Fibrotic Myofibroblasts Manifest Genome-Wide Derangements of Translational Control

Background: As a group, fibroproliferative disorders of the lung, liver, kidney, heart, vasculature and integument are common, progressive and refractory to therapy. They can emerge following toxic insults, but are frequently idiopathic. Their enigmatic propensity to resist therapy and progress to organ failure has focused attention on the myofibroblast–the primary effector of the fibroproliferative response. We have recently shown that aberrant beta 1 integrin signaling in fibrotic fibroblasts results in defective PTEN function, unrestrained Akt signaling and subsequent activation of the translation initiation machinery. How this pathological integrin signaling alters the gene expression pathway has not been elucidated. Results: Using a systems approach to study this question in a prototype fibrotic disease, Idiopathic Pulmonary Fibrosis (IPF); here we show organized changes in the gene expression pathway of primary lung myofibroblasts that persist for up to 9 sub-cultivations in vitro. When comparing IPF and control myofibroblasts in a 3-dimensional type I collagen matrix, more genes differed at the level of ribosome recruitment than at the level of transcript abundance, indicating pathological translational control as a major characteristic of IPF myofibroblasts. To determine the effect of matrix state on translational control, myofibroblasts were permitted to contract the matrix. Ribosome recruitment in control myofibroblasts was relatively stable. In contrast, IPF cells manifested large alterations in the ribosome recruitment pattern. Pathological studies suggest an epithelial origin for IPF myofibroblasts through the epithelial to mesenchymal transition (EMT). In accord wit

IPF fibroblasts are desensitized to type I collagen matrix-induced cell death by suppressing low autophagy via aberrant Akt/mTOR kinases.

Idiopathic pulmonary fibrosis (IPF) is a chronic, lethal interstitial lung disease in which the aberrant PTEN/Akt axis plays a major role in conferring a survival phenotype in response to the cell death inducing properties of type I collagen matrix. The underlying mechanism by which IPF fibroblasts become desensitized to polymerized collagen, thereby eluding collagen matrix-induced cell death has not been fully elucidated. We hypothesized that the pathologically altered PTEN/Akt axis suppresses autophagy via high mTOR kinase activity, which subsequently desensitizes IPF fibroblasts to collagen matrix induced cell death. We found that the autophagosome marker LC3-2 expression is suppressed, while mTOR activity remains high when IPF fibroblasts are cultured on collagen. However, LC3-2 expression increased in response to IPF fibroblast attachment to collagen in the presence of rapamycin. In addition, PTEN over-expression or Akt inhibition suppressed mTOR activity, thereby increasing LC3-2 expression in IPF fibroblasts. Furthermore, the treatment of IPF fibroblasts over-expressing PTEN or dominant negative Akt with autophagy inhibitors increased IPF fibroblast cell death. Enhanced p-mTOR expression along with low LC3-2 expression was also found in myofibroblasts within the fibroblastic foci from IPF patients. Our data show that the aberrant PTEN/Akt/mTOR axis desensitizes IPF fibroblasts from polymerized collagen driven stress by suppressing autophagic activity, which produces a viable IPF fibroblast phenotype on collagen. This suggests that the aberrantly regulated autophagic pathway may play an important role in maintaining a pathological IPF fibroblast phenotype in response to collagen rich environment

Advanced Therapeutic Strategies for Chronic Lung Disease Using Nanoparticle-Based Drug Delivery

Chronic lung diseases include a variety of obstinate and fatal diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), idiopathic pulmonary fibrosis (IPF), and lung cancers. Pharmacotherapy is important for the treatment of chronic lung diseases, and current progress in nanoparticles offers great potential as an advanced strategy for drug delivery. Based on their biophysical properties, nanoparticles have shown improved pharmacokinetics of therapeutics and controlled drug delivery, gaining great attention. Herein, we will review the nanoparticle-based drug delivery system for the treatment of chronic lung diseases. Various types of nanoparticles will be introduced, and recent innovative efforts to utilize the nanoparticles as novel drug carriers for the effective treatment of chronic lung diseases will also be discussed

IPF fibroblasts are desensitized to collagen rich matrix induced cell death in the presence of 3MA or CQ.

<p>A) 3×10⁴ control or IPF fibroblasts were cultured on polymerized collagen for 24 h in serum free medium in the presence of various doses of 3-methyladenine (3MA) or chloroquine (CQ), and cell viability was measured as described in the Materials and Methods. Shown is the % decrease in control and IPF fibroblasts in the presence of autophagic inhibitors on polymerized collagen (the viable IPF and control fibroblasts on collagen matrix in the absence of inhibitors (0 µM) are considered as 100% viable cells). B) 3×10⁴ control or IPF fibroblasts were cultured on 96 well tissue culture plates in the absence of polymerized collagen in serum free medium, and viable cells were measured in the presence of various doses of 3-methyl adenine (3MA, left panel) or chloroquine (CQ, right panel) for 24 h. Shown is the % decrease in viability in control and IPF fibroblasts in the presence of 3MA or CQ. All assays were performed in triplicate. C) 3×10⁴ control or IPF fibroblasts were cultured on polymerized collagen in serum free medium, and fibroblast proliferation was measured in the presence of 3MA (left panel) or CQ (right panel) for 24 h as described in the Materials and Methods. Shown is the % decrease in proliferation in control and IPF fibroblasts in the presence of 3MA or CQ. All assays were performed in three separate experiments to measure fibroblast viability or their proliferation using IPF cells in lane 5 and control fibroblasts in lane 3 in an upper panel in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094616#pone-0094616-g001" target="_blank">Fig 1A</a>.</p

