Lung Matrix Research Articles

Correlation Between MMP-7 and bFGF Expressions in Non-small Cell Lung Cancer Tissue and Clinicopathologic Features.

The present study intends to investigate the correlation between clinicopathologic features of non-small cell lung cancer and matrix metalloproteinase-7 (MMP-7) and basic fibroblast growth factor (bFGF) and to investigate the roles of MMP-7 and bFGF in detecting the course of disease of non-small cell lung cancer. Ninety cases of paraffin-embedded tissue samples from patient with primary non-small cell lung cancer and fifty cases of lung tissue samples from normal subjects were included in the present study. Immunohistochemical S-P method was used to detect proteins MMP-7 and bFGF. (1) The positive rate of MMP-7 protein was 14% in normal lung tissue section and 68.89% in non-small cell lung cancer tissue section, and the difference was statistically significant (χ (2)=38.774, P=0.000<0.05). There were 43 cases (43/56) with positive expression in patients with squamous cell carcinoma and 22 cases (22/34) with positive expression in patients with adenocarcinoma, and the difference was not statistically significant (χ (2)=1.539, P=0.215>0.05). There were 14 cases (14/51) with positive expression in patients with moderate- and well-differentiated lung carcinoma and 36 cases (36/39) with positive expression in patients with poor-differentiated lung carcinoma, and the difference was statistically significant (χ (2)=35.068, P=0.000<0.05). There were 37 cases (37/42) with positive expression in patients with lymphatic metastasis and 26 cases (26/48) with positive expression in patients without lymphatic metastasis, and the difference was statistically significant (χ (2) =12.279, P=0.000<0.05). (2) The mean intratumoral microvessel density (iMVD) was 46.2±6.77 in the field of lung cancer tissue at high magnification under MMP-7-positive condition and 30.8±7.54 in the field of lung cancer tissue at high magnification under MMP-7-negative condition, and the difference was statistically significant (t=9.641, P=0.000<0.05). (3) The positive rate of bFGF was 12% in normal tissue section and 63.3% in non-small cell lung cancer tissue section, and the difference was statistically significant (χ (2)=34.222, P=0.000<0.05). There were 41 cases (41/56) with positive expression in patients with squamous cell carcinoma and 20 cases (20/34) with positive expression in patients with adenocarcinoma, and the difference was not statistically significant (χ(2)=2.006, P=0.157>0.05). There were 29 cases (29/51) with positive expression in patients with moderate- and well-differentiated lung carcinoma and 35 cases (35/39) with positive expression in patients with poor-differentiated lung carcinoma, and the difference was statistically significant (χ (2)=10.085, P=0.001<0.05). There were 37 cases (37/42) with positive expression in patients with lymphatic metastasis and 25 cases (25/48) with positive expression in patients without lymphatic metastasis, and the difference was statistically significant (χ (2) =13.554, P=0.001<0.05). (4) The (iMVD) was 45.8±7.16 in the field at high magnification under bFGF-positive condition and 31.2±6.46 in the field at high magnification under bFGF-negative condition, and the difference was statistically significant (t=9.654, P=0.001<0.05). (5) A correlation was demonstrated between MMP-7 and bFGF in non-small cell lung cancer (r=0.353, P=0.000<0.05). Both MMP-7 and bFGF are participated in the progression of non-small cell lung cancer and exert a synergistic effect during physiological processes including pathogenesis, invasion, and metastasis of non-small cell lung cancer. Therefore, a combined detection of MMP-7 and bFGF for non-small cell lung cancer contributes to predict the progression and prognosis of non-small cell lung cancer, with significant clinical value.

