Tunable biomimetic materials elaborated by ice templating and self-assembly of collagen for tubular tissue engineering.

Synthetic tubular grafts currently used in clinical context fail frequently, and the expectations that biomimetic materials could tackle these limitations are high. However, developing tubular materials presenting structural, compositional and functional properties close to those of native tissues remains an unmet challenge. Here we describe a combination of ice templating and topotactic fibrillogenesis of type I collagen, the main component of tissues' extracellular matrix, yielding highly concentrated yet porous tubular collagen materials with controlled hierarchical architecture at multiple length scales, the hallmark of native tissues' organization. By modulating the thermal conductivity of the cylindrical molds, we tune the macroscopic porosity defined by ice. Coupling the aforementioned porosity patterns with two different fibrillogenesis routes results in a new family of tubular materials whose textural features and the supramolecular arrangement of type I collagen are achieved. The resulting materials present hierarchical elastic properties and are successfully colonized by human endothelial cells and alveolar epithelial cells on the luminal side, and by human mesenchymal stem cells on the external side. The proposed straightforward protocol is likely to be adapted for larger graft sizes that address ever-growing clinical needs, such as peripheral arterial disease or tracheal and bronchial reconstructions.

Multistep synthesis of a novel copper complex with potential for Alzheimer's disease diagnosis.

Positron emission tomography (PET) imaging of Aß plaques, is recognized as a tool for the diagnosis of Alzheimer's disease. As a contribution to the development of new strategies for early diagnosis of the disease, using PET medical imaging technique, a new copper complex, the [Cu(TE1PA-ONO)]+ was synthesized in ten steps. The key step of our strategy is the coupling of a monopicolinate-N-alkylated cyclam-based ligand with a moiety capable of recognizing Aß plaques via a successful and challenging Buchwald-Hartwig coupling reaction. To our knowledge, it is the first time that such a strategy is used to functionalize polyazamacrocyclic derivatives. The thermodynamic stability constants determined in MeOH/H2O solvent indicate that the attachment of this moiety does not weaken the chelating properties of TE1PA-ONO ligand in relation to parent HTE1PA. The novel complex described here is able to recognize amyloid plaques in brain sections from Alzheimer's disease patients and shows low toxicity to human neuronal cells.

Autoantibodies are associated with disease progression in idiopathic pulmonary fibrosis.

Several reports have highlighted a potential role of autoreactive B-cells and autoantibodies that correlates with increased disease severity in patients with idiopathic pulmonary fibrosis (IPF). Here we show that patients with IPF have an altered B-cell phenotype and that those subjects who have autoantibodies against the intermediate filament protein periplakin (PPL) have a significantly worse outcome in terms of progression-free survival. Using a mouse model of lung fibrosis, we demonstrate that introducing antibodies targeting the endogenous protein PPL (mimicking naturally occurring autoantibodies seen in patients) directly in the lung increases lung injury, inflammation, collagen and fibronectin expression through direct activation of follicular dendritic cells, which in turn activates and drives proliferation of fibroblasts. This fibrocyte population was also observed in fibrotic foci of patients with IPF and was increased in peripheral blood of IPF patients compared to aged-matched controls. This study reiterates the complex and heterogeneous nature of IPF, identifying new pathways that may prove suitable for therapeutic intervention.

Iron Oxide Nanoparticles Functionalized with Fucoidan: a Potential Theranostic Nanotool for Hepatocellular Carcinoma.

