Distinct niches within the extracellular matrix dictate fibroblast function in (cell free) 3D lung tissue cultures.

Cues from the extracellular matrix (ECM) and their functional interplay with cells play pivotal roles for development, tissue repair, and disease. However, the precise nature of this interplay remains elusive. We used an innovative 3D cell culture ECM model by decellularizing 300-µm-thick ex vivo lung tissue scaffolds (d3D-LTCs) derived from diseased and healthy mouse lungs, which widely mimics the native (patho)physiological in vivo ECM microenvironment. We successfully repopulated all d3D-LTCs with primary human and murine fibroblasts, and moreover, we demonstrated that the cells also populated the innermost core regions of the d3D-LTCs in a real 3D fashion. The engrafted fibroblasts revealed a striking functional plasticity, depending on their localization in distinct ECM niches of the d3D-LTCs, affecting the cells' tissue engraftment, cellular migration rates, cell morphologies, and protein expression and phosphorylation levels. Surprisingly, we also observed fibroblasts that were homing to the lung scaffold's interstitium as well as fibroblasts that were invading fibrotic areas. To date, the functional nature and even the existence of 3D cell matrix adhesions in vivo as well as in 3D culture models is still unclear and controversial. Here, we show that attachment of fibroblasts to the d3D-LTCs evidently occurred via focal adhesions, thus advocating for a relevant functional role in vivo. Furthermore, we found that protein levels of talin, paxillin, and zyxin and phosphorylation levels of paxillin Y118, as well as the migration-relevant small GTPases RhoA, Rac, and CDC42, were significantly reduced compared with their attachment to 2D plastic dishes. In summary, our results strikingly indicate that inherent physical or compositional characteristics of the ECM act as instructive cues altering the functional behavior of engrafted cells. Thus, d3D-LTCs might aid to obtain more realistic data in vitro, with a high relevance for drug discovery and mechanistic studies alike.

Classical transient receptor potential 6 (TRPC6) channels support myofibroblast differentiation and development of experimental pulmonary fibrosis.

Pulmonary fibrosis (PF) is a chronic progressive lung disease without effective medical treatment options leading to respiratory failure and death within 3-5years of diagnosis. The pathological process of PF is driven by aberrant wound-healing involving fibroblasts and myofibroblasts differentiated by secreted profibrotic transforming growth factor ß (TGF-ß1). Classical transient receptor potential 6 (TRPC6), a Na+- and Ca2+-permeable cation channel, is able to promote myofibroblast conversion of primary rat cardiac and human dermal fibroblasts and TRPC6-deficiency impaired wound healing after injury. To study a potential role of TRPC6 in the development of PF we analyzed lung function, gene and protein expression in wild-type (WT) and TRPC6-deficient (TRPC6-/-) lungs utilizing a bleomycin-induced PF-model. Fibrotic WT-mice showed a significant higher death rate while bleomycin-treated TRPC6-deficient mice were partly protected from fibrosis as a consequence of a lower production of collagen and an almost normal function of the respiratory system (reduced resistance and elastance compared to fibrotic WT-mice). On a molecular level TGF-ß1 induced TRPC6 up-regulation, increased Ca2+ influx and nuclear NFAT localization in WT primary murine lung fibroblasts (PMLFs) resulting in higher stress fiber formation and accelerated contraction rates as compared to treated TRPC6-deficient fibroblasts. Therefore, we conclude that TRPC6 is an important determinant for TGF-ß1-induced myofibroblast differentiation during fibrosis and specific channel inhibitors might be beneficial in a future treatment of PF.

BARD1 mediates TGF-ß signaling in pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a rapid progressive fibro-proliferative disorder with poor prognosis similar to lung cancer. The pathogenesis of IPF is uncertain, but loss of epithelial cells and fibroblast proliferation are thought to be central processes. Previous reports have shown that BARD1 expression is upregulated in response to hypoxia and associated with TGF-ß signaling, both recognized factors driving lung fibrosis. Differentially spliced BARD1 isoforms, in particular BARD1ß, are oncogenic drivers of proliferation in cancers of various origins. We therefore hypothesized that BARD1 and/or its isoforms might play a role in lung fibrosis. METHODS: We investigated BARD1 expression as a function of TGF-ß in cultured cells, in mice with experimentally induced lung fibrosis, and in lung biopsies from pulmonary fibrosis patients. RESULTS: FL BARD1 and BARD1ß were upregulated in response to TGF-ß in epithelial cells and fibroblasts in vitro and in vivo. Protein and mRNA expression studies showed very low expression in healthy lung tissues, but upregulated expression of full length (FL) BARD1 and BARD1ß in fibrotic tissues. CONCLUSION: Our data suggest that FL BARD1 and BARD1ß might be mediators of pleiotropic effects of TGF-ß. In particular BARD1ß might be a driver of proliferation and of pulmonary fibrosis pathogenesis and progression and represent a target for treatment.