Regional lung tissue changes with mechanical ventilation and fluid load

ABSTRACTPurpose: To investigate the regional gravity-dependent impact of mechanical ventilation and fluid overload on lung extracellular matrix (ECM) in healthy lungs. Materials and Methods: The glycosaminoglycans (GAGs) composition of the ventral and dorsal lung parenchyma was determined in anesthetized supine healthy rats mechanically ventilated for 4 hours in air: (a) at low (∼7.5 mL/kg) or high (∼ 23 mL /kg) tidal volume (VT) and 0 cmH2O positive end-expiratory pressure (PEEP); (b) at low or high VT at 5 cmH2O PEEP and (c) with or without 7 mL /(kg·h) intravenous saline infusion. Results: Mechanical ventilation degraded lung ECM, with alveolar septa thinning and structural GAGs disorganization. Low VT ventilation was associated with significant tissue structure changes in both ventral and dorsal lung regions, while high VT mainly affected the dependent ones. PEEP decreased ECM injury mainly in the ventral lung regions, although it did not prevent matrix fragmentation and washout at high VT. Intravascular fluid load increased lung damage prevalently in the ventral lung regions. Conclusion: Mechanical ventilation and fluid load may cause additive injuries in healthy lungs, mainly in ventral regions.

Extended Biomimetic Culture and Functional Assessment of Recellularized Human Lungs

RationaleNative lung scaffolds prepared by decellularization provide a novel platform for organ engineering. Recellularization with specific cell types requires ex vivo culture for evaluation and restoration of organ function prior to transplantation.MethodsHuman lung‐derived epithelial and endothelial cells were isolated and expanded in vitro. Immunostaining, flow cytometery, and qPCR confirmed cell identity. Epithelial and endothelial cells were delivered to lung scaffold airways and vasculature respectively (&gt;350x106).Constructs were cultured for 10 days in a custom automated, closed‐circuit bioreactor, providing physiologic perfusion and negative pressure ventilation. Perfusate was analyzed daily by iSTAT. Tissue was analyzed by histology and qPCR.ResultsDonor‐derived epithelium was a population of primarily basal‐cell phenotype (70% p63+), with 20% CC10+, and minor pneumocytes (&lt;10% SP‐B/C) by flow cytometry. Basal cell ciliogenesis was confirmed by ALI. Endothelium was sorted to CD31 purity.Biocompatibility to lung matrix confirmed maintenance of cell phenotype, viability, and proliferation (Ki67+) in slice co‐culture.Recellularized constructs were cultured for 6 days with constant perfusion, plus 4 days with additional fluid or air ventilation. Increasing rate of glucose metabolism and lactate production in the perfusate was quantified over time. Measured pO2, pCO2, and pH remained consistent.Cell distribution and viability was confirmed by Resazurin metabolism, and by histology/qPCR of serial tissue biopsies.ConclusionIntact acellular lung scaffolds can be repopulated with primary tissue‐derived cells and maintained in extended ex‐vivo biomimetic culture, supporting cell viability and function.image

Advancing host-directed therapy for tuberculosis.

Improved treatments are needed for nearly all forms of Mycobacterium tuberculosis infection. Adjunctive host-directed therapies have the potential to shorten tuberculosis treatment duration, prevent resistance and reduce lung injury by promoting autophagy, antimicrobial peptide production and other macrophage effector mechanisms, as well as by modifying specific mechanisms that cause lung inflammation and matrix destruction. The range of candidates is broad, including several agents approved for other clinical indications that are ready for evaluation in Phase II clinical trials. The promise of new and existing host-directed therapies that could accelerate response and improve tuberculosis treatment outcomes is discussed in this Opinion article.

Ex Vivo Four-Dimensional Lung Cancer Model Mimics Metastasis

We have developed a four-dimensional (4D) lung cancer model that forms perfusable tumor nodules. We determined if the model could be modified to mimic metastasis. We modified the 4D lung cancer model by seeding H1299, A549, or H460 cells through the trachea only to the left lobes of the acellular lung matrix. The model was modified so that the tumor cells can reach the right lobes of the acellular lung matrix only through the pulmonary artery as circulating tumor cells (CTC). We determined the gene expressions of the primary tumor, CTCs, and metastatic lesions using the Human OneArray chip. All cell lines formed a primary tumor in the left lobe of the exvivo 4D lung cancer model. The CTCs were identified in the media and increased over time. All cell lines formed metastatic lesions with H460 forming significantly more metastatic lesions than H1299 and A549 cells. The CTC gene signature predicted poor survival in lung cancer patients. Unique genes were significantly expressed in CTC compared with the primary tumor and metastatic lesion. The 4D lung cancer model can isolate tumor cells in 3 phases of tumor progression. This 4D lung cancer model may mimic the biology of lung cancer metastasis and may be used to determine its mechanism and potential therapy in the future.

