation from ES cells through regulation of smooth muscle-specific transcription factor, serum response factor (SRF) and its coactivator myocardin. Furthermore, we also demonstrated that another smooth muscle transcription factor, Dia1, functions as bridge protein between Cbx3 and SRF, through which Cbx3 modulates SRF activation, and mediates ultimately SMC differentiation from stem cells. Importantly, in vivo perivascular knockdown of Cbx3 significantly increased wire-injury-induced neointima formation in mice.

Conclusions Our findings demonstrated for the first time that Cbx3 has a crucial role in SMC differentiation and possesses an important protective function in vessel injury-induced neointima formation, indicating that Cbx3 could be a potential new therapeutic target for intervention in SMC proliferative-related vascular diseases.