Stephen Y. Chan

Translational Advances in the Field of Pulmonary Hypertension.Translating MicroRNA Biology in Pulmonary Hypertension. It Will Take More Than “miR” Words

TRANSLATIONAL ADVANCES IN THE FIELD OF PULMONARY HYPERTENSION Translating MicroRNA Biology in Pulmonary Hypertension It Will Take More Than “miR” Words Hyung J. Chun, Sébastien Bonnet, and Stephen Y. Chan Section of Cardiovascular Medicine, Department of Internal Medicine, Yale Cardiovascular Research Center, Yale University School of Medicine, New Haven, Connecticut; Pulmonary Hypertension Research Group, Quebec Heart and Lung Institute Research Centre, University of Laval, Quebec City, Quebec, Canada; and Center for Pulmonary Vascular Biology and Medicine, Pittsburgh Heart, Lung, Blood, and Vascular Medicine Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Negligible uptake and transfer of diet-derived pollen microRNAs in adult honey bees

ABSTRACT The putative transfer and gene regulatory activities of diet-derived miRNAs in ingesting animals are still debated. Importantly, no study to date has fully examined the role of dietary uptake of miRNA in the honey bee, a critical pollinator in both agricultural and natural ecosystems. After controlled pollen feeding experiments in adult honey bees, we observed that midguts demonstrated robust increases in plant miRNAs after pollen ingestion. However, we found no evidence of biologically relevant delivery of these molecules to proximal or distal tissues of recipient honey bees. Our results, therefore, support the premise that pollen miRNAs ingested as part of a typical diet are not robustly transferred across barrier epithelia of adult honey bees under normal conditions. Key future questions include whether other small RNA species in honey bee diets behave similarly and whether more specialized and specific delivery mechanisms exist for more efficient transport, particularly in the context of stressed barrier epithelia.

Endothelial dysfunction in pulmonary arterial hypertension: An evolving landscape (2017 Grover Conference Series)

Endothelial dysfunction is a major player in the development and progression of vascular pathology in pulmonary arterial hypertension (PAH), a disease associated with small vessel loss and obstructive vasculopathy that leads to increased pulmonary vascular resistance, subsequent right heart failure, and premature death. Over the past ten years, there has been tremendous progress in our understanding of pulmonary endothelial biology as it pertains to the genetic and molecular mechanisms that orchestrate the endothelial response to direct or indirect injury, and how their dysregulation can contribute to the pathogenesis of PAH. As one of the major topics included in the 2017 Grover Conference Series, discussion centered on recent developments in four areas of pulmonary endothelial biology: (1) angiogenesis; (2) endothelial-mesenchymal transition (EndMT); (3) epigenetics; and (4) biology of voltage-gated ion channels. The present review will summarize the content of these discussions and provide a perspective on the most promising aspects of endothelial dysfunction that may be amenable for therapeutic development.

Emerging Metabolic Therapies in Pulmonary Arterial Hypertension

Pulmonary hypertension (PH) is an enigmatic vascular disorder characterized by pulmonary vascular remodeling and increased pulmonary vascular resistance, ultimately resulting in pressure overload, dysfunction, and failure of the right ventricle. Current medications for PH do not reverse or prevent disease progression, and current diagnostic strategies are suboptimal for detecting early-stage disease. Thus, there is a substantial need to develop new diagnostics and therapies that target the molecular origins of PH. Emerging investigations have defined metabolic aberrations as fundamental and early components of disease manifestation in both pulmonary vasculature and the right ventricle. As such, the elucidation of metabolic dysregulation in pulmonary hypertension allows for greater therapeutic insight into preventing, halting, or even reversing disease progression. This review will aim to discuss (1) the reprogramming and dysregulation of metabolic pathways in pulmonary hypertension; (2) the emerging therapeutic interventions targeting these metabolic pathways; and (3) further innovation needed to overcome barriers in the treatment of this devastating disease.

