BackgroundMyofibroblasts play a crucial role in tissue repair. The functional similarities and differences between myofibroblasts and fibroblasts are not fully understood because they have not been separately isolated from a living body. The purpose of this study was to establish a method for the direct isolation of myofibroblasts and fibroblasts from injured lungs by using fluorescence-activated cell sorting and to compare their functions.ResultsWe demonstrated that lineage-specific cell surface markers (lin), such as CD31, CD45, CD146, EpCAM (CD326), TER119, and Lyve-1 were not expressed in myofibroblasts or fibroblasts. Fibroblasts of bleomycin-injured lungs and saline-treated lungs were shown to be enriched in linneg Sca-1high, and myofibroblasts of bleomycin-injured lungs were shown to be enriched in linneg Sca-1low CD49ehigh. Results from in-vitro proliferation assays indicated in-vitro proliferation of fibroblasts but not myofibroblasts of bleomycin-injured lungs and of fibroblasts of saline-treated lungs. However, fibroblasts and myofibroblasts might have a low proliferative capacity in vivo. Analysis of genes for collagen and collagen synthesis enzymes by qRT-PCR showed that the expression levels of about half of the genes were significantly higher in fibroblasts and myofibroblasts of bleomycin-injured lungs than in fibroblasts of saline-treated lungs. By contrast, the expression levels of 8 of 11 chemokine genes of myofibroblasts were significantly lower than those of fibroblasts.ConclusionsThis is the first study showing a direct isolation method of myofibroblasts and fibroblasts from injured lungs. We demonstrated functional similarities and differences between myofibroblasts and fibroblasts in terms of both their proliferative capacity and the expression levels of genes for collagen, collagen synthesis enzymes, and chemokines. Thus, this direct isolation method has great potential for obtaining useful information from myofibroblasts and fibroblasts.

Two new polybrominated diphenyl ethers (1 and 2) were isolated by bioassay-guided separations together with nine known compounds (3-11) from the marine sponge Phyllospongia dendyi collected from Palau. The structures were assigned on the basis of their spectral data. Compounds 3, 7, and 10 showed inhibitory activities to the assembly of microtubule proteins (IC(50): 29.6, 33.5, and 20.9 microM, respectively) and to the meiotic maturation of starfish oocytes (IC(50): 3.6, 4.2, and 4.2 microM, respectively), while 1, 2, 4-6, 8, 9, and 11 were not active at 100 microM. Two phenolic hydroxyls are required for their bioactivities.

Although differentiation of lung fibroblasts into α-smooth muscle actin (αSMA)-positive myofibroblasts is important in the progression of idiopathic pulmonary fibrosis (IPF), few biomarkers reflecting the fibrotic process have been discovered. We performed microarray analyses between FACS-sorted steady-state fibroblasts (lineage (CD45, TER-119, CD324, CD31, LYVE-1, and CD146)-negative and PDGFRα-positive cells) from untreated mouse lungs and myofibroblasts (lineage-negative, Sca-1-negative, and CD49e-positive cells) from bleomycin-treated mouse lungs. Amongst several genes up-regulated in the FACS-sorted myofibroblasts, we focussed on , the gene encoding latent transforming growth factor-β (TGF-β) binding protein-2 (LTBP2), because of the signal similarity to, which encodes αSMA, in the clustering analysis. The up-regulation was reproduced at the mRNA and protein levels in human lung myofibroblasts induced by TGF-β1. LTBP2 staining in IPF lungs was broadly positive in the fibrotic interstitium, mainly as an extracellular matrix (ECM) protein; however, some of the αSMA-positive myofibroblasts were also stained. Serum LTBP2 concentrations, evaluated using ELISA, in IPF patients were significantly higher than those in healthy volunteers (mean: 21.4 compared with 12.4 ng/ml) and showed a negative correlation with % predicted forced vital capacity (r = -0.369). The Cox hazard model demonstrated that serum LTBP2 could predict the prognosis of IPF patients (hazard ratio for death by respiratory events: 1.040, 95% confidence interval: 1.026-1.054), which was validated using the bootstrap method with 1000-fold replication. LTBP2 is a potential prognostic blood biomarker that may reflect the level of differentiation of lung fibroblasts into myofibroblasts in IPF.