Background/Purpose: Nearly all patients with Systemic sclerosis (SSc) suffer from vascular dysfunction as illustrated by the uniform presence of Raynaud’s phenomena. The role of vascular smooth muscle cells (VSMCs) in the development of vascular dysfunction is still unknown. In this study, we isolated VSMCs from skin biopsy, and we examined their functional phenotype.

Methods: We obtained 4 mm punch-skin-biopsy from 3 patients with diffuse cutaneous SSc and 3 matched healthy controls. Skin specimens were treated with a mixture of proteases, then after digestion we plated cells in culture media and harvested the primary cell culture after 10 days. VSMCs were isolated by magnetic microbeads using cell surface markers. First, we depleted total cells from CD31+ cells, followed by positive selection for CD146 + cells. To confirm the identity of this cell population (CD31- CD146+), we performed immunoflorocytometry staining for smooth muscle myosin heavy chain 11 (MYH11), Desmin and NG2. We investigated cell proliferation by using Bromodeoxyuridine (BrdU) assay, and cell viability in normal culture conditions as well as low serum conditions using MTT assay.  

Results: Out of the total cells obtained from primary cell culture, 15% were CD31- CD146 + (VSMCs). The majority of cells in this population stained for smooth muscle MYH11 (89.1%), in addition to Desmin and NG2, while the CD31+ and the fibroblast cell populations did not. This staining pattern differentiates VSMCs from pericytes. Next, we evaluated cell proliferation using BrdU, and we demonstrated uptake of BrdU by 19% of the control-VSMCs compared to 34% of SSc-VSMCs (P= 0.0031). The MTT assay showed increase cell proliferation of SSc-VSMCs compared to control-VSMCs (0.44 and 0.25, respectively. P= 7.38E-08). Under serum starvation conditions, SSc-VSMCs exhibited more proliferation capacity than control-VSMCs (0.30 and 0.21, respectively. P= 5.73E-13). Also, we performed immunoflorocytometry staining for B-catenin and we demonstrate a cytoplasmic to nuclear translocation of B-catenin in SSc-VSMCs but not in control-VSMCs. This finding may imply potential involvement of B-catenin in the induction of VSMCs activation in SSc.

Conclusion: This is the first report of the successful isolation and initial characterization of  SSc-VSMCs. We believe that increased proliferation of SSc-VSMCs in association with resistance to apoptosis may greatly impact the vascular lesion in SSc. Further studies are warranted to fully understand the trigger and maintenance of  the abnormal SSc-VSMCs phenotype.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/isolation-and-initial-characterization-of-dermal-vascular-smooth-muscle-cells-in-systemic-sclerosis/