Identification of new biomarkers for Acute Respiratory Distress Syndrome by expression-based genome-wide association study.

BACKGROUND: Accumulated to-date gene microarray data on Acute Respiratory Distress Syndrome (ARDS) in the Gene Expression Omnibus (GEO) represent a rich source for identifying new unsuspected targets and mechanisms of ARDS. The recently developed expression-based genome-wide association study (eGWAS) for analysis of GEO data was successfully used for analysis of gene expression of comparatively noncomplex adipose tissue, 75 % of which is represented by adipocytes. Although lung tissue is more heterogenic and does not possess a prevalent cell type for driving gene expression patterns, we hypothesized that eGWAS of ARDS samples will generate biologically meaningful results. METHODS: The eGWAS was conducted according to (Proc Natl Acad Sci U S A 109:7049-7054, 2012) and genes were ranked according to p values of chi-square test. RESULTS: The search of GEO retrieved 487 ARDS related entries. These entries were filtered for multiple qualitative and quantitative conditions and 219 samples were selected: mouse n sham/ARDS = 67/92, rat n = 13/13, human cells n = 11/11, canine n = 6/6 with the following ARDS model distributions: mechanical ventilation (MV)/cyclic stretch n = 11; endotoxin (LPS) treatment n = 8; MV + LPS n = 3; distant organ injury induced ARDS n = 3; chemically induced ARDS n = 2; Staphylococcus aureus induced ARDS n = 2; and one experiment each for radiation and shock induced ARDS. The eGWAS of this dataset identified 42 significant (Bonferroni threshold P < 1.55 × 10(-6)) genes. 66.6 % of these genes, were associated previously with lung injury and include the well known ARDS genes such as IL1R2 (P = 4.42 × 10(-19)), IL1ß (P = 3.38 × 10(-17)), PAI1 (P = 9.59 × 10(-14)), IL6 (P = 3.57 × 10(-12)), SOCS3 (P = 1.05 × 10(-10)), and THBS1 (P = 2.01 × 10(-9)). The remaining genes were new ARDS candidates. Expression of the most prominently upregulated genes, CLEC4E (P = 4.46 × 10(-14)) and CD300LF (P = 2.31 × 10(-16)), was confirmed by real time PCR. The former was also validated by in silico pathway analysis and the latter by Western blot analysis. CONCLUSIONS: Our first in the field application of eGWAS in ARDS and utilization of more than 120 publicly available microarray samples of ARDS not only justified applicability of eGWAS to complex lung tissue, but also discovered 14 new candidate genes which associated with ARDS. Detailed studies of these new candidates might lead to identification of unsuspected evolutionarily conserved mechanisms triggered by ARDS.

Meta-analysis of molecular response of kidney to ischemia reperfusion injury for the identification of new candidate genes.

BACKGROUND: Accumulated to-date microarray data on ischemia reperfusion injury (IRI) of kidney represent a powerful source for identifying new targets and mechanisms of kidney IRI. In this study, we conducted a meta-analysis of gene expression profiles of kidney IRI in human, pig, rat, and mouse models, using a new scoring method to correct for the bias of overrepresented species. The gene expression profiles were obtained from the public repositories for 24 different models. After filtering against inclusion criteria 21 experimental settings were selected for meta-analysis and were represented by 11 rat models, 6 mouse models, and 2 models each for pig and human, with a total of 150 samples. Meta-analysis was conducted using expression-based genome-wide association study (eGWAS). The eGWAS results were corrected for a rodent species bias using a new weighted scoring algorithm, which favors genes with unidirectional change in expression in all tested species. RESULTS: Our meta-analysis corrected for a species bias, identified 46 upregulated and 1 downregulated genes, of which 26 (55%) were known to be associated with kidney IRI or kidney transplantation, including LCN2, CCL2, CXCL1, HMOX1, ICAM1, ANXA1, and TIMP1, which justified our approach. Pathway analysis of our candidates identified "Acute renal failure panel" as the most implicated pathway, which further validates our new method. Among new IRI candidates were 10 novel (<5 published reports related to kidney IRI) and 11 new candidates (0 reports related to kidney IRI) including the most prominent candidates ANXA2, CLDN4, and TYROBP. The cross-species expression pattern of these genes allowed us to generate three workable hypotheses of kidney IRI, one of which was confirmed by an additional study. CONCLUSIONS: Our first in the field kidney IRI meta-analysis of 150 microarray samples, corrected for a species bias, identified 10 novel and 11 new candidate genes. Moreover, our new meta-analysis correction method improved gene candidate selection by identifying genes that are model and species independent, as a result, function of these genes can be directly extrapolated to the disease state in human and facilitate translation of potential diagnostic or therapeutic properties of these candidates to the bedside.

