Thoracic aortic aneurysms and dissections (TAAD) are the 15th most common cause of death in the U.S. They are frequently inherited in an autosomal dominant fashion, with the defective gene products including smooth muscle contractile proteins (e.g., MYH11), extracellular matrix (ECM) proteins, and TGF-? signaling components; loss of smooth muscle cells (SMCs) in the aortic media characterizes the disease. We recently identified a single gain-of-function mutation (R177Q) in protein kinase G1 (PKG1) as the cause of TAAD in families with early dissections; the mutation causes constitutive, cGMP-independent enzyme activation. We generated mice carrying the R177Q knock-in mutation (PKG1RQ), which are normotensive and have no overt phenotype up to 4 months of age; however, pressure overload by trans-aortic constriction (TAC) caused excessive mortality with aortic rupture and abnormal aortic and cardiac remodeling. Determining how sustained PKG1 activation affects aortic integrity is of broader clinical relevance, because case reports suggest that repeated use of the PKG-stimulating drug sildenafil (Viagra) can lead to TAAD, and treating mice with a MYH11 mutation with sildenafil increased development of TAAD under hypertensive stress. We found increased reactive oxygen species (ROS) and tissue markers of oxidative damage, excess TGF-? signaling, and altered contractile and ECM-related gene expression in SMCs and aortas of PKG1RQ-mutant compared to wild type mice. We hypothesize that excess PKG1 activity leads to increased oxidative stress and dysregulation of gene expression in SMCs, resulting in impaired aortic wall maintenance/repair, and that reducing ROS production may protect PKG1RQ-mutant mice from aortic aneurysms.
In Aim I, we will determine if hetero- and homozygous PKG1RQ mice develop aortic dilation and dissection spontaneously with age, or only under hypertensive stress, and examine the effect of pharmacological PKG activation on aortic wall integrity in wild type and heterozygous PKG1RQ mice.
In Aim II, we will compare ROS production by mitochondria, NOX isoforms, and uncoupled NO synthases in wild type and PKG1RQ SMCs and tissues. To determine if excess ROS production is causally linked to aortic pathology, we will treat PKG1RQ mice with cobinamide, a vitamin B12 analog that is a novel ROS neutralizing agent, or cross them with NOX4-deficient mice and mice over- expressing mitochondrial superoxide dismutase or catalase.
In Aim III, we will use RNA-seq and gene network analysis to compare a PKG1-induced gene signature in the aorta from wild type and PKG1RQ mice and from patients carrying the PKG1 mutation. We will analyze gene networks known to be involved in TAAD pathogenesis and determine ROS-induced transcriptome alterations.
In Aim I V, we will determine the molecular basis of PKG1 activation by the R?Q mutation using deuterium exchange/mass spectrometry. Since PKG1 is the pharmacological target of multiple, clinically-used drugs, a better understanding of the effects of long-term PKG activation and excess ROS on aortic wall integrity is important. The potent ROS- neutralizing agent cobinamide is a promising new agent for anti-oxidant therapy in vascular diseases.

Public Health Relevance

Rupture of the main artery in the body (the aorta) due to abnormal wall thinning (aneurysm) were the primary or contributing cause of over 17,500 deaths in the United States in 2009. Recently, a genetic mutation in cGMP-dependent protein kinase (PKG) was shown to cause aortic aneurysms in several families, and there are reports that use of drugs that work by activating PKG (i.e., Viagra) may contribute to aortic aneurysm formation in some people without known genetic predisposition. In this proposal we will determine how elevated PKG activity causes changes in the aorta that lead to aneurysm formation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL132141-03
Application #
9417078
Study Section
Atherosclerosis and Inflammation of the Cardiovascular System Study Section (AICS)
Program Officer
Tolunay, Eser
Project Start
2016-04-01
Project End
2020-01-31
Budget Start
2018-02-01
Budget End
2019-01-31
Support Year
3
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Kalyanaraman, Hema; Zhuang, Shunhui; Pilz, Renate B et al. (2017) The activity of cGMP-dependent protein kinase I? is not directly regulated by oxidation-induced disulfide formation at cysteine 43. J Biol Chem 292:8262-8268