Proposed model for the desensitization to polymerized collagen induced cell death in IPF fibroblasts.

<p>We previously found that when control fibroblasts are attached to polymerized collagen, cell death is increased <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094616#pone.0094616-Nho2" target="_blank">[44]</a>. In contrast, IPF fibroblasts are resistant to collagen matrix induced cell death. In this study, we further elucidate the underlying mechanism that bestows a highly viable IPF fibroblast phenotype in response to polymerized collagen. When IPF fibroblasts are allowed to interact with collagen matrix, Akt activity is inappropriately high due to PTEN suppression, which in turn increases mTOR kinase activity. Autophagy in IPF fibroblasts is then subsequently suppressed, and this pathological event desensitizes IPF fibroblasts to polymerized collagen induced cell death. Upwards arrow indicates enhanced activity. Downwards arrow indicates reduced activity.</p

LC3-2 expression was up-regulated in IPF fibroblasts in the presence of rapamycin on collagen.

<p>A) Left, 2×10⁵ serum starved 3 IPF and 3 control fibroblasts were attached to polymerized collagen as a function of time and LC3-2 and GAPDH was measured. Shown is a representative Western analysis IPF and control cells grown on polymerized collagen at 24 and 48 h. IPF and control fibroblasts were randomly selected for this assay. Right, shown is LC3-2 expression in 3 control and 3 IPF fibroblasts normalized to GAPDH. <i>p</i><0.03 versus control at 48 h. B) Left, equal number of control and IPF fibroblasts were cultured on polymerized collagen in serum free medium in the presence of various doses of rapamycin as indicated, and LC3-2 expression was measured at 24 h. GAPDH was used as a loading control. Shown is a representative Western analysis for LC3-2 expression. Right, shown is the LC3-2/GAPDH protein expression ratio in 4 control and 4 IPF fibroblasts in the presence of various doses of rapamycin. <i>p</i><0.03 versus DMSO, <i>p</i><0.05 versus DMSO, <i>p</i><0.01 versus control fibroblasts as indicated. Note that LC3-2 expression was highly up-regulated when IPF fibroblasts were cultured in the presence of rapamycin while LC3-2 expression was not significantly altered when control fibroblasts were treated with various doses of rapamycin. These control and IPF fibroblasts were randomly selected. C) Left, LC3-2 (red) expression was measured in randomly selected IPF and control fibroblasts cultured on cover slips coated with polymerized collagen in the presence of 100 nM of rapamycin using immunofluorescent microscopic analysis. Note that LC3-2 expression was high when rapamycin was used in IPF fibroblasts. DAPI (blue) was used as a nuclear counterstain. Rabbit IgG was used as a control and images were taken by an immunofluorescent microscope. Data are representative of 2 independent experiments. Original magnification, 63X, rabbit IgG isotype 60X. DMSO: DMSO control. Right, the quantification of fluorescence of autophagy in IPF and control fibroblasts on collagen. The fluorescence intensities of autophagy in IPF and control fibroblasts were quantified as described in the Materials and Methods. Shown is the relative fluorescence intensities of LC3-2 in two randomly selected IPF and control fibroblasts in the presence of DMSO or rapamycin. Y axis scale is presented in arbitrary units (a.u). <i>p</i><0.01 versus control, <i>p</i><0.02 versus DMSO as indicated.</p

mTOR activity is high while LC3-2 is low in cells within the fibroblastic foci of patients with IPF.

<p>IHC was performed with lung tissues from IPF patients or from histologically normal lungs (both n = 2) using p-mTOR and LC3-2 antibodies. These specimens were counterstained with hematoxylin. Shown is p-mTOR and LC3-2 expression in cells within the fibroblastic foci of IPF patients (left panels) or in normal lung alveoli tissue specimens (right panels). Lower panel, N/C is a negative control without IHC specific primary antibody. Arrows indicate p-mTOR positive cells. Note that p-mTOR level is high while LC3-2 expression is low in cells within the fibroblastic foci from IPF patients.</p

LC3-2 expression is low in IPF fibroblast attachment to polymerized collagen.

