Despite the origin of pulmonary arterial hypertension (PAH), pulmonary vascular resistance rises due to pulmonary vasoconstriction, arterial remodeling and polycythemia leading to right heart failure and death. Rodents exposed to chronic hypobaric hypoxia (CH) develop PAH. The complex process of developing PAH is driven, in part, by changes in gene expression. In PAH, smooth muscle intracellular Ca2+ is increased and endothelin 1 (ET-1) expression is up-regulated. Ca2+ regulates pulmonary arterial smooth muscle (PASMC) contraction and is linked to gene transcription through the nuclear factor of activated T cells (NFAT). NFATc3 isoform is specifically implicated in the development of the vasculature and maintenance of smooth muscle differentiate phenotype. The overall goal of this proposal is to determine the role of NFATc3 in the molecular mechanisms underlying the vascular changes associated with CH-PAH. The hypothesis is that CH activates NFATc3 in PASMC to mediate hypertrophy and enhance contractility of pulmonary arteries (PA) contributing to PAH.
Specific Aim 1 : To determine the role of NFATc3 in CH-induced PASMC hypertrophy and PAH. We will estimate PA pressure, measure mRNA and protein of the hypertrophic markers alpha-actin and myosin heavy chain in PA, NFATc3 binding to 1-actin and myosin heavy chain promoters, and determine structural changes of the pulmonary vasculature on wild type +/- calcineurin/NFAT inhibitor and NFATc3 knockout mice exposed to normoxia and hypobaric CH.
Specific Aim 2 : To establish the contribution of NFATc3 to CH-induced downregulation of Kv channel expression and increases in pulmonary vasoconstrictor reactivity. We will use the same animal models proposed in aim1 and determine mRNA and protein Kv isoforms;NFATc3 binding to KV 1.5 and 2.1 promoters and association to additional transcriptional regulators;PASMC membrane potential and agonist-induced vasoconstriction in isolated pressurized PA.
Specific Aim 3 : To determine the mechanisms by which CH increases NFATc3 transcriptional activity in PASMC. We will determine the mediators (ET-1, Ca2+, calcineurin and Rho-kinase) of CH-increased NFAT activity using NFAT-luciferase reporter mice and NFAT-luciferase crossed with NFATc3 KO mice. Findings from the proposed studies will provide novel information about the signaling mechanisms regulating changes in gene transcription in PAH. A better understanding of this mechanisms in PAH will lead to the development of novel therapeutic approaches to prevent and cure this debilitating disease.
. In the United States it is estimated that 300 new cases of primary pulmonary hypertension are diagnosed each year but the true prevalence and incidence is unknown (NHLBI, Facts About Primary Pulmonary Hypertension). Secondary pulmonary hypertension is much more common because it is caused by a variety of obstructive pulmonary diseases and living at high-altitude, two conditions associated with chronic hypoxia. Sustained high pulmonary arterial resistance to blood flow causes an increase in the right ventricular (RV) filling pressure, which will eventually cause RV hypertrophy, ischemia, failure, and sudden cardiac death. The most common causes of death among patients with pulmonary arterial hypertension are related to progressive right-sided heart failure and sudden cardiac death events. Thus there is a need to understand the impact of chronic hypoxia on physiological responses at the cellular, molecular, and genomic levels in order to develop appropriate treatment strategies for the large group of patients. Our long-term research goal is to define the mechanisms whereby chronic hypoxia leads to the pathologies of pulmonary hypertension to allow more rational design of pharmacological approaches to improve the quality of life of patients suffering lung diseases associated with chronic hypoxia, and to decrease the rate of morbidity and mortality linked to this condition. The complex process of developing pulmonary hypertension is driven, in part, by changes in gene expression. The role of the calcium-regulated transcription factor NFATc3 in pulmonary arterial hypertension has not been previously addressed, highlighting the impact of and need for the proposed research. The proposed studies will define for the first time the regulators and targets of NFATc3 signaling in pulmonary arterial smooth muscle cells in the devastating pathological condition of pulmonary arterial hypertension. NFATc3 has been previously linked to vascular development, regulation of vascular smooth muscle cell differentiation, proliferation and contractility. Therefore, a better understanding of the molecular mechanisms that underlie the vascular remodeling and increased vasoconstriction in pulmonary hypertension is expected to lead to the development of novel therapeutic approaches to prevent and treat this disease. The most novel aspects of this proposal are its ability to examine NFATc3 regulation of pulmonary vascular function in a truly integrated fashion. The planned experiments will utilize our expertise in molecular biology, vascular biology and integrated systems physiology.
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