Proceedings of The Physiological Society
University of Edinburgh (2011) Proc Physiol Soc 25, PC31
Characterisation of transcriptional and post transcriptional properties of vascular interstitial and cultured smooth muscle cells
C. L. Huggins1, O. V. Povstyan1, M. I. Harhun1
1. Division of Biomedical Sciences, St George's, University of London, London, United Kingdom.
Various in vitro models are used for studying mechanisms of phenotypic modulation of vascular smooth muscle cells (VSMCs) and the established culture of VSMCs (cVSMCs) is often used for this purpose. Vascular interstitial cell (VICs), recently found in wall of various blood vessels, likely represent resident phenotypically modulated VSMCs under normal physiological conditions1. In this study we compared the expression of proteins and corresponding genes for VSMCs-specific markers in freshly dispersed contractile VSMCs, VICs and cVSMCs from rat aorta using immunocytochemistry (30 cells for each protein, 3 animals) and comparative real-time RT-PCR from separately collected cells (~250 cells of each type, 3 animals). The data is presented as mean±S.E.M. ANOVA test for three variables was used for statistical analysis and differences of p<0.05 were considered significant. We observed that VICs, similarly to cVSMCs, display the presence of sparse αSM-actin-enriched stress fibres, while in VSMCs this protein is more tightly packed with higher density in superficial region of the cell. We found that compared to contractile VSMCs both VICs and cVSMCs show a decrease in gene and protein expression for smoothelin (0.30±0.06 in VICs and 0.34±0.15 in cVSMCs for gene and 0.39±0.05 in VICs and 0.53±0.04 in cVSMCs for immunofluorescence, all p<0.05), myosin light chain kinase (MLCK) (0.20±0.03 in VICs and 0.21±0.12 in cVSMCs for gene and 0.17±0.02 in VICs and 0.20±0.02 in cVSMCs for immunofluorescence, all p<0.05) and SM22α (0.22±0.00 in VICs and 0.20±0.08 in cVSMCs for gene and 0.18±0.03 in VICs and 0.40±0.02 in cVSMCs for immunofluorescence, all p<0.05). The expression of αSM-actin and smooth muscle myosin heavy chain (SM-MHC) was significantly decreased in cVSMCs compared to contractile VSMCs (αSM-actin 0.49±0.06 for gene, 0.27±0.03 for immunofluorescence; SM-MHC, 0.51±0.03 for gene, 0.41±0.06 for immunofluorescence, all p<0.05), however, compared to VSMCs it was not decreased in VICs (αSM-actin, 0.87±0.06 for gene (p>0.05), 1.42±0.11 for protein (p<0.05) and in SM-MHC (0.99±0.13 for gene and 1.03±0.06 for protein, both p>0.05). Our study demonstrates that both VICs and cVSMCs display the features of the putative phenotypically modulated VSMCs such as the presence of actin-enriched stress fibers and decrease in expression of contractile VSMCs markers such as smoothelin, MLCK and SM22α compared to contractile VSMCs. However, it was observed that the expression of two major contractile proteins SM-MHC and αSM-actin was decreased only in cVSMCs while it was not decreased in VICs compared VSMCs. These results suggest that mechanism of governing the phenotypic modulation may be different or altered in the cultured VSMCs comparing to the native phenotypically modulated VSMCs.
Where applicable, experiments conform with Society ethical requirements