RNA-seq analysis of transcriptomes in thrombin-treated and control human pulmonary microvascular endothelial cells.

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism. RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence. Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin," in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases. The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

The enterohemorrhagic Escherichia coli effector protein NleF binds mammalian Tmp21.

The human pathogens enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC), as well as the mouse pathogen Citrobacter rodentium encode type III secretion system (T3SS) effector proteins to promote their survival in the infected host. The mechanisms of action and the host targets of T3SS effectors are under active investigation because of their importance to bacterial virulence. The non-locus of enterocyte effacement (LEE)-encoded protein F, NleF, contributes to E. coli and C. rodentium colonization of piglets and mice, respectively. Here we sought to characterize the host binding partners of NleF. Using a yeast two-hybrid screen, we identified Tmp21, a type-I integral membrane protein and COPI-vesicle receptor involved in trans-Golgi network function, as an NleF-binding partner. We confirmed this interaction using immunoprecipitation and bimolecular fluorescence complementation (BiFC). We expressed a temperature-sensitive vesicular stomatitis virus glycoprotein (tsVSVG) to monitor protein trafficking and determined that NleF slows the intracellular trafficking of tsVSVG from the endoplasmic reticulum to the Golgi.

Pleiotropic functions of pre-B-cell colony-enhancing factor (PBEF) revealed by transcriptomics of human pulmonary microvascular endothelial cells treated with PBEFsiRNA.

This study profiled transcriptomes of human pulmonary microvascular endothelial cells (HMVEC-L) treated with pre-B-cell colony-enhancing factor (PBEF) siRNA or scrambled RNA to gain insight into transcriptional regulations of PBEF on the endothelial function using the Affymetrix GeneChips HG-U133 plus 2. Several important themes are emerged from this study. First, PBEF affected expressions of multiple genes in the endothelium. Expression of 373 genes was increased and 64 genes decreased by at least 1.3-fold in the PBEFsiRNA-treated HMVEC-L versus the scramble RNA control. Second, the microarray results confirmed previous reports of PBEF-mediated gene expressions in some pathways but provided a more complete repertoire of molecules in those pathways. Third, most of the affected canonical pathways have not previously been reported to be PBEF responsive. Fourth, network analysis supports that PBEF has pleiotropic functions. Our first transcriptome analysis of human pulmonary microvascular endothelial cells treated with PBEFsiRNA has provided important insights into the transcriptional regulation of gene expression in HMVEC-L cells by PBEF. Further in-depth analysis of these transcriptional regulations may shed light on molecular mechanisms underlying PBEF-mediated endothelial functions and dysfunctions in various diseases and provide new leads of therapeutic targets to those diseases.

RNA-seq reveals novel transcriptome of genes and their isoforms in human pulmonary microvascular endothelial cells treated with thrombin.

The dysregulation of vascular endothelial cells by thrombin has been implicated in the development of a number of pathologic disorders such as inflammatory conditions, cancer, diabetes, coronary heart disease. However, transcriptional regulation of vascular endothelial cells by thrombin is not completely understood. In the present study, Illumina RNA-seq was used to profile the transcriptome in human pulmonary microvascular endothelial cells (HMVEC-L) treated with thrombin for 6 h to gain insight into thrombin's direct effects on the endothelial function. Out of 100 million total reads from a paired end sequencing assay, 91-94% of the reads were aligned to over 16,000 genes in the reference human genome. Thrombin upregulated 150 known genes and 480 known isoforms, and downregulated 2,190 known genes and 3,574 known isoforms by at least 2 fold. Of note, thrombin upregulated 1,775 previously unknown isoforms and downregulated 12,202 previously unknown isoforms by at least 2 fold. Many genes displayed isoform specific differential expression levels and different usage of transcriptional start sites after the thrombin treatment. The cross comparisons between our RNA-seq data and those of DNA microarray analysis of either 6 h thrombin treated HUVEC or 5 h TNFα treated HMVEC have provided a significant overlapping list of differentially expressed genes, supporting the robust utility of our dataset. Further in-depth follow-up analysis of the transcriptional regulation reported in this study may shed light on molecular pathogenic mechanisms underlying thrombin mediated endothelial dysfunction in various diseases and provide new leads of potential therapeutic targets.