Pulmonary hypertension (PH) is a progressive disorder associated with a variety of diseases commonly afflicting veteran patients. Emerging evidence demonstrates that PH and excessive proliferation of pulmonary artery smooth muscle cells (PASMC) are caused by derangements in mitochondrial function that impair mitochondrial respiration and alter mitochondrial metabolic programming in a manner that enhances glycolytic metabolism and diminishes oxidative phosphorylation. However, abnormalities in mitochondrial quality control mechanisms that regulate mitophagy have not been investigated as a source of mitochondrial dysfunction and PASMC hyperproliferation in PH, and will be explored in detail in the proposed studies. The proposed studies will investigate the role of the mitophagy initiator protein, PTEN-induced putative kinase-1 (PINK1) in the pathogenesis of PH and PASMC proliferation. Our preliminary data demonstrate that PINK1 is reduced in hypoxia-exposed PASMC in vitro and in the lungs and pulmonary artery tissues of rodents that develop PH in hypoxic conditions. In the proposed studies, we will investigate the hypothesis that loss of PINK1 causes impairments in mitophagy and mitochondrial metabolic programming that lead to excessive PASMC proliferation in PH. Additional studies will define the mechanisms that regulate PINK1 loss in hypoxic conditions. We will test our hypothesis through the execution of the following Specific Aims:
Aim 1 will investigate transcriptional and posttranscriptional mechanisms that regulate PINK1 through microRNA (miR). Among several miRs identified by in silico analysis, miR-27a and miR-516a exhibited the greatest differential expression changes in the hypoxic rodent lung and in hypoxic PASMC. Therefore, initial studies will determine if PINK1 loss occurs through miR-27a- or miR-516a-mediated posttranscriptional suppression of PINK1. To identify factors that regulate PINK1 transcription, we employed bioinformatics tools to define predicted transcription factor (TF) binding sites in the PINK1 promoter. Among several TFs identified, regulatory interactions between cAMP response element-binding protein (CREB) and PINK1 had not been previously validated. Therefore, we will conduct studies using a PINK1 promoter luciferase reporter vector to determine whether putative CREB binding sites are functional. To enhance the translational impact, we will determine mechanisms that regulate PINK1 expression and activity using idiopathic pulmonary artery hypertension (IPAH) PASMC and lung tissue.
Aim 2 will characterize the functional consequences of PINK1 alterations on mitochondrial metabolic reprogramming, mitophagy, and PASMC proliferation in PH. In isolated rat PASMC, the impact of hypoxia or PINK1 gain and loss of function on mitochondrial oxygen consumption and glycolysis will be analyzed using the Seahorse XF96 Bioanalyzer?. Additional studies will determine the effects of hypoxia or PINK1 alterations on mitophagy in the lung in vivo using Mt-Keima transgenic mice and in PASMC isolated from rats and IPAH donors using the mKeima-Red mitophagy detection vector in vitro. The fundamental goals of this proposal are to provide novel insights into aspects of mitochondrial dysfunction in PH that contribute to PASMC hyperproliferation and PH through alterations in PINK1 and mitophagy. The successful execution of this work will broaden our understanding of the role of mitophagy in PH and lay the groundwork to investigate novel therapies that target PINK1.

Public Health Relevance

Pulmonary hypertension (PH) is a common disease that increases patient morbidity, mortality, hospital costs, and length of stay. PH often arises as a comorbid complication in lung diseases that are associated with chronic hypoxemia. For example, the smoking-related lung disease, chronic obstructive pulmonary disease disproportionately affects our veteran population and is associated with increased mortality in patients who develop PH. Despite advancements in therapies in PH that improve function, relieve symptoms, and prolong survival, morbidity remains unacceptably high and disease prognosis remains poor, indicating the need for novel therapeutic approaches. The research described in this proposal seeks to better understand pathways that contribute to PH pathogenesis.

National Institute of Health (NIH)
Veterans Affairs (VA)
Non-HHS Research Projects (I01)
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Special Emphasis Panel (ZRD1)
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Veterans Health Administration
United States
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