Uptake and impact of natural diet-derived small RNA in invertebrates: Implications for ecology and agriculture

ABSTRACT The putative transfer and gene regulatory activities of diet-derived small RNAs (sRNAs) in ingesting animals are still debated. The existence of natural uptake of diet-derived sRNA by invertebrate species could have significant implication for our understanding of ecological relationships and could synergize with efforts to use RNA interference (RNAi) technology in agriculture. Here, we synthesize information gathered from studies in invertebrates using natural or artificial dietary delivery of sRNA and from studies of sRNA in vertebrate animals and plants to review our current understanding of uptake and impact of natural diet-derived sRNA on invertebrates. Our understanding has been influenced and sometimes confounded by the diversity of invertebrates and ingested plants studied, our limited insights into how gene expression may be modulated by dietary sRNAs at the mechanistic level, and the paucity of studies focusing directly on natural uptake of sRNA. As such, we suggest 2 strategies to investigate this phenomenon more comprehensively and thus facilitate the realization of its potentially broad impact on ecology and agriculture in the future.

Integration of Complex Data Sources to Provide Biologic Insight into Pulmonary Vascular Disease (2015 Grover Conference Series)

The application of complex data sources to pulmonary vascular diseases is an emerging and promising area of investigation. The use of -omics platforms, in silico modeling of gene networks, and linkage of large human cohorts with DNA biobanks are beginning to bear biologic insight into pulmonary hypertension. These approaches to high-throughput molecular phenotyping offer the possibility of discovering new therapeutic targets and identifying variability in response to therapy that can be leveraged to improve clinical care. Optimizing the methods for analyzing complex data sources and accruing large, well-phenotyped human cohorts linked to biologic data remain significant challenges. Here, we discuss two specific types of complex data sources—gene regulatory networks and DNA-linked electronic medical record cohorts—that illustrate the promise, challenges, and current limitations of these approaches to understanding and managing pulmonary vascular disease.

p62/SQSTM1 cooperates with hyperactive mTORC1 to regulate glutathione production, maintain mitochondrial integrity and promote tumorigenesis

p62/sequestosome-1 (SQSTM1) is a multifunctional adaptor protein and autophagic substrate that accumulates in cells with hyperactive mTORC1, such as kidney cells with mutations in the tumor suppressor genes tuberous sclerosis complex (TSC)1 or TSC2. Here we report that p62 is a critical mediator of TSC2-driven tumorigenesis, as Tsc2+/- and Tsc2f/f Ksp-CreERT2+ mice crossed to p62-/- mice were protected from renal tumor development. Metabolic profiling revealed that depletion of p62 in Tsc2-null cells decreased intracellular glutamine, glutamate, and glutathione (GSH). p62 positively regulated the glutamine transporter Slc1a5 and increased glutamine uptake in Tsc2-null cells. We also observed p62-dependent changes in Gcl, Gsr, Nqo1, and Srxn1, which were decreased by p62 attenuation and implicated in GSH production and utilization. p62 attenuation altered mitochondrial morphology, reduced mitochondrial membrane polarization and maximal respiration, and increased mitochondrial reactive oxygen species and mitophagy marker PINK1. These mitochondrial phenotypes were rescued by addition of exogenous GSH and overexpression of Sod2, which suppressed indices of mitochondrial damage and promoted growth of Tsc2-null cells. Finally, p62 depletion sensitized Tsc2-null cells to both oxidative stress and direct inhibition of GSH biosynthesis by buthionine sulfoximine. Our findings show how p62 helps maintain intracellular pools of GSH needed to limit mitochondrial dysfunction in tumor cells with elevated mTORC1, highlighting p62 and redox homeostasis as nodal vulnerabilities for therapeutic targeting in these tumors. Cancer Res; 77(12); 3255-67. ©2017 AACR.

Outcomes of persistent pulmonary hypertension following transcatheter aortic valve replacement

