Vascular dysfunction is a major pathologic component of systemic sclerosis (SSc). The role of vascular smooth muscle cells (vSMCs) in the development of vascular dysfunction is still unknown. In this study, we describe a method for isolation of dermal vSMCs from skin biopsies, and we outline their functional phenotype.
Methods
We obtained 4 mm punch-skin biopsies from three SSc patients and matched controls. After trypsin digestion, cells were cultured for 14 days. vSMCs were isolated by first depleting CD31+ cells (endothelial cells), followed by positive selection of CD146+ cells. The CD31- CD146+ cells were then cultured in media optimized for SMCs proliferation. We evaluated cell proliferation, viability and apoptosis in normal and in low serum culture conditions. Cytoplasmic and nuclear expression levels of β-catenin were also investigated.
Results
The CD31- CD146+ cell population expressed smooth muscle MYH11, Desmin and Vimentin but did not express NG2. Flow cytometry confirmed the high purity of CD31- CD146+ MYHC11+ cell population that was maintained for up to the eleventh passage. SSc-vSMCs exhibited increased cell proliferation and viability compared to control cells. Under serum starvation conditions, SSc-vSMCs exhibited more proliferative capacity, and resistance to apoptosis compared to control-vSMCs. Furthermore, a cytoplasmic to nuclear translocation of β-catenin was seen in SSc-vSMCs but not in control-vSMCs.
Conclusions
This is the first report of successful isolation and initial characterization of SSc-vSMCs. It is likely that increased proliferation of SSc-vSMCs in association with resistance to apoptosis can adversely impact the vascular lesion in SSc.
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