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Principal Investigator: Robert Wirka
Organization: UNIV OF NORTH CAROLINA CHAPEL HILL
Fiscal Year: 2024
Award: $122,700
Funding agency: National Heart Lung and Blood Institute
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.
Terms: <Adopted><Affect><Aorta><ApoE knockout mouse><Apoptosis><Apoptosis Pathway><Area><Arterial Fatty Streak><Arterial Fatty Streaks><Arteries><Atheroma><Atheromatous><Atheromatous degeneration><Atheromatous plaque><Atherosclerosis><Atherosclerotic Cardiovascular Disease><Automobile Driving><Award><Basal Transcription Factor><Basal transcription factor genes><Bayesian Modeling><Bayesian Prediction><Bayesian adaptive designs><Bayesian adaptive models><Bayesian belief network><Bayesian belief updating model><Bayesian framework><Bayesian hierarchical model><Bayesian network model><Bayesian nonparametric models><Bayesian spatial data model><Bayesian spatial image models><Bayesian spatial models><Bayesian statistical models><Bayesian tracking algorithms><Binding><Biologic Models><Biological Models><Biology><Blood Vessels><Cardiac artery><Cell Body><Cell Death><Cell Differentiation><Cell Differentiation process><Cell Function><Cell Lineage><Cell Locomotion><Cell Migration><Cell Movement><Cell Physiology><Cell Process><Cells><Cellular Function><Cellular Migration><Cellular Motility><Cellular Physiology><Cellular Process><Cessation of life><Chromatin><Coronary><Coronary Arteriosclerosis><Coronary Artery Disease><Coronary Artery Disorder><Coronary Atherosclerosis><Coronary artery><Data><Data Set><Death><Development><Diabetes Mellitus><Differentation Markers><Differentiation Antigens><Differentiation Markers><Differentiation in cell culture><Disease><Disorder><E12 protein><E1A Binding Protein p300><EP300><EP300 gene><Fellowship><Fibroblasts><Gene Expression><Gene Transcription><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic Transcription><Genetic study><Goals><HLH protein E12><Heart artery><Homolog of Drosophila TWIST 1><Homolog of Drosophila TWIST1><Human><Human Genetics><Hypertension><Immunofluorescence><Immunofluorescence Immunologic><Immunohistochemistry><Immunohistochemistry Cell/Tissue><Immunohistochemistry Staining Method><In Situ><In vitro cell differentiation><Injury><Invaded><Investigators><KAT3B><KO mice><Knock-out><Knock-out Mice><Knockout><Knockout Mice><Learning><Leiomyocyte><Lesion><Link><Maps><Marker Antigens><Medicine><Mentors><Mice><Mice Mammals><Model System><Modeling><Modern Man><Molecular><Molecular Interaction><Monitor><Murine><Mus><Nature><Network Analysis><Null Mouse><Paper><Pathway Analysis><Pathway interactions><Phenotype><Position><Positioning Attribute><Process><Programmed Cell Death><Proliferating><RNA Expression><Regulation><Research><Research Personnel><Researchers><Risk Factors><Role><Rupture><Sampling><Serum Response Factor><Shapes><Smooth Muscle Cells><Smooth Muscle Myocytes><Smooth Muscle Tissue Cell><Subcellular Process><TWIST gene><TWIST1><TWIST1 gene><Time><Training><Transcription><Transcription Factor Proto-Oncogene><Transcription Factor SRF><Transcription Factor TWIST><Transcription Regulation><Transcription factor genes><Transcriptional Control><Transcriptional Regulation><United States><Variant><Variation><Vascular Diseases><Vascular Disorder><Vascular Hypertensive Disease><Vascular Hypertensive Disorder><Vascular Smooth Muscle><Work><aggressive therapy><aggressive treatment><atheromatosis><atherosclerosis plaque><atherosclerotic coronary disease><atherosclerotic disease><atherosclerotic lesions><atherosclerotic plaque><atherosclerotic vascular disease><blood lipid><blood vessel disorder><c-fos Serum Response Factor><career development><causal allele><causal gene><causal mutation><causal variant><causative mutation><causative variant><cell dedifferentiation><cell motility><cell type><cellular differentiation><conditional knock-out><conditional knockout><coronary arterial disease><design><designing><developmental><diabetes><differential expression><differentially expressed><differentiation in culture><differentiation in vitro><disease risk><disorder risk><driving><genome scale><genome-wide><genomewide><helix-loop-helix protein E12><high blood pressure><histone acetyltransferase p300><human data><human disease><hyperpiesia><hyperpiesis><hypertensive disease><hypertensive disorder><in vitro cellular differentiation><in vivo><injuries><insight><migration><myocardin><necrocytosis><novel><p300><pathway><promoter><promotor><response><scRNA-seq><single cell RNA-seq><single cell RNAseq><single cell expression profiling><single cell transcriptomic profiling><single-cell RNA sequencing><skills><social role><targeted drug therapy><targeted drug treatments><targeted therapeutic><targeted therapeutic agents><targeted therapy><targeted treatment><transcription factor><transcriptional differences><twist protein><vascular><vascular dysfunction><vasculopathy><vulnerable plaque>