POTENTIAL DIAGNOSTIC AND PROGNOSTIC BIOMARKERS OF IDIOPATHIC PULMONARY FIBROSIS AND THE IMPACT OF E2F8 IN COLLAGEN 1 SYNTHESIS

Idiopathic pulmonary Fibrosis (IPF) is a chronic lung disease with median survival about 2-3.5 years (1). Lung transplant is the most effective therapy although pirfenidone (2) and nintetanib (3) delay mortality or disease progression. Biomarkers are used for diagnosis and management of diseases (4) but for IPF, a reliable biomarker is not established (5, 6). In this project, we proposed to investigate lung transcript expressions and plasma proteins as diagnostic or prognostic biomarkers and their implication in Pathogenesis of IPF. First, we found 44 transcripts qualified after t-test, Benjamini and Hochberg multiple correction, and 1.5-fold change cutoff. We observed 33 transcripts that commonly distinguished IPF from control or COPD. The scoring coefficients of the principal components (COMP) distinguished IPF from control or COPD by using Classification Tree model. The receiver operating characteristic (ROC) curves for scoring coefficients of COMP of IPF vs control was with Area Under Curve (AUC) = 0.90 (95% CI), sensitivity 85% and specificity 91% and IPF or COPD was AUC= 0.88 (95% CI), sensitivity 86 % and specificity 90%. Second, we measured 24 biomarkers in IPF plasma using Searchlight Protein Array. In cross-sectional study, intercellular adhesion molecule 1 (ICAM), vascular cell adhesion molecule 1 (VCAM1), and S100 calcium binding protein A12 (S100A12), and surfactant protein D (SFTPD) were predictors of IPF mortality (P < 0.05). In serial studies, previously identified proteins ICAM1, VCAM1, S100A12, interleukin 8 (IL8) and novel proteins matrix metallopeptidase 10 (MMP10) and tissue inhibitor of metalloproteinase 1 (TIMP1) were predictors of IPF mortality. Third, we discovered in lung fibroblasts from IPF a deficiency of E2F transcription factor 8 (E2F8), which is eliminated by an F-box protein 16 (FBXO16). Biologically, E2F8 reduces fibroblast proliferation, and E2F8 also decreases collagen 1A1 mRNA synthesis. Hence, we found a model of lung fibrosis focusing on two novel molecular inputs (E2F8 and FBXO16) linked to fibroblast behavior in subjects with IPF. In summary, our transcriptomic and plasma proteins analyses revealed the utility of biomarker discovery in advancing precision medicine in IPF. Together these finding provide novel insights that may improve IPF diagnosis, prognostic and potential drug discovery

Cartilage oligomeric matrix protein in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and life threatening disease with median survival of 2.5-3 years. The IPF lung is characterized by abnormal lung remodeling, epithelial cell hyperplasia, myofibroblast foci formation, and extracellular matrix deposition. Analysis of gene expression microarray data revealed that cartilage oligomeric matrix protein (COMP), a non-collagenous extracellular matrix protein is among the most significantly up-regulated genes (Fold change 13, p-value <0.05) in IPF lungs. This finding was confirmed at the mRNA level by nCounter® expression analysis in additional 115 IPF lungs and 154 control lungs as well as at the protein level by western blot analysis. Immunohistochemical analysis revealed that COMP was expressed in dense fibrotic regions of IPF lungs and co-localized with vimentin and around pSMAD3 expressing cells. Stimulation of normal human lung fibroblasts with TGF-β1 induced an increase in COMP mRNA and protein expression. Silencing COMP in normal human lung fibroblasts significantly inhibited cell proliferation and negatively impacted the effects of TGF-β1 on COL1A1 and PAI1. COMP protein concentration measured by ELISA assay was significantly increased in serum of IPF patients compared to controls. Analysis of serum COMP concentrations in 23 patients who had prospective blood draws revealed that COMP levels increased in a time dependent fashion and correlated with declines in force vital capacity (FVC). Taken together, our results should encourage more research into the potential use of COMP as a biomarker for disease activity and TGF-β1 activity in patients with IPF. Hence, studies that explore modalities that affect COMP expression, alleviate extracellular matrix rigidity and lung restriction in IPF and interfere with the amplification of TGF-β1 signaling should be persuaded. © 2013 Vuga et al

VCAM-1 is a TGF-β1 inducible gene upregulated in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic lethal interstitial lung disease of unknown etiology. We previously reported that high plasma levels of vascular cell adhesion molecule 1 (VCAM-1) predict mortality in IPF subjects. Here we investigated the cellular origin and potential role of VCAM-1 in regulating primary lung fibroblast behavior. VCAM-1 mRNA was significantly increased in lungs of subjects with IPF compared to lungs from control subjects (p=0.001), and it negatively correlated with two markers of lung function, forced vital capacity (FVC) and pulmonary diffusion capacity for carbon monoxide (DLCO). VCAM-1 protein levels were highly expressed in IPF subjects where it was detected in fibrotic foci and blood vessels of IPF lung. Treatment of human lung fibroblasts with TGF-β1 significantly increased steady-state VCAM1 mRNA and protein levels without affecting VCAM1 mRNA stability. Further, cellular depletion of VCAM-1 inhibited fibroblast cell proliferation and reduced G2/M and S phases of the cell cycle suggestive of cell cycle arrest. These effects on cell cycle progression triggered by VCAM1 depletion were associated with reductions in levels of phosphorylated extracellular regulated kinase 1/2 and cyclin D1. Thus, these observations suggest that VCAM-1 is a TGF-β1 responsive mediator that partakes in fibroblast proliferation in subjects with IPF

Functional Genomics of Chlorine-induced Acute Lung Injury in Mice

Acute lung injury can be induced indirectly (e.g., sepsis) or directly (e.g., chlorine inhalation). Because treatment is still limited to supportive measures, mortality remains high (∼74,500 deaths/yr). In the past, accidental (railroad derailments) and intentional (Iraq terrorism) chlorine exposures have led to deaths and hospitalizations from acute lung injury. To better understand the molecular events controlling chlorine-induced acute lung injury, we have developed a functional genomics approach using inbred mice strains. Various mouse strains were exposed to chlorine (45 ppm × 24 h) and survival was monitored. The most divergent strains varied by more than threefold in mean survival time, supporting the likelihood of an underlying genetic basis of susceptibility. These divergent strains are excellent models for additional genetic analysis to identify critical candidate genes controlling chlorine-induced acute lung injury. Gene-targeted mice then could be used to test the functional significance of susceptibility candidate genes, which could be valuable in revealing novel insights into the biology of acute lung injury

COMP induction by TGF β1 is dose dependent and Hypoxia induces COMP.

<p>A) COMP mRNA was determined by qRT-PCR in NHLF treated with 2, 5, and 10 ng/ml TGF β1. B) PAI induction by 2, 5, and 10 ng/ml TGF β1. C) Exposure of cells to extreme hypoxia (1% O₂) for 24 hours causes induction of COMP mRNA (as measured by qRT-PCR), but not accompanied by a similar increase in PAI1 (D).</p

COMP modulates TGF-β signaling.

<p>NHLF were transfected with 70(SCR), treated for 6 hours with TGF-β1 (5 ng/mL) and RNA extracted after 24 hours. (A–B) qRT-PCR was used to determine mRNA levels of PAI1 and COL1A1 in NHLF. (C) The effect of COMP inhibition using siRNA on TGF-β1 induced NHLF proliferation.</p