Ramin Zargham1, Gaétan Thibault. 1. Institut de recherches cliniques de Montréal, Université de Montréal and Department of Experimental Medicine, McGill University, 110, avenue des Pins ouest, Montréal, Québec, Canada H2W 1R7.
Abstract
OBJECTIVE: Vascular smooth muscle cell (VSMC) de-differentiation is a prerequisite for migration from the tunica media to the intima after vascular injury. Integrin cell adhesion molecules participate in VSMC phenotype modulation. Alpha 8 beta 1 integrin is a differentiation marker of VSMCs and its knockdown heightens migration. In the present study, we examined whether or not alpha 8 integrin is required for the maintenance of VSMC differentiated phenotype. METHODS: Alpha 8 integrin in rat VSMC was knocked down by short interference RNA (siRNA) targeting alpha 8 integrin in comparison to a non-silencing siRNA. Cytoskeletal and morphological changes in VSMC were examined by immunofluorescence staining. The expression of phenotype-dependent markers was analyzed by immunoblotting. RESULTS: Alpha 8 integrin gene silencing evoked drastic changes in characteristics of the VSMC differentiated phenotype, including VSMC morphology, actin fibre organization, focal adhesion assembly and the expression of phenotype-dependent markers in favor of de-differentiation. Then, we investigated whether or not phenotype modulation induced by alpha 8 integrin gene silencing could be reversed by an inducer of VSMC differentiation. Transforming growth factor-beta (TGF-beta) failed to upregulate smooth muscle-myosin heavy chain as well as the assembly of parallel actin fibres in VSMCs transfected by siRNA-alpha 8. In addition, TGF-beta-induced vinculin localization at the tip of the cells was impaired by alpha 8 integrin gene silencing. CONCLUSION: These data suggest that alpha 8 integrin expression is required for maintenance of the VSMC differentiated phenotype, a state that is crucial for non-motile VSMCs.
OBJECTIVE: Vascular smooth muscle cell (VSMC) de-differentiation is a prerequisite for migration from the tunica media to the intima after vascular injury. Integrin cell adhesion molecules participate in VSMC phenotype modulation. Alpha 8 beta 1 integrin is a differentiation marker of VSMCs and its knockdown heightens migration. In the present study, we examined whether or not alpha 8 integrin is required for the maintenance of VSMC differentiated phenotype. METHODS: Alpha 8 integrin in rat VSMC was knocked down by short interference RNA (siRNA) targeting alpha 8 integrin in comparison to a non-silencing siRNA. Cytoskeletal and morphological changes in VSMC were examined by immunofluorescence staining. The expression of phenotype-dependent markers was analyzed by immunoblotting. RESULTS: Alpha 8 integrin gene silencing evoked drastic changes in characteristics of the VSMC differentiated phenotype, including VSMC morphology, actin fibre organization, focal adhesion assembly and the expression of phenotype-dependent markers in favor of de-differentiation. Then, we investigated whether or not phenotype modulation induced by alpha 8 integrin gene silencing could be reversed by an inducer of VSMC differentiation. Transforming growth factor-beta (TGF-beta) failed to upregulate smooth muscle-myosin heavy chain as well as the assembly of parallel actin fibres in VSMCs transfected by siRNA-alpha 8. In addition, TGF-beta-induced vinculin localization at the tip of the cells was impaired by alpha 8 integrin gene silencing. CONCLUSION: These data suggest that alpha 8 integrin expression is required for maintenance of the VSMC differentiated phenotype, a state that is crucial for non-motile VSMCs.
Authors: Hironobu Fujiwara; Manuela Ferreira; Giacomo Donati; Denise K Marciano; James M Linton; Yuya Sato; Andrea Hartner; Kiyotoshi Sekiguchi; Louis F Reichardt; Fiona M Watt Journal: Cell Date: 2011-02-18 Impact factor: 41.582