Objectives To determine the prevalence and factors associated with persistent pulmonary hypertension (PH) following transcatheter aortic valve replacement (TAVR) and its relationship with long-term mortality. Methods Consecutive patients who underwent TAVR from July 2011 through January 2016 were studied. The prevalence of baseline PH (mean pulmonary artery pressure ≥25 mm Hg on right heart catheterisation) and the prevalence and the predictors of persistent≥moderate PH (pulmonary artery systolic pressure (PASP)>45 mm Hg on 1 month post-TAVR transthoracic Doppler echocardiography) were collected. Cox models quantified the effect of persistent PH on subsequent mortality while adjusting for confounders. Results Of the 407 TAVR patients, 273 (67%) had PH at baseline. Of these, 102 (25%) had persistent≥moderate PH. Mortality at 2 years in patients with no baseline PH versus those with PH improvement (follow-up PASP≤45 mm Hg) versus those with persistent≥moderate PH was 15.4%, 16.6% and 31.3%, respectively (p=0.049). After adjusting for Society of Thoracic Surgeons Predicted Risk of Mortality and baseline right ventricular function (using tricuspid annular plane systolic excursion), persistent≥moderate PH remained associated with all-cause mortality (HR=1.82, 95% CI 1.06 to 3.12, p=0.03). Baseline characteristics associated with increased likelihood of persistent≥moderate PH were ≥moderate tricuspid regurgitation, ≥moderate mitral regurgitation, atrial fibrillation/flutter, early (E) to late (A) ventricular filling velocities (E/A ratio) and left atrial volume index. Conclusions Persistency of even moderate or greater PH at 1 month post-TAVR is common and associated with higher all-cause mortality.

Rapamycin-induced miR-21 promotes mitochondrial homeostasis and adaptation in mTORC1 activated cells

mTORC1 hyperactivation drives the multi-organ hamartomatous disease tuberous sclerosis complex (TSC). Rapamycin inhibits mTORC1, inducing partial tumor responses; however, the tumors regrow following treatment cessation. We discovered that the oncogenic miRNA, miR-21, is increased in Tsc2-deficient cells and, surprisingly, further increased by rapamycin. To determine the impact of miR-21 in TSC, we inhibited miR-21 in vitro. miR-21 inhibition significantly repressed the tumorigenic potential of Tsc2-deficient cells and increased apoptosis sensitivity. Tsc2-deficient cells’ clonogenic and anchorage independent growth were reduced by ∼50% (p<0.01) and ∼75% (p<0.0001), respectively, and combined rapamycin treatment decreased soft agar growth by ∼90% (p<0.0001). miR-21 inhibition also increased sensitivity to apoptosis. Through a network biology-driven integration of RNAseq data, we discovered that miR-21 promotes mitochondrial adaptation and homeostasis in Tsc2-deficient cells. miR-21 inhibition reduced mitochondrial polarization and function in Tsc2-deficient cells, with and without co-treatment with rapamycin. Importantly, miR-21 inhibition limited Tsc2-deficient tumor growth in vivo, reducing tumor size by approximately 3-fold (p<0.0001). When combined with rapamcyin, miR-21 inhibition showed even more striking efficacy, both during treatment and after treatment cessation, with a 4-fold increase in median survival following rapamycin cessation (p=0.0008). We conclude that miR-21 promotes mTORC1-driven tumorigenesis via a mechanism that involves the mitochondria, and that miR-21 is a potential therapeutic target for TSC-associated hamartomas and other mTORC1-driven tumors, with the potential for synergistic efficacy when combined with rapalogs.

Formidable challenges to the notion of biologically important roles for dietary small RNAs in ingesting mammals

The notion of uptake of active diet-derived small RNAs (sRNAs) in recipient organisms could have significant implications for our understanding of oral therapeutics and nutrition, for the safe use of RNA interference (RNAi) in agricultural biotechnology, and for ecological relationships. Yet, the transfer and subsequent regulation of gene activity by diet-derived sRNAs in ingesting mammals are still heavily debated. Here, we synthesize current information based on multiple independent studies of mammals, invertebrates, and plants. Rigorous assessment of these data emphasize that uptake of active dietary sRNAs is neither a robust nor a prevalent mechanism to maintain steady-state levels in higher organisms. While disagreement still continues regarding whether such transfer may occur in specialized contexts, concerns about technical difficulties and a lack of consensus on appropriate methods have led to questions regarding the reproducibility and biologic significance of some seemingly positive results. For any continuing investigations, concerted efforts should be made to establish a strong mechanistic basis for potential effects of dietary sRNAs and to agree on methodological guidelines for realizing such proof. Such processes would ensure proper interpretation of studies aiming to prove dietary sRNA activity in mammals and inform potential for application in therapeutics and agriculture.