Acellular Lung Scaffolds Direct Differentiation of Endoderm to Functional Airway Epithelial Cells: Requirement of Matrix-Bound HS Proteoglycans

SummaryEfficient differentiation of pluripotent cells to proximal and distal lung epithelial cell populations remains a challenging task. The 3D extracellular matrix (ECM) scaffold is a key component that regulates the interaction of secreted factors with cells during development by often binding to and limiting their diffusion within local gradients. Here we examined the role of the lung ECM in differentiation of pluripotent cells in vitro and demonstrate the robust inductive capacity of the native lung matrix alone. Extended culture of stem cell-derived definitive endoderm on decellularized lung scaffolds in defined, serum-free medium resulted in differentiation into mature airway epithelia, complete with ciliated cells, club cells, and basal cells with morphological and functional similarities to native airways. Heparitinase I, but not chondroitinase ABC, treatment of scaffolds revealed that the differentiation achieved is dependent on heparan sulfate proteoglycans and its bound factors remaining on decellularized scaffolds.

Localization and stretch-dependence of lung elastase activity in development and compensatory growth.

Synthesis and remodeling of the lung matrix is necessary for primary and compensatory lung growth. Because cyclic negative force is applied to developing lung tissue during the respiratory cycle, we hypothesized that stretch is a critical regulator of lung matrix remodeling. By using quantitative image analysis of whole-lung and whole-lobe elastin in situ zymography images, we demonstrated that elastase activity increased twofold during the alveolar stage of postnatal lung morphogenesis in the mouse. Remodeling was restricted to alveolar walls and ducts and was nearly absent in dense elastin band structures. In the mouse pneumonectomy model of compensatory lung growth, elastase activity increased threefold, peaking at 14 days postpneumonectomy and was higher in the accessory lobe compared with other lobes. Remodeling during normal development and during compensatory lung growth was different with increased major airway and pulmonary arterial remodeling during development but not regeneration, and with homogenous remodeling throughout the parenchyma during development, but increased remodeling only in subpleural regions during compensatory lung growth. Left lung wax plombage prevented increased lung elastin during compensatory lung growth. To test whether the adult lung retains an innate capacity to remodel elastin, we developed a confocal microscope-compatible stretching device. In ex vivo adult mouse lung sections, lung elastase activity increased exponentially with strain and in peripheral regions of lung more than in central regions. Our study demonstrates that lung elastase activity is stretch-dependent and supports a model in which externally applied forces influence the composition, structure, and function of the matrix during periods of alveolar septation.

Senescent Cells Contribute to the Physiological Remodeling of Aged Lungs.

Age-associated decline in organ function governs life span. We determined the effect of aging on lung function and cellular/molecular changes of 8- to 32-month old mice. Proteomic analysis of lung matrix indicated significant compositional changes with advanced age consistent with a profibrotic environment that leads to a significant increase in dynamic compliance and airway resistance. The excess of matrix proteins deposition was associated modestly with the activation of myofibroblasts and transforming growth factor-beta signaling pathway. More importantly, detection of senescent cells in the lungs increased with age and these cells contributed toward the excess extracellular matrix deposition observed in our aged mouse model and in elderly human samples. Mechanistic target of rapamycin (mTOR)/AKT activity was enhanced in aged mouse lungs compared with those from younger mice associated with the increased expression of the histone variant protein, MH2A, a marker for aging and potentially for senescence. Introduction in the mouse diet of rapamycin, significantly blocked the mTOR activity and limited the activation of myofibroblasts but did not result in a reduction in lung collagen deposition unless it was associated with prevention of cellular senescence. Together these data indicate that cellular senescence significantly contributes to the extracellular matrix changes associated with aging in a mTOR 1-dependent mechanism.

Automated decellularization of intact, human-sized lungs for tissue engineering.

