Abstract 5852: Nitric Oxide Determines Mesodermic Differentiation of Mouse Embryonic Stem Cells Activating Class II Histone Deacetylases: Consequences for Post-ischemia Hindlimb Regeneration
INTRODUCTION: Nitric Oxide is a pleiotropic regulator of core histone tail modification. Its effect on human endothelial cells results in a marked deacetylation as consequence of class II Histone deacetilases (HDACs) activation. Whether similar epigenetic events occur in undifferentiated embryonic stem cells (ES) treated with NO is currently unknown.
METHODS & RESULTS: The present study reports that NO induced global deacetylation of H3 histones in ES cells is depending on class II HDACs sub-cellular localization and activity (1.6 fold increase vs ctr; n=3; p<0.05) and that this phenomenon is associated to the inhibition of Oct4, Nanog and KLF4 expression (mRNA level: Oct4 65% decrease vs ctr; n=5; p<0.004; Nanog 90% decrease vs ctr; n=4; p<0.05; KLF4: 80% decrease vs ctr; n=5; p<0,05). In this condition NO induced formation of macromolecular complexes made of HDAC3, 4, 7 and the protein phosphatase 2A (PP2A). These events correlated well with the expression of the mesodermal specific protein Brachyury (Bry) (3.0 fold increase vs ctr; n=3) and the appearance of several vascular and skeletal muscle differentiation markers which were abolished in the presence of the class II-specific inhibitor MC1568 or in cells transfected with HDAC4 or HDAC7 short interfering RNA (siRNA). The regulation of mesodermic/cardiovascular gene expression determined by NO treatment prompted us to evaluate the therapeutic effect of these cells in the context of ischemic hind-limb regeneration. We found that NO-treated ES efficiently localized in the ischemic tissue contributing to regeneration of muscular and vascular structures. This effect was abolished by the MC1568 inhibitor.
CONCLUSIONS: These findings provide new insights about the relevance of the epigenetic mechanism involved in the NO-dependent ES differentiation and represent the first evidence of a direct and specific role of class II HDACs activation in the early mesodermal commitment and regeneration capacity of mouse embryonic stem cells.