Fucoidan is a natural sulfated polysaccharide with a large range of biological activities including anticancer and anti-oxidation activities. Hepatocellular carcinoma is the fourth most common aggressive cancer type. The aim of this study was to investigate the bioactivity of free fucoidan versus its vectorization using nanoparticles (NPs) in human hepatoma cells, Huh-7. Iron oxide NPs were functionalized with fucoidan by a one-step surface complexation. NP cellular uptake was quantified by magnetic measurement at various extracellular iron concentrations. Cell invasion and migration were reduced with NPs while free fucoidan increases these events at low fucoidan concentration (≤0.5 µM). Concomitantly, a high decrease of reactive oxygen species production related with a decrease of the matrix metalloproteinase-9 activity and an increase of its expression was observed with NPs compared to free fucoidan. A proteomic analysis evidenced that some fucoidan regulated proteins appeared, which were related to protein synthesis, N-glycan processing, and cellular stress. To our knowledge, this is the first study which reveals such activity induced by fucoidan. These results pave the way for USPIO-fucoidan-NPs as potential theranostic nanotools for hepatocellular carcinoma treatment.

Hypoxia Promotes a Mixed Inflammatory-Fibrotic Macrophages Phenotype in Active Sarcoidosis.

Background: Macrophages are pivotal cells in sarcoidosis. Monocytes-derived (MD) macrophages have recently been demonstrated to play a major role especially in pulmonary sarcoidosis. From inflammatory tissues to granulomas, they may be exposed to low oxygen tension environments. As hypoxia impact on sarcoidosis immune cells has never been addressed, we designed the present study to investigate MD-macrophages from sarcoidosis patients in this context. We hypothesized that hypoxia may induce functional changes on MD-macrophages which could have a potential impact on the course of sarcoidosis. Methods: We studied MD-macrophages, from high active sarcoidosis (AS) (n=26), low active or inactive sarcoidosis (IS) (n=24) and healthy controls (n=34) exposed 24 hours to normoxia (21% O2) or hypoxia (1.5% O2). Different macrophage functions were explored: hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-κB) activation, cytokines secretion, phagocytosis, CD80/CD86/HLA-DR expression, profibrotic response. Results: We observed that hypoxia, with a significantly more pronounced effect in AS compared with controls and IS, increased the HIF-1α trans-activity, promoted a proinflammatory response (TNFα, IL1ß) without activating NF-κB pathway and a profibrotic response (TGFß1, PDGF-BB) with PAI-1 secretion associated with human lung fibroblast migration inhibition. These results were confirmed by immunodetection of HIF-1α and PAI-1 in granulomas observed in pulmonary biopsies from patients with sarcoidosis. Hypoxia also decreased the expression of CD80/CD86 and HLA-DR on MD-macrophages in the three groups while it did not impair phagocytosis and the expression of CD36 expression on cells in AS and IS at variance with controls. Conclusions: Hypoxia had a significant impact on MD-macrophages from sarcoidosis patients, with the strongest effect seen in patients with high active disease. Therefore, hypoxia could play a significant role in sarcoidosis pathogenesis by increasing the macrophage proinflammatory response, maintaining phagocytosis and reducing antigen presentation, leading to a deficient T cell response. In addition, hypoxia could favor fibrosis by promoting profibrotic cytokines response and by sequestering fibroblasts in the vicinity of granulomas.

IL-33-ST2 axis regulates myeloid cell differentiation and activation enabling effective club cell regeneration.

Evidence points to an indispensable function of macrophages in tissue regeneration, yet the underlying molecular mechanisms remain elusive. Here we demonstrate a protective function for the IL-33-ST2 axis in bronchial epithelial repair, and implicate ST2 in myeloid cell differentiation. ST2 deficiency in mice leads to reduced lung myeloid cell infiltration, abnormal alternatively activated macrophage (AAM) function, and impaired epithelial repair post naphthalene-induced injury. Reconstitution of wild type (WT) AAMs to ST2-deficient mice completely restores bronchial re-epithelialization. Central to this mechanism is the direct effect of IL-33-ST2 signaling on monocyte/macrophage differentiation, self-renewal and repairing ability, as evidenced by the downregulation of key pathways regulating myeloid cell cycle, maturation and regenerative function of the epithelial niche in ST2-/- mice. Thus, the IL-33-ST2 axis controls epithelial niche regeneration by activating a large multi-cellular circuit, including monocyte differentiation into competent repairing AAMs, as well as group-2 innate lymphoid cell (ILC2)-mediated AAM activation.