<p>A) 2×10⁵ serum starved control (n = 11) and IPF fibroblasts (n = 10) were attached to type I polymerized collagen (2 mg/ml) in serum free medium and LC3-2 and GAPDH expression was measured at 24 h. B) Shown is a box-and-whisker plot that LC3-2 is normalized to GAPDH in control and IPF fibroblasts as described in the Materials and Methods. <i>p</i><0.03 IPF versus control fibroblasts. All control and IPF fibroblasts were randomly selected for this assay.</p

Suppression of autophagic activity in IPF fibroblasts over-expressing PTEN or dominant negative Akt increases IPF fibroblast cell death on collagen.

<p>A) 3×10⁴ IPF fibroblasts infected with adenovirus expressing wild type PTEN (WP), dominant negative Akt (DA) or empty vector (GFP) were cultured on polymerized collagen in the presence of 100 µM of 3MA or DMSO in serum free medium and viable cells were measured at 24 h. <i>p</i><0.01 versus GFP, <i>p</i><0.02 versus GFP. B) IPF fibroblasts infected with adenovirus expressing wild type PTEN (WP), dominant negative Akt (DA) or empty vector (GFP) were cultured on polymerized collagen in the presence of 100 µM of CQ or water in serum free medium and viable cells were measured at 24 h. <i>p</i><0.01 versus GFP, <i>p</i><0.02 versus GFP. C) 3x10⁴ control or IPF fibroblasts grown on 96 well plates coated with polymerized collagen were treated with 100 µM of 3 methyl adenine (MA) and chloroquine (CQ) together, and viable cells were measured at 24 h in serum free medium. Shown is the % change in viable control or IPF fibroblasts treated with 3MA and CQ on collagen matrix. <i>p</i><0.01 versus control fibroblasts. D) IPF fibroblasts infected with adenovirus expressing wild type PTEN (WP), dominant negative Akt (DA) or empty vector (GFP) cultured on polymerized collagen matrix were treated with both 100 µM of 3MA and CQ together (3MA+CQ), and viable cells were measured at 24 h. <i>p</i><0.01 versus GFP, <i>p</i><0.03 versus GFP, <i>p</i><0.03 versus DMSO/water as indicated. All assays were performed in triplicate using IPF cells shown in lane 5 or control fibroblasts in lane 3 in an upper panel in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094616#pone-0094616-g001" target="_blank">Fig 1A</a>.</p

mTOR kinase activity remains high when IPF fibroblasts are cultured on collagen matrix.

<p>A) Left, 2×10⁵ serum starved control lung (n = 11) and IPF fibroblasts (n = 10) were cultured on polymerized collagen in serum free medium for 24 h and ser 2448 p-mTOR (p-mTOR) levels were measured by Western analysis. Shown is a Western analysis for p-mTOR levels in all IPF and control fibroblasts grown in serum free medium on polymerized collagen. GAPDH was used as a loading control. Right, Shown is a box-and-whisker plot of p-mTOR protein expression in 11 control and 10 IPF fibroblasts normalized to GAPDH on collagen matrix. <i>p</i><0.001 IPF versus control fibroblasts. B) Upper, randomly selected 2×10⁵ of 3 control or of 3 IPF fibroblasts were cultured on polymerized collagen in serum free medium as a function of time and ser 2448 p-mTOR levels were measured. GAPDH was used as a loading control. Lower, shown is the p-mTOR/GAPDH protein expression ratio in randomly selected control and IPF fibroblasts as a function of time. <i>p</i><0.01 IPF versus control fibroblasts at 24 h. C) Upper, control and IPF fibroblasts (n = 5 each) were cultured in the presence of various doses of rapamycin on collagen gel for 24 h and p-mTOR level was then measured. Shown is the representative p-mTOR protein level in control and IPF fibroblasts on polymerized collagen. GAPDH level was used as a loading control. Lower, p-mTOR/GAPDH expression ratio in control (n = 5) and IPF fibroblasts (n = 5) in the presence of various doses of rapamycin on collagen are shown. <i>p</i> = 0.01 versus DMSO control. <i>p</i><0.01 versus DMSO control as indicated. Cells were randomly selected for this assay. D) IPF fibroblasts infected with adenovirus expressing dominant negative Akt (DA) or empty vector GFP were cultured on collagen gel for 24 h in serum free medium and p-mTOR, Akt, phosphorylated Akt and GAPDH levels were measured. IPF fibroblasts expressing enhanced p-mTOR level was selected to examine whether Akt inhibition suppresses p-mTOR level (lane 5 in upper panel of IPF in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094616#pone-0094616-g002" target="_blank">Fig 2A</a>). This assay was repeated twice. E) Control fibroblasts infected with adenovirus expressing hyperactive Akt (HA) or empty vector (GFP) were cultured on collagen for 24 h in serum free medium and p-mTOR, total Akt, phosphorylated Akt and GAPDH levels were measured. Control fibroblasts expressing low p-mTOR level were selected to examine whether hyperactive Ak increases mTOR kinase activity (lane 3 in upper panel of control fibroblasts in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094616#pone-0094616-g002" target="_blank">Fig 2A</a>). This assay was repeated twice.</p