Résumé :
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Vascular smooth muscle cells (VSMCs) anomalies are directly involved in different forms of myopathies including Duchene myopathy and desminopathies. The alteration of cytoskeletal mechanostransduction and transcriptional pathways play an important role in the evolution of the vasculopathies. Serum response factor (SRF) is a key transcription factor in SMCs. The activity of SRF is modulated by the RhoA-actin pathway and growth factors signaling pathways. Our aim is to study the role of SRF in adult VSMCs in vivo and in vitro by conditional invalidation of SRF gene using a tamoxifen-inducible and smooth muscle-specific Cre recombinase, SM22-CREERT2(ki). In order to identify the targets of SRF in VSMCs in vivo, RNA was isolated from aorta 8 days after tamoxifen injection in control and mutant mice. Preliminary analysis of gene expression microarrays shows that more than 100 genes are differentially expressed in the aorta of the SRF mutants. The validation of these data by Q-PCR is ongoing. In order to evaluate the role of SRF in the transcriptional response of VSMCs to experimental cyclic stretch, we isolated VSMCs from the aorta of floxed SRF mice, and optimized the conditions for maintaining VSMCs differentiation in culture with a modified serum-free medium. We infected primary VSMCs cultures with a recombinant adenovirus expressing the Cre recombinase to inactivate SRF in vitro. More than 95% decrease of SRF mRNA expression was obtained by this approach. The effect of stretch on SMC-specific genes expression is currently evaluated using the FlexerCell system in control and SRF-deficient VSMCs. Chemical inhibition or dominant negative approach will be used to decipher the pathways linking biomechanical stretch to SRF activation.
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