Models Contribution to the Understanding of Sarcoidosis Pathogenesis: "Are There Good Models of Sarcoidosis?"

Sarcoidosis is a systemic, granulomatous, and noninfectious disease of unknown etiology. The clinical heterogeneity of the disease (targeted tissue(s), course of the disease, and therapy response) supports the idea that a multiplicity of trigger antigens may be involved. The pathogenesis of sarcoidosis is not yet completely understood, although in recent years, considerable efforts were put to develop novel experimental research models of sarcoidosis. In particular, sarcoidosis patient cells were used within in vitro 3D models to study their characteristics compared to control patients. Likewise, a series of transgenic mouse models were developed to highlight the role of particular signaling pathways in granuloma formation and persistence. The purpose of this review is to put in perspective the contributions of the most recent models in the understanding of sarcoidosis.

Intermittent Hypoxia Increases the Severity of Bleomycin-Induced Lung Injury in Mice.

BACKGROUND: Severe obstructive sleep apnea (OSA) with chronic intermittent hypoxia (IH) is common in idiopathic pulmonary fibrosis (IPF). Here, we evaluated the impact of IH on bleomycin- (BLM-) induced pulmonary fibrosis in mice. METHODS: C57BL/6J mice received intratracheal BLM or saline and were exposed to IH (40 cycles/hour; FiO2 nadir: 6%; 8 hours/day) or intermittent air (IA). In the four experimental groups, we evaluated (i) survival; (ii) alveolar inflammation, pulmonary edema, lung oxidative stress, and antioxidant enzymes; (iii) lung cell apoptosis; and (iv) pulmonary fibrosis. RESULTS: Survival at day 21 was lower in the BLM-IH group (p < 0.05). Pulmonary fibrosis was more severe at day 21 in BLM-IH mice, as assessed by lung collagen content (p = 0.02) and histology. At day 4, BLM-IH mice developed a more severe neutrophilic alveolitis, (p < 0.001). Lung oxidative stress was observed, and superoxide dismutase and glutathione peroxidase expression was decreased in BLM-IH mice (p < 0.05 versus BLM-IA group). At day 8, pulmonary edema was observed and lung cell apoptosis was increased in the BLM-IH group. CONCLUSION: These results show that exposure to chronic IH increases mortality, lung inflammation, and lung fibrosis in BLM-treated mice. This study raises the question of the worsening impact of severe OSA in IPF patients.

G908R NOD2 variant in a family with sarcoidosis.

BACKGROUND: Sarcoidosis is a systemic disease characterized by the formation of immune granulomas in various organs, mainly the lungs and the lymphatic system. Exaggerated granulomatous reaction might be triggered in response to unidentified antigens in individuals with genetic susceptibility. The present study aimed to determine the genetic variants implicated in a familial case of sarcoidosis. METHODS: Sarcoidosis presentation and history, NOD2 profile, NF-κB and cytokine production in blood monocytes/macrophages were evaluated in individuals from a family with late appearance of sarcoidosis. RESULTS: In the present study, we report a case of familial sarcoidosis with typical thoracic sarcoidosis and carrying the NOD2 2722G > C variant. This variant is associated with the presence of three additional SNPs for the IL17RA, KALRN and EPHA2 genes, which discriminate patients expressing the disease from others. Despite a decrease in NF-κB activity, IL-8 and TNF-A mRNA levels were increased at baseline and in stimulated conditions. CONCLUSIONS: Combination of polymorphisms in the NOD2, IL17RA, EPHA2 and KALRN genes could play a significant role in the development of sarcoidosis by maintaining a chronic pro-inflammatory status in macrophages.

Identification of periplakin as a major regulator of lung injury and repair in mice.