We developed an automated system that can be used to decellularize whole human-sized organs and have shown lung as an example. Lungs from 20 to 30 kg pigs were excised en bloc with the trachea and decellularized with our established protocol of deionized water, detergents, sodium chloride, and porcine pancreatic DNase. A software program was written to control a valve manifold assembly that we built for selection and timing of decellularization fluid perfusion through the airway and the vasculature. This system was interfaced with a prototypic bioreactor chamber that was connected to another program, from a commercial source, which controlled the volume and flow pressure of fluids. Lung matrix that was decellularized by the automated method was compared to a manual method previously used by us and others. Automation resulted in more consistent acellular matrix preparations as demonstrated by measuring levels of DNA, hydroxyproline (collagen), elastin, laminin, and glycosaminoglycans. It also proved highly beneficial in saving time as the decellularization procedure was reduced from days down to just 24 h. Developing a rapid, controllable, automated system for production of reproducible matrices in a closed system is a major step forward in whole-organ tissue engineering.

Pursuing novel molecular mechanisms and treatment strategies to enhance lung growth in neonatal chronic lung disease (CLD)

s of the 51st Workshop for Pediatric Research 51st Workshop for Pediatric Research Gottingen, Germany 16-17 April 2015 This supplement has not been sponsored. Meeting abstracts Rationale Infants born prematurely or with intrauterine growth restriction (IUGR) are often afflicted with CLD, a debilitating disorder marked by diminished alveoli and lung micro-vessels. Despite advances in neonatology, CLD's incidence remains high and its prevention elusive. Mechanical ventilation (MV), high inspired O2, and IUGR can disrupt key signaling pathways, reduce cell survival and disrupt matrix elements, thus inhibiting lung growth [1–6]. Aims (1) To define the impact of MV, O2 and IUGR on lung structure and function in newborn rodents. (2) identify altered signaling pathways of neonatal lung injury, and test novel treatment strategies. (3) mechanistic studies to define how such aberrant signaling may impact cell survival in culture. Methods (1) Exposure of newborn mice to prolonged MV or hyperoxia; IUGR in rats induced by maternal low protein diet. (2) Prolonged MV-O2 in pups treated with a serine elastase inhibitor Elafin. (3) Studies with mouse alveolar epithelial (AEC) and lung micro-vascular endothelial (LMVEC) cells. Results MV, O2, and IUGR inhibit alveolar and micro-vessel formation and disrupt matrix components in lung, resulting in reduced respiratory system compliance and increased airway resistance. TGFβ signaling is disrupted after MV, O2, and IUGR which adversely affects homeostasis of AEC and LMVEC, contributing to impaired lung growth with disordered lung matrix. Moreover, MV-O2 inhibited EGF-receptor activation and reduced Kruppel-like factor 4 (Klf4), a regulator of cell differentiation and survival. We identified this novel pEGFR-Klf4 axis as a key regulator of AEC survival, and discovered that pEGFR-Klf4 signaling is protected by treatment with Elafin during MV-O2. Conclusion These results identify critical pathways that can impact lung growth arrest, as seen in CLD, and uncover novel mechanisms that could promote alveolarization, thus pointing the way toward new treatments to improve the outcomes of infants with CLD.

Alpha-1 proteinase inhibitors for the treatment of alpha-1 antitrypsin deficiency: safety, tolerability, and patient outcomes.

Alpha-1 antitrypsin (AAT) deficiency remains an underrecognized genetic disease with predominantly pulmonary and hepatic manifestations. AAT is derived primarily from hepatocytes; however, macrophages and neutrophils are secondary sources. As the natural physiological inhibitor of several proteases, most importantly neutrophil elastase (NE), it plays a key role in maintaining pulmonary protease–antiprotease balance. In deficient states, unrestrained NE activity promotes damage to the lung matrix, causing structural defects and impairing host defenses. The commonest form of AAT deficiency results in a mutated Z AAT that is abnormally folded, polymerized, and aggregated in the liver. Consequently, systemic levels are lower, resulting in diminished pulmonary concentrations. Hepatic disease occurs due to liver aggregation of the protein, while lung destruction ensues from unopposed protease-mediated damage. In this review, we will discuss AAT deficiency, its clinical manifestations, and augmentation therapy. We will address the safety and tolerability profiles of AAT replacement in the context of patient outcomes and cost-effectiveness and outline future directions for work in this field.

