# Role of the TLR4 signaling in smooth muscle cell phenotypic transition

> **NIH NIH R01** · CLEVELAND CLINIC LERNER COM-CWRU · 2022 · $402,500

## Abstract

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...

## Key facts

- **NIH application ID:** 10448240
- **Project number:** 5R01HL150193-02
- **Recipient organization:** CLEVELAND CLINIC LERNER COM-CWRU
- **Principal Investigator:** Olga Cherepanova
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $402,500
- **Award type:** 5
- **Project period:** 2021-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10448240

## Citation

> US National Institutes of Health, RePORTER application 10448240, Role of the TLR4 signaling in smooth muscle cell phenotypic transition (5R01HL150193-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10448240. Licensed CC0.

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