Periplakin is a component of the desmosomes that acts as a cytolinker between intermediate filament scaffolding and the desmosomal plaque. Periplakin is strongly expressed by epithelial cells in the lung and is a target antigen for autoimmunity in idiopathic pulmonary fibrosis. The aim of this study was to determine the role of periplakin during lung injury and remodeling in a mouse model of lung fibrosis induced by bleomycin. We found that periplakin expression was downregulated in the whole lung and in alveolar epithelial cells following bleomycin-induced injury. Deletion of the Ppl gene in mice improved survival and reduced lung fibrosis development after bleomycin-induced injury. Notably, Ppl deletion promoted an antiinflammatory alveolar environment linked to profound changes in type 2 alveolar epithelial cells, including overexpression of antiinflammatory cytokines, decreased expression of profibrotic mediators, and altered cell signaling with a reduced response to TGF-ß1. These results identify periplakin as a previously unidentified regulator of the response to injury in the lung.

FGF9 prevents pleural fibrosis induced by intrapleural adenovirus injection in mice.

Fibroblast growth factor 9 (FGF9) is necessary for fetal lung development and is expressed by epithelium and mesothelium. We evaluated the role of FGF9 overexpression on adenoviral-induced pleural injury in vivo and determined the biological effects of FGF9 on mesothelial cells in vitro. We assessed the expression of FGF9 and FGF receptors by mesothelial cells in both human and mouse lungs. Intrapleural injection of an adenovirus expressing human FGF9 (AdFGF9) or a control adenovirus (AdCont) was performed. Mice were euthanized at days 3, 5, and 14 Expression of FGF9 and markers of inflammation and myofibroblastic differentiation was studied by qPCR and immunohistochemistry. In vitro, rat mesothelial cells were stimulated with FGF9 (20 ng/ml), and we assessed its effect on proliferation, survival, migration, and differentiation. FGF9 was expressed by mesothelial cells in human idiopathic pulmonary fibrosis. FGF receptors, mainly FGFR3, were expressed by mesothelial cells in vivo in humans and mice. AdCont instillation induced diffuse pleural thickening appearing at day 5, maximal at day 14 The altered pleura cells strongly expressed α-smooth muscle actin and collagen. AdFGF9 injection induced maximal FGF9 expression at day 5 that lasted until day 14 FGF9 overexpression prevented pleural thickening, collagen and fibronectin accumulation, and myofibroblastic differentiation of mesothelial cells. In vitro, FGF9 decreased mesothelial cell migration and inhibited the differentiating effect of transforming growth factor-ß1. We conclude that FGF9 has a potential antifibrotic effect on mesothelial cells.

Experimental models of sarcoidosis.

PURPOSE OF REVIEW: Sarcoidosis is a disease caused by a complex combination of genetic susceptibility, immune networks and infectious and/or environmental agents. The onset and phenotypic variability of sarcoidosis remain poorly elucidated, not only due to the lack of clearly identified causes, but also because it is widely considered that no reliable model of this disease is available. In this review, we discuss the various models of granulomatous diseases in order to challenge this assertion. RECENT FINDINGS: A large number of models of granulomatous diseases are available, both cellular models used to study the natural history of granulomas and experimental animal models mostly developed in rodents. SUMMARY: Although none of the available models fully reproduces sarcoidosis, most of them generate various data supporting key concepts. Selected models with a high level of confidence among those already published may provide various pieces of the sarcoidosis jigsaw puzzle, whereas clinical data can provide other elements. A 'systems biology' approach for modelling may be a way of piecing together the various pieces of the puzzle. Finally, experimental models and a systemic approach should be considered to be tools for preclinical evaluation of the efficacy of drugs prior to testing in clinical trials.

Serum Amyloid P Contained in Alveolar Fluid From Patients With Acute Respiratory Distress Syndrome Mediates the Inhibition of Monocyte Differentiation into Fibrocyte.

