Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary artery pressure and vascular resistance, typically leading to right heart failure and death. Current therapies improve quality of life of the patients but have a modest effect on long-term survival. A detailed transcriptomics and systems biology view of the PAH lung is expected to provide new testable hypotheses for exploring novel treatments. We completed transcriptomics analysis of PAH and control lung tissue to develop disease-specific and clinical data/tissue pathology gene expression classifiers from expression datasets. Gene expression data were integrated into pathway analyses. Gene expression microarray data were collected from 58 PAH and 25 control lung tissues. The strength of the dataset and its derived disease classifier was validated using multiple approaches. Pathways and upstream regulators analyses was completed with standard and novel graphical approaches. The PAH lung dataset identified expression patterns specific to PAH subtypes, clinical parameters, and lung pathology variables. Pathway analyses indicate the important global role of TNF and transforming growth factor signaling pathways. In addition, novel upstream regulators and insight into the cellular and innate immune responses driving PAH were identified. Finally, WNT-signaling pathways may be a major determinant underlying the observed sex differences in PAH. This study provides a transcriptional framework for the PAH-diseased lung, supported by previously reported findings, and will be a valuable resource to the PAH research community. Our investigation revealed novel potential targets and pathways amenable to further study in a variety of experimental systems.
Genome-wide association studies of schizophrenia (GWAS) have revealed the role of rare and common genetic variants, but the functional effects of the risk variants remain to be understood. Protein interactome-based studies can facilitate the study of molecular mechanisms by which the risk genes relate to schizophrenia (SZ) genesis, but protein–protein interactions (PPIs) are unknown for many of the liability genes. We developed a computational model to discover PPIs, which is found to be highly accurate according to computational evaluations and experimental validations of selected PPIs. We present here, 365 novel PPIs of liability genes identified by the SZ Working Group of the Psychiatric Genomics Consortium (PGC). Seventeen genes that had no previously known interactions have 57 novel interactions by our method. Among the new interactors are 19 drug targets that are targeted by 130 drugs. In addition, we computed 147 novel PPIs of 25 candidate genes investigated in the pre-GWAS era. While there is little overlap between the GWAS genes and the pre-GWAS genes, the interactomes reveal that they largely belong to the same pathways, thus reconciling the apparent disparities between the GWAS and prior gene association studies. The interactome including 504 novel PPIs overall, could motivate other systems biology studies and trials with repurposed drugs. The PPIs are made available on a webserver, called Schizo-Pi at http://severus.dbmi.pitt.edu/schizo-pi with advanced search capabilities.
Pulmonary arterial hypertension (PAH) is a deadly vascular disease, characterized by increased pulmonary arterial pressures and right heart failure. Considering prior non-US studies of atrial arrhythmias in PAH, this retrospective, regional multi-center US study sought to define more completely the risk factors and impact of paroxysmal and non-paroxysmal forms of atrial fibrillation and flutter (AF/AFL) on mortality in this disease. We identified patients seen between 2010 and 2014 at UPMC (Pittsburgh) hospitals with hemodynamic and clinical criteria for PAH or chronic thromboembolic pulmonary hypertension (CTEPH) and determined those meeting electrocardiographic criteria for AF/AFL. We used Cox proportional hazards regression with time-varying covariates to analyze the association between AF/AFL occurrence and survival with adjustments for potential cofounders and hemodynamic severity. Of 297 patients with PAH/CTEPH, 79 (26.5%) suffered from AF/AFL at some point. AF/AFL was first identified after PAH diagnosis in 42 (53.2%), identified prior to PAH diagnosis in 27 (34.2%), and had unclear timing in the remainder. AF/AFL patients were older, more often male, had lower left ventricular ejection fractions, and greater left atrial volume indices and right atrial areas than patients without AF/AFL. AF/AFL (whether diagnosed before or after PAH) was associated with a 3.81-fold increase in the hazard of death (95% CI 2.64–5.52, p < 0.001). This finding was consistent with multivariable adjustment of hemodynamic, cardiac structural, and heart rate indices as well as in sensitivity analyses of patients with paroxysmal versus non-paroxysmal arrhythmias. In these PAH/CTEPH patients, presence of AF/AFL significantly increased mortality risk. Mortality remained elevated in the absence of a high burden of uncontrolled or persistent arrhythmias, thus suggesting additional etiologies beyond rapid heart rate as an explanation. Future studies are warranted to confirm this observation and interrogate whether other therapies beyond rate and rhythm control are necessary to mitigate this risk.
p62/sequestosome-1 (SQSTM1) is a multifunctional adaptor protein and autophagic substrate which accumulates in cells with hyperactive mTORC1, such as kidney cells with mutations in the tumor suppressor genes TSC1 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 ROS 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 glutathione biosynthesis by buthionine sulfoximine (BSO). Our findings show how p62 helps maintain intracellular pools of glutathione 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.
