Pulmonary Arterial Hypertension (PAH) is a fatal disease caused by elevated vascular resistance due to lumen-obliterating processes in small pulmonary arteries. Recently we reported that chronic exposure of interleukin-13 (IL-13) in murine lungs, using a transgenic approach (IL-13 Tg), induced impressive smooth muscle cell (SMC)-driven pulmonary vascular remodeling leading to pulmonary hypertension (PH). We further showed that IL-13-induced proliferation of pulmonary artery SMC (PASMC) in vivo and in vitro is mediated through Arginase2 (Arg2) via IL-13 receptor alpha2 (IL-13?2). Our preliminary studies have further identified a novel link between IL-13?2-Arg2 and HIF1?, with increased protein levels of HIF1? in IL-13 Tg lungs and IL-13-stimulated hPASMC. Given that HIF1? is a metabolic switch from oxidative phosphorylation to glycolysis, these findings suggest that IL-13 might induce the Warburg type effect (increased aerobic glycolysis and decreased oxidative phosphorylation in cells leading to proliferation) to stimulate proliferation of vascular SMC. We also found serine727-phosphorylated STAT3, which was regulated by Arg2 via IL-13R?2 in our previous study, was activated in the mitochondrial fractions in IL-13 Tg lungs. Arg2 is a mitochondrial enzyme and HIF1? has also been localized to mitochondria recently. Taken together, this serine727-phosphorylated STAT3 might be the molecule that links the IL-13R?2-Arg2-HIF1? pathway to regulate mitochondrial metabolism of the mitochondria of hPASMC. Based on these findings, we propose the following hypothesis and Specific Aims: Hypothesis 1. HIF1? plays a critical role in IL-13-stimulated proliferation of hPASMC. 2. IL-13 regulates the metabolic shift from oxidative phosphorylation to glycolysis via 13R?2, HIF1? and Arg2 thereby inducing PASMC proliferation. 3. IL-13-induced metabolic shift from oxidative phosphorylation to glycolysis is necessary to the development of PH.
Our Aims are to:
Aim 1. Define the role of HIF1? in the generation of the IL-13-IL-13R?2-induced proliferative phenotypes in PASMC.
Aim 2. Define the role of IL-13 in the development of the metabolic shift from oxidative phosphorylation to glycolysis in PASMC.
Aim 3. Define the potential therapeutic roles of targeting IL-13-induced metabolic shift in PASMC in the development of PH. Experimental Approach To address Aim 1, we will address transcriptional and translational regulation of HIF1? by IL-13 using human PASMC (hPASMC). Also, the pulmonary vascular phenotype of IL-13 Tg with in vivo inhibition of HIF1? by intranasal lentiviral delivery of shRNA against HIF1? and crossin IL-13 Tg with HIF1? conditional knockout mice will be assessed. A possible interaction between Arg2 and HIF1? will be addressed by conducting immunoprecipitation and Co-localization technique. To address Aim 2, mitochondrial function including measuring oxygen consumption rate, lactate level, etc., will be assessed in IL-13-stimulated hPASMC before and after inhibition of IL-13R?2, HIF1? and Arg2 gene expression using specific siRNA or primarily cultured PASMC from knockout mice of target gene. The role of serine727-phosphorylated STAT3 in the regulation of mitochondrial function will be addressed using specific chemical inhibitor and siRNA. To address Aim 3, a metabolic modifier to enhance mitochondrial oxidative phosphorylation (to reverse Warburg type phenotype), dichloroacetate, will be given to IL-13 Tg, then pulmonary vascular phenotype of these mice will be assessed. To address the IL-13R?2-Arg2-HIF1? axis in other animal models of PH, knockout mice of target gene will be exposed to chronic hypoxia model with or without SU5416 (VEGF receptor blocker). Pulmonary vascular phenotype will then be assessed. This work will provide us with a novel insight into the pathogenesis of pulmonary hypertension induced by a complex interaction between immune and metabolic processes in addition to a novel regulatory pathway of HIF1?. This grant proposes a research mentorship program at Yale University under the primary sponsorship of Dr. Hyung Joon Chun, an expert in pulmonary vascular signaling and pharmacology, and Dr. Patty Lee, an expert in immune-mediated vascular injury and repair in the lungs as co-mentor. We have also enlisted the expertise of Yale and outside Yale investigators, Dr. Jack Elias, Gerald Shadel, William Sessa, Frank Giordano and Chun Geun Lee, as advisory committee members to provide scientific and career counseling. The proposed career and research program as outlined will provide an extraordinary scientific environment wherein Dr. Cho can launch her future independent career as a physician-scientist.
Pulmonary arterial hypertension is a fatal disease caused by elevated pulmonary vascular resistance. Our preliminary experiments demonstrated that IL-13, an inflammatory mediator, could be a critical contributor to the developmen of pulmonary hypertension by proliferating vascular smooth muscle cells, leading to obliteration of pulmonary artery. We also found that IL-13 possibly induces the metabolc change in these proliferating vascular smooth muscle cells, and in these studies we wil examine whether these metabolic changes are related to the development of IL-13-induced pulmonary hypertension.