OBJECTIVE: Alveolar fibrocytes are monocyte-derived mesenchymal cells associated with poor prognosis in patients with acute respiratory distress syndrome. Our aims were to determine the following: 1) the ability of monocytes from acute respiratory distress syndrome patients to differentiate into fibrocytes; 2) the influence of the acute respiratory distress syndrome alveolar environment on fibrocyte differentiation; and 3) mediators involved in this modulation, focusing on serum amyloid P. DESIGN: Experimental in vitro investigation. SETTING: Two ICUs of a teaching hospital. PATIENTS: Twenty-five patients (19 mild-to-severe acute respiratory distress syndrome and six matched ventilated controls without acute respiratory distress syndrome) were enrolled. Six healthy volunteers served as non-ventilated controls. INTERVENTIONS: Peripheral blood mononuclear cells were isolated from acute respiratory distress syndrome, ventilated controls, and non-ventilated controls blood and cultured in vitro. Fibrocytes were counted at basal condition and after culture with broncho-alveolar lavage fluid. Plasma and broncho-alveolar lavage fluid serum amyloid P contents were determined by western blot and enzyme-linked immunosorbent assay. Serum amyloid P was located in normal and acute respiratory distress syndrome lung by immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: Acute respiratory distress syndrome peripheral blood mononuclear cells had a three-fold increased ability to differentiate into fibrocytes compared to ventilated controls or non-ventilated controls. Acute respiratory distress syndrome broncho-alveolar lavage fluid inhibited by 71% (55-94) fibrocyte differentiation compared to saline control. Ventilated controls' broncho-alveolar lavage fluid was a less potent inhibitor (51% [23-66%] of inhibition), whereas non-ventilated controls' broncho-alveolar lavage fluid had no effect on fibrocyte differentiation. Serum amyloid P concentration was decreased in plasma and dramatically increased in broncho-alveolar lavage fluid during acute respiratory distress syndrome. Alveolar serum amyloid P originated, in part, from the release of serum amyloid P associated with lung connective tissue during acute respiratory distress syndrome. Serum amyloid P depletion decreased the inhibitory effect of acute respiratory distress syndrome broncho-alveolar lavage fluid by 60%, whereas serum amyloid P replenishment of serum amyloid P-depleted acute respiratory distress syndrome broncho-alveolar lavage fluid restored their full inhibitory effect. CONCLUSIONS: The presence of fibrocytes in the lung during acute respiratory distress syndrome could result in a balance between higher ability of monocytes to differentiate into fibrocytes and the inhibitory effect of the alveolar environment, mainly dependent on serum amyloid P.

FGF9 and FGF18 in idiopathic pulmonary fibrosis promote survival and migration and inhibit myofibroblast differentiation of human lung fibroblasts in vitro.

Idiopathic pulmonary fibrosis (IPF) is characterized by an accumulation of extracellular matrix proteins and fibroblasts in the distal airways. Key developmental lung signaling pathways are reactivated in IPF. For instance, fibroblast growth factor 9 (FGF9) and FGF18, involved in epithelial-mesenchymal interactions, are critical for lung development. We evaluated the expression of FGF9, FGF18, and FGF receptors (FGFRs) in lung tissue from controls and IPF patients and assessed their effect on proliferation, survival, migration, and differentiation of control and IPF human lung fibroblasts (HLFs). FGF9, FGF18, and all FGFRs were present in the remodeled alveolar epithelium close to the fibroblast foci in IPF lungs. FGFR3 was generally detected in fibroblast foci by immunohistochemistry. In vitro, HLFs mainly expressed mesenchyme-associated FGFR isoforms (FGFR1c and FGFR3c) and FGFR4. FGF9 did not affect fibroblast proliferation, whereas FGF18 inhibited cell growth in control fibroblasts. FGF9 and FGF18 decreased Fas-ligand-induced apoptosis in control but not in IPF fibroblasts. FGF9 prevented transforming growth factor ß1-induced myofibroblast differentiation. FGF9 and FGF18 increased the migratory capacities of HLF, and FGF9 actively modulated matrix metalloproteinase activity. In addition, FGFR3 inhibition by small interfering RNA impacted p-ERK activation by FGF9 and FGF18 and their effects on differentiation and migration. These results identify FGF9 as an antiapoptotic and promigratory growth factor on HLF, maintaining fibroblasts in an undifferentiated state. The biological effects of FGF9 and FGF18 were partially driven by FGFR3. FGF18 was a less potent molecule. Both growth factors likely contribute to the fibrotic process in vivo.