Aims Endothelial cell dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension (PAH). We aimed to characterise endothelial cell (EC) dynamics in PAH at single-cell resolution. Methods and Results We carried out single-cell RNA sequencing (scRNA-seq) of lung ECs isolated from an EC lineage-tracing mouse model in Control and SU5416/Hypoxia-induced PAH conditions. EC populations corresponding to distinct lung vessel types, including two discrete capillary populations, were identified in both Control and PAH mice. Differential gene expression analysis revealed global PAH-induced EC changes that were confirmed by bulk RNA-seq. This included upregulation of the major histocompatibility complex class II pathway, supporting a role for ECs in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population including upregulation of genes involved in cell death, cell motility and angiogenesis. Interestingly, four genes with genetic variants associated with PAH were dysregulated in mouse ECs in PAH. To compare relevance across PAH models and species, we performed a detailed analysis of EC heterogeneity and response to PAH in rats and humans through whole-lung PAH scRNA-seq datasets, revealing that 51% of up-regulated mouse genes were also up-regulated in rat or human PAH. We identified promising new candidates to target endothelial dysfunction including CD74, the knockdown of which regulates EC proliferation and barrier integrity in vitro. Finally, with an in silico cell ordering approach, we identified zonation-dependent changes across the arteriovenous axis in mouse PAH and showed upregulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and micro-vasculature. Conclusions This study uncovers PAH-induced EC transcriptomic changes at a high resolution, revealing novel targets for potential therapeutic candidate development.
Pulmonary arterial hypertension (PAH) is characterized by occlusion of lung arterioles, leading to marked increases in pulmonary vascular resistance. Although heritable forms of PAH are known to be driven by genetic mutations that share some commonality of function, the extent to which these effectors converge to regulate shared processes in this disease is unknown. We have causally connected extracellular matrix (ECM) remodeling and mechanotransduction to the miR-130/301 family in a feedback loop that drives vascular activation and downstream PAH. However, the molecular interconnections between factors genetically associated with PAH and this mechano-driven feedback loop remain undefined. We performed systematic manipulation of matrix stiffness, the miR-130/301 family, and factors genetically associated with PAH in primary human pulmonary arterial cells and assessed downstream and reciprocal consequences on their expression. We found that a network of factors linked to heritable PAH converges upon the matrix stiffening-miR-130/301-PPARγ-LRP8 axis in order to remodel the ECM. Furthermore, we leveraged a computational network biology approach to predict a number of additional molecular circuits functionally linking this axis to the ECM. These results demonstrate that multiple genes associated with heritable PAH converge to control the miR-130/301 circuit, triggering a self-amplifying feedback process central to pulmonary vascular stiffening and disease.
BackgroundNetwork analysis is a common approach for the study of genetic view of diseases and biological pathways. Typically, when a set of genes are identified to be of interest in relation to a disease, say through a genome wide association study (GWAS) or a different gene expression study, these genes are typically analyzed in the context of their protein-protein interaction (PPI) networks. Further analysis is carried out to compute the enrichment of known pathways and disease-associations in the network. Having tools for such analysis at the fingertips of biologists without the requirement for computer programming or curation of data would accelerate the characterization of genes of interest. Currently available tools do not integrate network and enrichment analysis and their visualizations, and most of them present results in formats not most conducive to human cognition.ResultsWe developed the tool Lens for Enrichment and Network Studies of human proteins (LENS) that performs network and pathway and diseases enrichment analyses on genes of interest to users. The tool creates a visualization of the network, provides easy to read statistics on network connectivity, and displays Venn diagrams with statistical significance values of the network's association with drugs, diseases, pathways, and GWASs. We used the tool to analyze gene sets related to craniofacial development, autism, and schizophrenia.ConclusionLENS is a web-based tool that does not require and download or plugins to use. The tool is free and does not require login for use, and is available at http://severus.dbmi.pitt.edu/LENS.
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