The purpose of this award is to provide Dr. Robert Wirka protected time, allowing him to obtain the training necessary to become an independent investigator using human genetic findings to define novel mechanisms driving coronary artery disease (CAD). Career development activities are designed to strengthen Dr. Wirka's skills in three key areas necessary for achieving this research paradigm: i) Linking disease-associated variation to causal genes; ii) Determining how causal genes affect cellular and vascular biology; and iii) Defining the molecular pathways and networks affected by causal genes. His mentor, Dr. Quertermous, will guide and monitor his progress towards independence. During atherosclerosis, vascular smooth muscle cells (SMCs) de- differentiate, proliferate and migrate into the lesion in a process known as ?phenotypic modulation?. In a recent first-author paper in Nature Medicine, Dr. Wirka generated single-cell gene expression datasets from diseased mouse and human arteries and characterized the process of SMC phenotypic modulation in unprecedented detail. In the current proposal, Dr. Wirka has used these data to identify the transcription factor TWIST1 as a potential novel regulator of SMC phenotypic modulation. In pathway analysis of the mouse and human gene expression data, TWIST1 is strongly predicted to promote SMC phenotypic modulation. Consistent with this predicted role, TWIST1 gene expression increases in SMCs during phenotypic modulation in the mouse and human single-cell data, and preliminary studies show that TWIST1 inhibits expression of SMC differentiation markers. Importantly, TWIST1 is also strongly associated with multiple vascular diseases, including CAD, in human genetic studies. The proposed studies will determine the role of TWIST1 in vascular SMCs during disease, and determine the cellular and molecular mechanisms by which this occurs.
In Aim 1, SMC- specific conditional Twist1 knockout mice will be used to determine how Twist1 affects SMC phenotype within the atherosclerotic lesion: i) assessment of single-cell gene expression will be used to determine the effect of SMC-specific Twist1 knockout on the ability of SMCs to undergo phenotypic modulation during disease and to map the molecular pathways affected by Twist1, and ii) in situ studies of the diseased artery wall will examine the effect of SMC-specific Twist1 knockout on classical aspects of lesion phenotype and the contribution of SMCs to the lesion.
In Aim 2, cultured human coronary artery SMCs (HCASMCs) will be used to determine the cellular and molecular effects of TWIST1: i) the effect of TWIST1 perturbation on SMC phenotypes relevant to atherosclerosis such as differentiation, proliferation, migration, invasion, and cell death will be determined, and ii) the detailed molecular mechanism by which TWIST1 inhibits expression of the SMC differentiation marker ACTA2 will be explored. These studies will determine the cellular and molecular mechanisms by which a CAD- associated gene and potential novel SMC master regulator influences disease risk. Importantly, these studies will also provide Dr. Wirka with the final skills necessary to achieve scientific independence.

Public Health Relevance

- Despite aggressive treatment of known risk factors, coronary artery disease (CAD) is still responsible for one in every six deaths in the United States. Evidence from gene expression in diseased human coronary arteries and human genetic studies strongly implicates the gene TWIST1 in CAD. This project will determine the mechanisms by which TWIST1 affects vascular wall biology and thus CAD risk, with the goal of developing novel treatments to reduce the burden of CAD.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
1K08HL152308-01
Application #
9950624
Study Section
NHLBI Mentored Clinical and Basic Science Review Committee (MCBS)
Program Officer
Huang, Li-Shin
Project Start
2020-05-04
Project End
2025-04-30
Budget Start
2020-05-04
Budget End
2021-04-30
Support Year
1
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Stanford University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305