CORRECTION

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung with feweffective therapeutic options. Structural remodelling of the extracellular matrix [i.e. collagen cross-linkingmediated by the lysyl oxidase (LO) family of enzymes (LOX, LOXL1-4)] might contribute to disease pathogenesis and represent a therapeutic target. This study aimed to further our understanding of the mechanisms by which LO inhibitors might improve lung fibrosis. Lung tissues from IPF and non-IPF subjects were examined for collagen structure (second harmonic generation imaging) and LO gene (microarray analysis) and protein (immunohistochemistry and western blotting) levels. Functional effects (collagen structure and tissue stiffness using atomic force microscopy) of LO inhibitors on collagen remodelling were examined in two models, collagen hydrogels and decellularized human lung matrices. LOXL1/LOXL2 gene expression and protein levels were increased in IPF versus non-IPF. Increased collagen fibril thickness in IPF versus non-IPF lung tissues correlated with increased LOXL1/LOXL2, and decreased LOX, protein expression. beta-Aminoproprionitrile (beta-APN; pan-LO inhibitor) but not Compound A (LOXL2-specific inhibitor) interfered with transforming growth factor-beta-induced collagen remodelling in both models. The beta-APN treatment group was tested further, and beta-APN was found to interfere with stiffening in the decellularized matrix model. LOXL1 activity might drive collagen remodelling in IPF lungs. The interrelationship between collagen structural remodelling and LOs is disrupted in IPF lungs. Inhibition of LO activity alleviates fibrosis by limiting fibrillar collagen cross-linking, thereby potentially impeding the formation of a pathological microenvironment in IPF

Lysyl oxidases in idiopathic pulmonary fibrosis: A key participant in collagen I matrix remodelling

Introduction: The fibrotic element in Idiopathic Pulmonary Fibrosis (IPF) is a key feature and is associated with Usual Interstitial Pneumonia (UIP) pattern. Fibrillar collagen I (COL1) has second harmonic generation (SHG) properties, with signals both in the forward (F) (organized collagen) & backward (B) (disorganized collagen) directions. Collagen fibre organisation is governed by the degree of cross-linking & catalysed by enzymes from the Lysyl Oxidase family (LOX, LOXL1-4). This study aimed to investigate COL1 structural remodelling & Lysyl Oxidase levels in IPF. Methods: Formalin-fixed parenchymal tissues from two cohorts; (1) non-diseased donors (ND) (n=8) & IPF explanted lungs (n=8), (2) diagnostic biopsies (UIP=26; non-UIP= 9) were analysed for SHG; F/B SHG signal ratio represented the proportion of organized to disorganized collagen. Tissue sections were immunostained for LOX, LOXL1 & LOXL2. Whole tissue images were captured & quantified by computerized imageanalysis. Results: Increased F/B ratio in IPF vs ND (

Visualizing lipid structure and raft domains in living cells with two-photon microscopy.

The lateral organization of cellular membranes is formed by the clustering of specific lipids, such as cholesterol and sphingolipids, into highly condensed domains (termed lipid rafts). Hence such domains are distinct from the remaining membrane by their lipid structure (liquid-ordered vs. -disordered domains). Here, we directly visualize membrane lipid structure of living cells by using two-photon microscopy. In macrophages, liquid-ordered domains are particularly enriched on membrane protrusions (filopodia), adhesion points and cell-cell contacts and cover 10-15% of the cell surface at 37 degrees C. By deconvoluting the images, we demonstrate the existence of phase separation in vivo. We compare the properties of microscopically visible domains (<1 microm2), with those of isolated detergent-resistant membranes and provide evidence that membrane coverage by lipid rafts and their fluidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis

Atomized human amniotic mesenchymal stromal cells for direct delivery to the airway for treatment of lung injury

Background: Current treatment regimens for inhalation injury are mainly supportive and rely on self-regeneration processes for recovery. Cell therapy with mesenchymal stromal cells (MSCs) is increasingly being investigated for the treatment of inhalation injury. Human amniotic MSCs (hAMSCs) were used in this study due to their potential use in inflammatory and fibrotic conditions of the lung. This study aimed at demonstrating that hAMSCs can be atomized with high viability, for the purpose of achieving a more uniform distribution of cells throughout the lung. Another aim of this study was to set ground for future application to healthy and diseased lungs by demonstrating that hAMSCs were able to survive after being sprayed onto substrates with different stiffness. Methods: Two methods of atomization were evaluated, and the LMA MAD780 device was selected for atomizing hAMSCs for optimized delivery. To mimic the stiffness of healthy and diseased lungs, gelatin gel (10% w/v) and tissue culture plastic were used as preliminary models. Poly-l-lysine (PLL) and collagen I coatings were used as substrates on which the hAMSCs were cultured after being sprayed. Results: The feasibility of atomizing hAMSCs was demonstrated with high cell viability (81 +/- 3.1% and 79 +/- 11.6% for cells sprayed onto plastic and gelatin, respectively, compared with 85 +/- 4.8% for control/non-sprayed cells) that was unaffected by the different stiffness of substrates. The presence of the collagen I coating on which the sprayed cells were cultured yielded higher cell proliferation compared with both PLL and no coating. The morphology of sprayed cells was minimally compromised in the presence of the collagen I coating. Conclusions: This study demonstrated that hAMSCs are able to survive after being sprayed onto substrates with different stiffness, especially in the presence of collagen I. Further studies may advance the effectiveness of cell therapy for lung regeneration