Desmuslin gene knockdown causes altered expression of phenotype markers and differentiation of saphenous vein smooth muscle cells Ying Xiao, PhD, Zhibin.

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Desmuslin gene knockdown causes altered expression of phenotype markers and differentiation of saphenous vein smooth muscle cells Ying Xiao, PhD, Zhibin Huang, MD, Henghui Yin, PhD, Hui Zhang, PhD, Shenming Wang, PhD Journal of Vascular Surgery Volume 52, Issue 3, Pages 684-690 (September 2010) DOI: 10.1016/j.jvs.2010.03.069 Copyright © 2010 Terms and Conditions

Fig 1 Desmuslin downregulation by short interfering RNA (siRNA) in vascular smooth muscle cells (VSMCs). VSMCs were transfected with desmuslin siRNA oligos 1 or 2 or siRNA scramble. A subset of cells was also mock transfected. Desmuslin protein levels were assessed by Western blot and normalized with reference to α-tubulin levels. Representative blots are shown. The results are presented as mean ± standard deviation as determined from three independent experiments (ie, cells were obtained from three different patients). *P < .05 vs scramble; **P < .05 vs mock; ***P < .05 vs oligo 1. Journal of Vascular Surgery 2010 52, 684-690DOI: (10.1016/j.jvs.2010.03.069) Copyright © 2010 Terms and Conditions

Fig 2 Expression of vascular smooth muscle cell phenotype-dependent markers (smooth muscle [SM] α-actin, smooth muscle myosin heavy chain [SM-MHC], and smoothelin) following desmuslin gene knockdown by short interfering RNA (siRNA). Control cells were transfected with siRNA scramble. Protein levels were assessed by Western blot and normalized with reference to α-tubulin levels. Representative blots are shown. The results are presented as mean ± standard deviation as determined from five independent experiments (ie, cells were obtained from five different patients). *P < .05 vs scramble. Journal of Vascular Surgery 2010 52, 684-690DOI: (10.1016/j.jvs.2010.03.069) Copyright © 2010 Terms and Conditions

Fig 3 Expression of matrix metalloproteinase-2 (MMP-2) in vascular smooth muscle cells following desmuslin gene knockdown by short interfering RNA (siRNA). Control cells were transfected with siRNA scramble. Protein levels were assessed by Western blot and normalized with reference to α-tubulin levels. Representative blots are shown. The results are presented as mean ± standard deviation from five independent experiments (ie, cells were obtained from five different patients). *P < .05 vs scramble. Journal of Vascular Surgery 2010 52, 684-690DOI: (10.1016/j.jvs.2010.03.069) Copyright © 2010 Terms and Conditions

Fig 4 Collagen synthesis in vascular smooth muscle cells after desmuslin gene knockdown by short interfering RNA (siRNA) as indicated by incorporation of L-[3,4-3H] proline into the cell layer (A) and media (B). Control cells were transfected with siRNA scramble. The results are presented as the median and interquartile range as determined from four independent experiments (ie, cells were obtained from four different patients). *P < .05 vs scramble. Journal of Vascular Surgery 2010 52, 684-690DOI: (10.1016/j.jvs.2010.03.069) Copyright © 2010 Terms and Conditions

Fig 5 Representative immunofluorescence micrographs of vascular smooth muscle cells transfected with scramble small inhibitory RNA (siRNA) and desmuslin siRNA. Cells transfected with siRNA scramble exhibited parallel and dense actin filaments, while cells treated with the desmuslin siRNA had dispersed and thinner filaments. Cells transfected with siRNA scramble retained a normal spindle-like morphology, while cells transfected with desmuslin siRNA were more polygonal in shape. Desmuslin-knockdown cells were larger than control cells. Journal of Vascular Surgery 2010 52, 684-690DOI: (10.1016/j.jvs.2010.03.069) Copyright © 2010 Terms and Conditions