Abstract 1005: Vascular Cells Derived from Mouse Embryonic Stem Cells Display a Low Inflammatory Response due to Decreased TLR4 Expression
Embryonic stem (ES) cells and ES cell-derived differentiated vascular cells represent a promising potential in cardiovascular diseases as they can be used in tissue regeneration approaches. However, inflammation may pose a major hurdle. LPS consists a strong proinflammatory stimulus and most cell types respond by inducing elevated levels of cytokines. In order to assess the inflammatory response of embryonic stem cells (ES), endothelial (esEC) and smooth muscle cells (esSMC) derived from ES cells, we treated these cells with LPS and analysed the mRNA levels of various cytokines using RNase protection assay (RPA). Suprisingly, with the exception of MIF no significant cytokine mRNA levels were observed either at baseline or after stimulation. On the contrary, sca-1+ adult stem cells isolated from the adventitia showed significant levels of cytokine mRNA at baseline and a marked increase in cytokine expression after endotoxin challenge. Since TLR4 is the signal transducing receptor for LPS, we studied its expression in the three cell types and found that TLR4 mRNA and protein were not expressed in these cells. Analysis of the DNA methylation status of the TLR4 upstream region revealed that TLR4 promoter is methylated in ES ansd ES derived cells. Moreover, in vitro methylation suppressed TLR4 promoter activity in reporter gene assays. Chromatin immunoprecipitation assays showed that in this region histones H3 and H4 are hypoacetylated in ES cells. Interestingly, treatment with a histone deacetylase inhibitor (TSA) could partially relieve gene repression induced by methylation. Finally, the increased levels of TLR4 observed in ES cells after treatment with an inhibitor of DNA methylation or deacetylase activity, confer responsiveness to LPS, as significant induction of IL-6 mRNA was detected in endotoxin stimulated ES cells. In conclusion, we have shown for the first time that mouse embryonic stem cells and stem cell-derived endothelial and smooth muscle cells are unresponsive to LPS due to low levels of TLR4. Epigenetic modifications, DNA methylation and histone deacetylation, on the TLR4 promoter contribute to this suppression.