# Molecular mechanisms of vascular calcification and their connection to coronary disease risk

> **NIH NIH R01** · STANFORD UNIVERSITY · 2023 · $589,233

## Abstract

The combination of lineage tracing and single cell RNA sequencing (scRNAseq) in mouse atherosclerosis
models has created a paradigm shift in our understanding of vascular disease, showing that lesion smooth
muscle cells (SMC) undergo phenotypic transitions into derivative cells with multiple complex phenotypes. We
identified TCF21 as a coronary artery disease (CAD) associated gene mapped by genome-wide association
studies (GWAS) and showed that this gene regulates a disease-related transition of SMC to a fibroblast like
phenotype, producing cells we term “fibromyocytes.” Further, we and others have shown that medial SMC can
also transition to a second SMC-derived cellular phenotype, characterized by expression of genes known for
their role in endochondral bone formation, substantiating and expanding previous work investigating this
process that is linked to intimal vascular calcification. We showed that this chondrogenic process, which gives
rise to cells we term “chondromyocytes” (CMC), is actively inhibited by two CAD associated genes, one
encoding the TGFB1 signaling molecule SMAD3, and the other encoding the environmental sensing aryl
hydrocarbon receptor (AHR). Knockout (KO) of both genes in mouse models showed increased transition to
CMC, larger lesion size and increased vascular calcification. These studies identified SOX9 as a primary driver
of the phenotypic transition to the CMC phenotype. Our longterm goal is to elucidate the molecular
mechanisms that mediate the detrimental CMC transition. Our Central Hypothesis postulates that SOX9 is
a key initiator of this chondrogenic process in the vascular wall, as it is in endochondral bone
formation, and regulation of its expression and function in SMC is intimately linked to vascular
calcification and disease risk. Our objective is thus to determine the upstream epigenetic signals that
modulate SOX9 expression, and how SOX9 expression contributes to CMC development and vascular
calcification. Specifically, in Aim 1 we will employ Sox9 KO and SMC lineage tracing in the ApoE KO mouse
atherosclerosis model to characterize the effect of this gene on SMC cell state transitions, and the impact of
perturbing these transitions on disease morphology and cellular anatomy. In Aim 2, we will conduct scRNAseq
in these mice to characterize the SMC gene expression program downstream of Sox9 in this cell type. Single
cell assay of transposase accessible chromatin sequencing (scATACseq) in the same animals will map
enhancers genome-wide that are differentially regulated in CMC phenotypic transition, and identify specific
transcription factors (TFs) that bind these enhancers to regulate expression of CMC genes. Studies proposed
in Aim 3 will employ in vitro studies to characterize the transcriptional and epigenetic mechanism by which
SOX9 interacts with the inhibitory factors SMAD3 and AHR, and novel TFs that promote transition to the CMC
phenotype. The proposed studies will identify cellular and molecular mec...

## Key facts

- **NIH application ID:** 10673742
- **Project number:** 5R01HL158525-02
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** THOMAS QUERTERMOUS
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $589,233
- **Award type:** 5
- **Project period:** 2022-08-01 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10673742

## Citation

> US National Institutes of Health, RePORTER application 10673742, Molecular mechanisms of vascular calcification and their connection to coronary disease risk (5R01HL158525-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10673742. Licensed CC0.

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