Forkhead Box F1 represses cell growth and inhibits COL1 and ARPC2 expression in lung fibroblasts in vitro.

Aberrant expression of master phenotype regulators or alterations in their downstream pathways in lung fibroblasts may play a central role in idiopathic pulmonary fibrosis (IPF). Interrogating IPF fibroblast transcriptome datasets, we identified Forkhead Box F1 (FOXF1), a DNA-binding protein required for lung development, as a candidate actor in IPF. Thus we determined FOXF1 expression levels in fibroblasts cultured from normal or IPF lungs in vitro, and explored FOXF1 functions in these cells using transient and stable loss-of-function and gain-of-function models. FOXF1 mRNA and protein were expressed at higher levels in IPF fibroblasts compared with normal fibroblasts (mRNA: +44%, protein: +77%). Immunohistochemistry showed FOXF1 expression in nuclei of bronchial smooth muscle cells, endothelial cells, and lung fibroblasts including fibroblastic foci of IPF lungs. In normal lung fibroblasts, FOXF1 repressed cell growth and expression of collagen-1 (COL1) and actin-related protein 2/3 complex, subunit 2 (ARPC2). ARPC2 knockdown inhibited cell growth and COL1 expression, consistent with FOXF1 acting in part through ARPC2 repression. In IPF fibroblasts, COL1 and ARPC2 repression by FOXF1 was blunted, and FOXF1 did not repress growth. FOXF1 expression was induced by the antifibrotic mediator prostaglandin E2 and repressed by the profibrotic cytokine transforming growth factor-ß1 in both normal and IPF lung fibroblasts. Ex vivo, FOXF1 knockdown conferred CCL-210 lung fibroblasts the ability to implant in uninjured mouse lungs. In conclusion, FOXF1 functions and regulation were consistent with participation in antifibrotic pathways. Alterations of pathways downstream of FOXF1 may participate to fibrogenesis in IPF fibroblasts.

Epithelial SCAP/INSIG/SREBP signaling regulates multiple biological processes during perinatal lung maturation.

Pulmonary surfactant is required for lung function at birth and throughout postnatal life. Defects in the surfactant system are associated with common pulmonary disorders including neonatal respiratory distress syndrome and acute respiratory distress syndrome in children and adults. Lipogenesis is essential for the synthesis of pulmonary surfactant by type II epithelial cells lining the alveoli. This study sought to identify the role of pulmonary epithelial SREBP, a transcriptional regulator of cellular lipid homeostasis, during a critical time period of perinatal lung maturation in the mouse. Genome wide mRNA expression profiling of lung tissue from transgenic mice with epithelial-specific deletions of Scap (Scap(Δ/Δ), resulting in inactivation of SREBP signaling) or Insig1 and Insig2 (Insig1/2(Δ/Δ), resulting in activation of SREBP signaling) was assessed. Differentially expressed genes responding to SREBP perturbations were identified and subjected to functional enrichment analysis, pathway mapping and literature mining to predict upstream regulators and transcriptional networks regulating surfactant lipid homeostasis. Through comprehensive data analysis and integration, time dependent effects of epithelial SCAP/INSIG/SREBP deletion and defined SCAP/INSIG/SREBP-associated genes, bioprocesses and downstream pathways were identified. SREBP signaling influences epithelial development, cell death and cell proliferation at E17.5, while primarily influencing surfactant physiology, lipid/sterol synthesis, and phospholipid transport after birth. SREBP signaling integrated with the Wnt/ß-catenin and glucocorticoid receptor signaling pathways during perinatal lung maturation. SREBP regulates perinatal lung lipogenesis and maturation through multiple mechanisms by interactions with distinct sets of regulatory partners.