186: Forecasting patient recruitment for time-to-event analysis in National Lung Matrix Trial

186: Forecasting patient recruitment for time-to-event analysis in National Lung Matrix Trial

187: National Lung Matrix Trial: multi-drug, genetic marker-directed, non-comparative, multi-centre, multi-arm phase II trial in non-small cell lung cancer

187: National Lung Matrix Trial: multi-drug, genetic marker-directed, non-comparative, multi-centre, multi-arm phase II trial in non-small cell lung cancer

Lung matrix and vascular remodeling in mechanically ventilated elastin haploinsufficient newborn mice.

Elastin plays a pivotal role in lung development. We therefore queried if elastin haploinsufficient newborn mice (Eln(+/-)) would exhibit abnormal lung structure and function related to modified extracellular matrix (ECM) composition. Because mechanical ventilation (MV) has been linked to dysregulated elastic fiber formation in the newborn lung, we also asked if elastin haploinsufficiency would accentuate lung growth arrest seen after prolonged MV of neonatal mice. We studied 5-day-old wild-type (Eln(+/+)) and Eln(+/-) littermates at baseline and after MV with air for 8-24 h. Lungs of unventilated Eln(+/-) mice contained ∼50% less elastin and ∼100% more collagen-1 and lysyl oxidase compared with Eln(+/+) pups. Eln(+/-) lungs contained fewer capillaries than Eln(+/+) lungs, without discernible differences in alveolar structure. In response to MV, lung tropoelastin and elastase activity increased in Eln(+/+) neonates, whereas tropoelastin decreased and elastase activity was unchanged in Eln(+/-) mice. Fibrillin-1 protein increased in lungs of both groups during MV, more in Eln(+/-) than in Eln(+/+) pups. In both groups, MV caused capillary loss, with larger and fewer alveoli compared with unventilated controls. Respiratory system elastance, which was less in unventilated Eln(+/-) compared with Eln(+/+) mice, was similar in both groups after MV. These results suggest that elastin haploinsufficiency adversely impacts pulmonary angiogenesis and that MV dysregulates elastic fiber integrity, with further loss of lung capillaries, lung growth arrest, and impaired respiratory function in both Eln(+/+) and Eln(+/-) mice. Paucity of lung capillaries in Eln(+/-) newborns might help explain subsequent development of pulmonary hypertension previously reported in adult Eln(+/-) mice.

P202 Studying The Relationship Between Matrix Metalloproteinases And Lung Tissue Damage During A Clinical Exacerbation Of Cystic Fibrosis

Background Cystic Fibrosis (CF) is the commonest fatal autosomal recessive condition of Caucasians. The lung in CF is characterised by chronic bacterial colonisation, inflammation, and lung tissue destruction. Matrix Metalloproteinases (MMPs) are proteases that are expressed in inflammatory states that degrade lung matrix. Whilst MMPs are known to be raised in many inflammatory lung conditions, their role in CF is poorly understood. Aims The study aimed to compare lung function and MMP levels throughout an exacerbation. Methods Sputum samples and clinical information were obtained with written consent from 23 adult subjects with CF. Samples were collected at day 0, day 14 and week 6 of an exacerbation of CF. Samples were frozen, before Luminex bioassay and IL-8 ELISA was performed. The sputum concentration of MMP 1, 2, 3, 7, 8, 9,10,12,13 and IL-8 were measured. Results A statistically significant increase in IL-8 concentration (pg/ml) occurred between day 14 and week 6 (p = 0.0387). The concentration of MMP-9 decreased from day 0 to day 14 (p = 0.006), and then rose significantly between day 14 and week 6 (p = 0.0412). However, the concentration of MMP-9 at week 6 was significantly less than at day 0 (0.0453). A significant decrease in the concentration of MMP-8 occurred between day 0 and day 14 (p = 0.0124); and between day 0 to week 6 (p = 0.0426). Conclusions MMPs and other inflammatory markers are raised during exacerbations, and fall with treatment. High proteolytic activity may lead to a worsening lung function and contribute significantly to structural changes within the CF lung. Furthering our understanding of this diverse group of proteases could lead to potential novel therapeutic targets which could help prevent irreversible lung damage.