Enolase 1 (ENO1) and protein disulfide-isomerase associated 3 (PDIA3) regulate Wnt/ß-catenin-driven trans-differentiation of murine alveolar epithelial cells.

The alveolar epithelium represents a major site of tissue destruction during lung injury. It consists of alveolar epithelial type I (ATI) and type II (ATII) cells. ATII cells are capable of self-renewal and exert progenitor function for ATI cells upon alveolar epithelial injury. Cell differentiation pathways enabling this plasticity and allowing for proper repair, however, are poorly understood. Here, we applied proteomics, expression analysis and functional studies in primary murine ATII cells to identify proteins and molecular mechanisms involved in alveolar epithelial plasticity. Mass spectrometry of cultured ATII cells revealed a reduction of carbonyl reductase 2 (CBR2) and an increase in enolase 1 (ENO1) and protein disulfide-isomerase associated 3 (PDIA3) protein expression during ATII-to-ATI cell trans-differentiation. This was accompanied by increased Wnt/ß-catenin signaling, as analyzed by qRT-PCR and immunoblotting. Notably, ENO1 and PDIA3, along with T1α (podoplanin; an ATI cell marker), exhibited decreased protein expression upon pharmacological and molecular Wnt/ß-catenin inhibition in cultured ATII cells, whereas CBR2 levels were stabilized. Moreover, we analyzed primary ATII cells from mice with bleomycin-induced lung injury, a model exhibiting activated Wnt/ß-catenin signaling in vivo. We observed reduced CBR2 significantly correlating with surfactant protein C (SFTPC), whereas ENO1 and PDIA3 along with T1α were increased in injured ATII cells. Finally, siRNA-mediated knockdown of ENO1, as well as PDIA3, in primary ATII cells led to reduced T1α expression, indicating diminished cell trans-differentiation. Our data thus identified proteins involved in ATII-to-ATI cell trans-differentiation and suggest a Wnt/ß-catenin-driven functional role of ENO1 and PDIA3 in alveolar epithelial cell plasticity in lung injury and repair.

Multidimensional immunolabeling and 4D time-lapse imaging of vital ex vivo lung tissue.

During the last decades, the study of cell behavior was largely accomplished in uncoated or extracellular matrix (ECM)-coated plastic dishes. To date, considerable cell biological efforts have tried to model in vitro the natural microenvironment found in vivo. For the lung, explants cultured ex vivo as lung tissue cultures (LTCs) provide a three-dimensional (3D) tissue model containing all cells in their natural microenvironment. Techniques for assessing the dynamic live interaction between ECM and cellular tissue components, however, are still missing. Here, we describe specific multidimensional immunolabeling of living 3D-LTCs, derived from healthy and fibrotic mouse lungs, as well as patient-derived 3D-LTCs, and concomitant real-time four-dimensional multichannel imaging thereof. This approach allowed the evaluation of dynamic interactions between mesenchymal cells and macrophages with their ECM. Furthermore, fibroblasts transiently expressing focal adhesions markers incorporated into the 3D-LTCs, paving new ways for studying the dynamic interaction between cellular adhesions and their natural-derived ECM. A novel protein transfer technology (FuseIt/Ibidi) shuttled fluorescently labeled α-smooth muscle actin antibodies into the native cells of living 3D-LTCs, enabling live monitoring of α-smooth muscle actin-positive stress fibers in native tissue myofibroblasts residing in fibrotic lesions of 3D-LTCs. Finally, this technique can be applied to healthy and diseased human lung tissue, as well as to adherent cells in conventional two-dimensional cell culture. This novel method will provide valuable new insights into the dynamics of ECM (patho)biology, studying in detail the interaction between ECM and cellular tissue components in their natural microenvironment.

Preclinical validation and imaging of Wnt-induced repair in human 3D lung tissue cultures.

Chronic obstructive pulmonary disease (COPD) is characterised by a progressive loss of lung tissue. Inducing repair processes within the adult diseased lung is of major interest and Wnt/ß-catenin signalling represents a promising target for lung repair. However, the translation of novel therapeutic targets from model systems into clinical use remains a major challenge.We generated murine and patient-derived three-dimensional (3D) ex vivo lung tissue cultures (LTCs), which closely mimic the 3D lung microenvironment in vivo. Using two well-known glycogen synthase kinase-3ß inhibitors, lithium chloride (LiCl) and CHIR 99021 (CT), we determined Wnt/ß-catenin-driven lung repair processes in high spatiotemporal resolution using quantitative PCR, Western blotting, ELISA, (immuno)histological assessment, and four-dimensional confocal live tissue imaging.Viable 3D-LTCs exhibited preserved lung structure and function for up to 5 days. We demonstrate successful Wnt/ß-catenin signal activation in murine and patient-derived 3D-LTCs from COPD patients. Wnt/ß-catenin signalling led to increased alveolar epithelial cell marker expression, decreased matrix metalloproteinase-12 expression, as well as altered macrophage activity and elastin remodelling. Importantly, induction of surfactant protein C significantly correlated with disease stage (per cent predicted forced expiratory volume in 1 s) in patient-derived 3D-LTCs.Patient-derived 3D-LTCs represent a valuable tool to analyse potential targets and drugs for lung repair. Enhanced Wnt/ß-catenin signalling attenuated pathological features of patient-derived COPD 3D-LTCs.

