Abstract There is a growing body of evidence that smooth muscle cell (SMC) phenotypic transitions play an important role in the pathogenesis of atherosclerosis. However, there is still little understanding of molecular mechanisms responsible for these transitions in vivo. Recently, we found that the embryonic stem cell/induced pluripotency stem cell (iPSC) factor OCT4, which was believed to be silenced in somatic cells, plays an atheroprotective role in SMC, in that genetic inactivation of Oct4 in SMC led to marked increases in lesion size and multiple indices of plaque instability in Apoe‒/‒ mice. While we showed that OCT4 is required for SMC migration and investment into the protective fibrous cap, we also have evidence that its loss in SMC was associated with SMC transition toward a macrophage (MФ)-like state, including increased MФ marker expression, lipid accumulation, and phagocytosis. Given the difficulty in overexpressing targeting iPSC factors, it is crucial to identify endogenous molecular mechanisms responsible for OCT4 activation in SMC that can be potentially used to mediate beneficial SMC-OCT4-dependent effects. It has been reported that activation of the innate immune Toll-like receptor (TLR)/TRIF-dependent IRF3/NFkB signaling is required for efficient reprogramming to iPSC, as well as in cell trans-differentiation to other cell lineages by driving epigenetic plasticity of cells. While mechanisms and the critical role of the innate immunity in the progression of atherosclerosis are well characterized in the immune cells, virtually nothing is known about the role of TLRs in the phenotypic transition of SMC in atherosclerosis. Our data demonstrate that TLR4 signaling is responsible for hydroxymethylation of the Oct4 promoter and reactivation of Oct4 in SMC. Intriguingly, Oct4 deficiency in SMC, in turn, up-regulates Tlr4, and leads to dysregulation of both classical TLR4-downstream mechanisms, including MyD88-dependent (NFkB-mediated inflammatory response) and TRIF-dependent (IRF3/LXRa-mediated cholesterol efflux), in vitro and in vivo. In addition, Chromatin Immunoprecipitation (ChIP)-seq analysis on specimens isolated from mouse atherosclerotic arteries identified Tlr4 as a putative target of OCT4. Together, these results demonstrate the importance of TLR4- signaling in SMC both upstream and downstream of OCT4. Our central hypothesis is that TLR4 signaling mediates atheroprotective changes in SMC by regulating levels of the pluripotency factors OCT4 via a feedback mechanism. To test this hypothesis, we propose the following aims: Aim 1 will test the hypothesis that TLR4 signaling regulates levels of OCT4 in SMC via a feedback mechanism. Aim 2 will test the hypothesis that TLR4 signaling in SMC is atheroprotective. This Aim will utilize genetic and pharmacological approaches to define the role of TLR4 signaling in SMC at different stages of atherosclerosis. Aim 3 will test the hypothesis that the up- regulation of TLR4 in Oct4 defic...