529 The Cancer Research UK Stratified Medicine Programme: From national screening to national trial

Background: The increasing demand for analysis of multiple predictive genetic markers in tumour samples to guide therapeutic decision-making requires the rapid validation and uptake of new knowledge and technology, as well as collaboration between clinical teams, cellular pathology and molecular genetics laboratories. Material and Methods: Between 2011 and 2013, formalin-fixed paraffinembedded tumour samples from patients consented through a network of hospitals underwent targeted mutation analysis. This was initially performed using Sanger sequencing and similar methods but following cross-validation an amplicon-based targeted next generation sequencing panel was implemented. Clinical information based on the requirements of the national cancer registration dataset was also centrally collated for each participant. The current second phase of the initiative has a sole focus on patients with advanced lung cancer, and aims to expand the geographical reach to perform molecular pre-screening of samples to help determine patient eligibility for the National Lung Matrix Trial, a multi-arm stratified phase II clinical trial of novel therapeutics. Results: 10,754 patients (98% of those approached) consented to analysis of material during phase one, with 9010 samples submitted for analysis. The table shows the overall results of analysis for the first 7850 samples. Analysis by histological subtype enriches for certain mutations, with 925 pulmonary adenocarcinomas showing 36.5% KRAS mutations, 11.6% EGFR mutations and 2.5% ALK gene rearrangements and 56 mucinous colorectal adenocarcinomas showing 43.5% BRAF mutations and 50% KRAS mutations. Conclusions: A centralised national testing network for genetic analysis of formalin-fixed paraffin-embedded tumour samples is feasible and acceptable to patients and clinicians, with applications beyond genotyping for currently licensed oncologic therapies. Acknowledgements: Funding for the Stratified Medicine Programme is acknowledged from Cancer Research UK and programme founding partners AstraZeneca and Pfizer.

Liver growth factor treatment reverses emphysema previously established in a cigarette smoke exposure mouse model.

Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease largely associated with cigarette smoke exposure (CSE) and characterized by pulmonary and extrapulmonary manifestations, including systemic inflammation. Liver growth factor (LGF) is an albumin-bilirubin complex with demonstrated antifibrotic, antioxidant, and antihypertensive actions even at extrahepatic sites. We aimed to determine whether short LGF treatment (1.7 μg/mouse ip; 2 times, 2 wk), once the lung damage was established through the chronic CSE, contributes to improvement of the regeneration of damaged lung tissue, reducing systemic inflammation. We studied AKR/J mice, divided into three groups: control (air-exposed), CSE (chronic CSE), and CSE + LGF (LGF-treated CSE mice). We assessed pulmonary function, morphometric data, and levels of various systemic inflammatory markers to test the LGF regenerative capacity in this system. Our results revealed that the lungs of the CSE animals showed pulmonary emphysema and inflammation, characterized by increased lung compliance, enlargement of alveolar airspaces, systemic inflammation (circulating leukocytes and serum TNF-α level), and in vivo lung matrix metalloproteinase activity. LGF treatment was able to reverse all these parameters, decreasing total cell count in bronchoalveolar lavage fluid and T-lymphocyte infiltration in peripheral blood observed in emphysematous mice and reversing the decrease in monocytes observed in chronic CSE mice, and tends to reduce the neutrophil population and serum TNF-α level. In conclusion, LGF treatment normalizes the physiological and morphological parameters and levels of various systemic inflammatory biomarkers in a chronic CSE AKR/J model, which may have important pathophysiological and therapeutic implications for subjects with stable COPD.

