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BackgroundIdiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). In this project we sought to determine whether genome scale transcript profiling using RNA Sequencing (RNA-Seq) could be applied to archived Formalin-Fixed Paraffin-Embedded (FFPE) IPF tissues.ResultsWe isolated total RNA from 7 IPF and 5 control FFPE lung tissues and performed 50 base pair paired-end sequencing on Illumina 2000 HiSeq. TopHat2 was used to map sequencing reads to the human genome. On average ~62 million reads (53.4% of ~116 million reads) were mapped per sample. 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR < 0.05). We compared our results to differentially expressed genes calculated from a previously published dataset generated from FF tissues analyzed on Agilent microarrays (GSE47460). The overlap of differentially expressed genes was very high (760 increased and 1,413 decreased, FDR < 0.05). Only 92 differentially expressed genes changed in opposite directions. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Gene network analysis of MMP7, a highly differentially expressed gene in both datasets, revealed the same canonical pathways and gene network candidates in RNA-Seq and microarray data. For validation by NanoString nCounter® we selected 35 genes that had a fold change of 2 in at least one dataset (10 discordant, 10 significantly differentially expressed in one dataset only and 15 concordant genes). High concordance of fold change and FDR was observed for each type of the samples (FF vs FFPE) with both microarrays (r = 0.92) and RNA-Seq (r = 0.90) and the number of discordant genes was reduced to four.ConclusionsOur results demonstrate that RNA sequencing of RNA obtained from archived FFPE lung tissues is feasible. The results obtained from FFPE tissue are highly comparable to FF tissues. The ability to perform RNA-Seq on archived FFPE IPF tissues should greatly enhance the availability of tissue biopsies for research in IPF.Electronic supplementary materialThe online version of this article (doi:10.1186/s12890-016-0356-4) contains supplementary material, which is available to authorized users.

Type I collagen is the most abundant protein in the human body and is composed of two ␣1(I) and one ␣2(I) polypeptides which assemble into a triple helix. For the proper assembly of the collagen triple helix, the individual polypeptides must be translated in coordination. Here, we show that serine-threonine kinase receptor-associated protein (STRAP) is tethered to collagen mRNAs by interaction with LARP6. LARP6 is a protein which directly binds the 5= stem-loop (5=SL) present in collagen ␣1(I) and ␣2(I) mRNAs, but it interacts with STRAP with its C-terminal domain, which is not involved in binding 5=SL. Being tethered to collagen mRNAs, STRAP prevents unrestricted translation, primarily that of collagen ␣2(I) mRNAs, by interacting with eukaryotic translation initiation factor 4A (eIF4A). In the absence of STRAP, more collagen ␣2(I) mRNA can be pulled down with eIF4A, and collagen ␣2(I) mRNA is unrestrictedly loaded onto the polysomes. This results in an imbalance of synthesis of ␣1(I) and ␣2(I) polypeptides, in hypermodifications of ␣1(I) polypeptide, and in inefficient assembly of the polypeptides into a collagen trimer and their secretion as monomers. These defects can be partially restored by supplementing STRAP. Thus, we discovered STRAP as a novel regulator of the coordinated translation of collagen mRNAs. Collagen type I is a heterotrimeric protein composed of two ␣1 and one ␣2 polypeptides which are folded into a triple helix and secreted into the extracellular matrix (1). Type I collagen forms fibrils and provides strength and elasticity to connective tissues (2) and is normally expressed at a high level in bone, tendon, skin, and arterial adventitia (1). Fibroproliferative disorders are characterized by excessive and persistent synthesis of collagen I in parenchymal organs. Excessive deposition of type I collagen forms a fibrotic scar, resulting in organ failure. Fibroproliferative disorders are a major medical problem, associated with 45% of deaths in the United States (3, 4). Conversely, lack of type I collagen expression is incompatible with life, and an impaired ability to assemble collagen I heterotrimer leads to osteogenesis imperfecta and Ehlers-Danlos syndromes (5, 6). Currently, there is no cure for fibroproliferative diseases due to the lack of understanding of complex mechanism of collagen I biosynthesis.Expression of type I collagen is predominantly regulated at the posttranscriptional level. Stabilization of collagen mRNAs is one of the mechanisms by which collagen-producing cells increase collagen expression upon stimulation (7-13). Additionally, collagen expression is regulated by coordinating translation of collagen ␣1(I) and ␣2(I) mRNAs. Translation of collagen mRNAs is regulated by a unique structural element in the 5= untranslated regions (UTRs) of collagen mRNAs, the 5= stem-loop (5=SL) (10, 11, 14-16). La ribonucleoprotein domain family member 6 (LARP6) binds 5=SL with high affinity and specificity (17, 18). Knockdown of LARP6 or mutation of 5=SL reduces type I collagen expression a...

Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease characterized by aberrant remodeling of the lung parenchyma with extensive changes to the phenotypes of all lung resident cells. The introduction of transcriptomics, genome scale profiling of thousands of RNA transcripts, caused a significant inversion in IPF research. Instead of generating hypotheses based on animal models of disease, or biological plausibility, with limited validation in humans, investigators were able to generate hypotheses based on unbiased molecular analysis of human samples and then use animal models of disease to test their hypotheses. In this review, we describe the insights made from transcriptomic analysis of human IPF samples. We describe how transcriptomic studies led to identification of novel genes and pathways involved in the human IPF lung such as: matrix metalloproteinases, WNT pathway, epithelial genes, role of microRNAs among others, as well as conceptual insights such as the involvement of developmental pathways and deep shifts in epithelial and fibroblast phenotypes. The impact of lung and transcriptomic studies on disease classification, endotype discovery, and reproducible biomarkers is also described in detail. Despite these impressive achievements, the impact of transcriptomic studies has been limited because they analyzed bulk tissue and did not address the cellular and spatial heterogeneity of the IPF lung. We discuss new emerging technologies and applications, such as single-cell RNAseq and microenvironment analysis that may address cellular and spatial heterogeneity. We end by making the point that most current tissue collections and resources are not amenable to analysis using the novel technologies. To take advantage of the new opportunities, we need new efforts of sample collections, this time focused on access to all the microenvironments and cells in the IPF lung.

Type I collagen is the most abundant protein in the human body. Its excessive synthesis results in fibrosis of various organs. Fibrosis is a major medical problem without an existing cure. Excessive synthesis of type I collagen in fibrosis is primarily due to stabilization of collagen mRNAs. We recently reported that intermediate filaments composed of vimentin regulate collagen synthesis by stabilizing collagen mRNAs. Vimentin is a primary target of Withaferin-A (WF-A). Therefore, we hypothesized that WF-A may reduce type I collagen production by disrupting vimentin filaments and decreasing the stability of collagen mRNAs. This study is to determine if WF-A exhibits anti-fibrotic properties in vitro and in vivo and to elucidate the molecular mechanisms of its action. In lung, skin and heart fibroblasts WF-A disrupted vimentin filaments at concentrations of 0.5–1.5 µM and reduced 3 fold the half-lives of collagen α1(I) and α2(I) mRNAs and protein expression. In addition, WF-A inhibited TGF-β1 induced phosphorylation of TGF-β1 receptor I, Smad3 phosphorylation and transcription of collagen genes. WF-A also inhibited in vitro activation of primary hepatic stellate cells and decreased their type I collagen expression. In mice, administration of 4 mg/kg WF-A daily for 2 weeks reduced isoproterenol-induced myocardial fibrosis by 50%. Our findings provide strong evidence that Withaferin-A could act as an anti-fibrotic compound against fibroproliferative diseases, including, but not limited to, cardiac interstitial fibrosis.