Enhanced Lung Epithelial Specification of Human Induced Pluripotent Stem Cells on Decellularized Lung Matrix

Whole-lung scaffolds can be created by perfusion decellularization of cadaveric donor lungs. The resulting matrices can then be recellularized to regenerate functional organs. This study evaluated the capacity of acellular lung scaffolds to support recellularization with lung progenitors derived from human induced pluripotent stem cells (iPSCs). Whole rat and human lungs were decellularized by constant-pressure perfusion with 0.1% sodium dodecyl sulfate solution. Resulting lung scaffolds were cryosectioned into slices or left intact. Human iPSCs were differentiated to definitive endoderm, anteriorized to a foregut fate, and then ventralized to a population expressing NK2 homeobox 1 (Nkx2.1). Cells were seeded onto slices and whole lungs, which were maintained under constant perfusion biomimetic culture. Lineage specification was assessed by quantitative polymerase chain reaction and immunofluorescent staining. Regenerated left lungs were transplanted in an orthotopic position. Activin-A treatment, followed by transforming growth factor-β inhibition, induced differentiation ofhuman iPSCs to anterior foregut endoderm as confirmed by forkhead box protein A2 (FOXA2), SRY (Sex Determining Region Y)-Box 17 (SOX17), and SOX2 expression. Cells cultured on decellularized lung slices demonstrated proliferation and lineage commitment after5 days. Cells expressing Nkx2.1 were identified at 40%to 60% efficiency. Within whole-lung scaffolds andunder perfusion culture, cells further upregulated Nkx2.1 expression. After orthotopic transplantation, grafts were perfused and ventilated by host vasculature and airways. Decellularized lung matrix supports the culture and lineage commitment of human iPSC-derived lung progenitor cells. Whole-organ scaffolds and biomimetic culture enable coseeding of iPSC-derived endothelial and epithelial progenitors and enhance early lung fate. Orthotopic transplantation may enable further invivo graft maturation.

Evaluation of small molecule FRET reporter for the diagnosis and monitoring of proteolytic activity in a chronic obstructive lung disease model

Proteases such as neutrophil elastase (NE) and matrix metalloprotease 12 (MMP-12) are key factors in inflammatory processes and contribute the gradual destruction of extracellular lung matrix in chronic inflammation. We now hypothesize that activity levels of inflammation-relevant proteases may be useful indicators for the onset and progression of obstructive lung diseases such as cystic fibrosis or COPD. With the recently published small molecule FRET protease reporters NEmo and LaRee, for detection of NE and MMP-12 activity, respectively, it is possible to monitor protease activity at the single cell level.

Regulation of TGF-β1-driven Differentiation of Human Lung Fibroblasts: EMERGING ROLES OF CATHEPSIN B AND CYSTATIN C

Lung matrix homeostasis partly depends on the fine regulation of proteolytic activities. We examined the expression of human cysteine cathepsins (Cats) and their relative contribution to TGF-β1-induced fibroblast differentiation into myofibroblasts. Assays were conducted using both primary fibroblasts obtained from patients with idiopathic pulmonary fibrosis and human lung CCD-19Lu fibroblasts. Pharmacological inhibition and genetic silencing of Cat B diminished α-smooth muscle actin expression, delayed fibroblast differentiation, and led to an accumulation of intracellular 50-kDa TGF-β1. Moreover, the addition of Cat B generated a 25-kDa mature form of TGF-β1 in Cat B siRNA-pretreated lysates. Inhibition of Cat B decreased Smad 2/3 phosphorylation but had no effect on p38 MAPK and JNK phosphorylation, indicating that Cat B mostly disturbs TGF-β1-driven canonical Smad signaling pathway. Although mRNA expression of cystatin C was stable, its secretion, which was inhibited by brefeldin A, increased during TGF-β1-induced differentiation of idiopathic pulmonary fibrosis and CCD-19Lu fibroblasts. In addition, cystatin C participated in the control of extracellular Cats, because its gene silencing restored their proteolytic activities. These data support the notion that Cat B participates in lung myofibrogenesis as suggested for stellate cells during liver fibrosis. Moreover, we propose that TGF-β1 promotes fibrosis by driving the effective cystatin C-dependent inhibition of extracellular matrix-degrading Cats.