Upregulation of TGF-ß1 in experimental proliferative vitreoretinopathy is accompanied by epithelial to mesenchymal transition.

BACKGROUND: Proliferative vitreoretinopathy (PVR) is characterized by epithelial to mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells and consecutive formation of fibrous membranes, leading to retinal redetachment. Transforming growth factor beta (TGF-ß) has been suggested to play an important role in this process, but the role of TGF-ß isoforms is unknown. METHODS: In pigmented rabbits (n = 14), PVR was induced by cryopexy and a full-thickness limbus-parallel incision. PVR was evaluated by indirect ophthalmoscopy. Concentrations of TGF-ß isoforms were determined by multiplex bead assay analysis in aqueous humor (AH) and vitreous samples. EMT marker vimentin was analyzed by western blot. Masson's-trichrome, haematoxilin and eosine (H&E), and immunohistochemical analysis for EMT marker alpha SMA were performed on cross-sections of eyes. RESULTS: PVR was induced in all treated eyes. The number of quadrants affected by PVR was 1 (n = 5), 2 (n = 2), 3 (n = 2), 4 (n = 5). Vimentin and alpha SMA were expressed during PVR development. During PVR development, both TGF-ß1 levels (AH: p = 0.001; vitreous: p = 0.002) and TGF-ß2 levels increased (AH: p = 0.027; vitreous: p = 0.02), while TGF-ß3 was not detected at any timepoint. The increase was more pronounced for TGF-ß1 than for TGF- ß2 (AH: p = 0.002; vitreous: p = 0.0005), and only TGF-ß1 correlated with the amount of PVR (p = 0.024, r = 0,723). CONCLUSIONS: Development of PVR membranes was accompanied by a pronounced upregulation of TGF-ß1, rather than TGF-ß2. Therefore TGF-ß1 could be a promising target for inhibition of PVR.

In-vivo and ex-vivo characterization of laser-induced choroidal neovascularization variability in mice.

BACKGROUND: Retinal argon laser coagulation is an established procedure for induction of choroidal neovascularization (CNV) in rodents. This study aimed to evaluate the in-vivo and ex-vivo morphology and variability of laser-induced CNV spots over time. METHODS: Female C57/Bl/6 mice, 3-6 months of age, were treated with five spots of retinal argon laser coagulation per eye (150 mW, 100 ms, 50 µm). In-vivo fluorescein angiography (FA) and standard high-resolution spectral-domain optical coherence tomography (SD-OCT) were performed on day (d) 0, d1, d4, d7, d14 and d21. Ex-vivo histology, CD31 immunostaining, flatmount and confocal microscopy were also conducted. CNV size in the retinal and choroidal focus, CNV morphology, central retinal thickness (CRT) and FA CNV activity grading were assessed in-vivo at all times and compared to the ex-vivo assessments. RESULTS: SD-OCT revealed sub-retinal and intra-retinal fluid, and permitted evaluation of longitudinal morphologic changes of the induced CNV. Laser spot area in FA and CRT in SD-OCT did not differ in longitudinal evaluation. CNV could not be consistently outlined on SD-OCT images, and CNV volume as assessed on SD-OCT did not change over time. Significant CNV activity changes were only found in FA CNV activity grading, peaking on d4 and decreasing by d7. CONCLUSIONS: Non-invasive SD-OCT provides additional morphological information on laser-induced CNV. However, reliable evaluation of CNV requires FA. Spontaneous regression of CNV activity within 14 days after induction has to be taken into account when utilizing this model for testing the efficacies of potential future treatments.

Overexpression of HTRA1 leads to ultrastructural changes in the elastic layer of Bruch's membrane via cleavage of extracellular matrix components.

Variants in the chromosomal region 10q26 are strongly associated with an increased risk for age-related macular degeneration (AMD). Two potential AMD genes are located in this region: ARMS2 and HTRA1 (high-temperature requirement A1). Previous studies have suggested that polymorphisms in the promotor region of HTRA1 result in overexpression of HTRA1 protein. This study investigated the role of HTRA1 overexpression in the pathogenesis of AMD. Transgenic Htra1 mice overexpressing the murine protein in the retinal pigment epithelium (RPE) layer of the retina were generated and characterized by transmission electron microscopy, immunofluorescence staining and Western Blot analysis. The elastic layer of Bruch's membrane (BM) in the Htra1 transgenic mice was fragmented and less continuous than in wild type (WT) controls. Recombinant HTRA1 lacking the N-terminal domain cleaved various extracellular matrix (ECM) proteins. Subsequent Western Blot analysis revealed an overexpression of fibronectin fragments and a reduction of fibulin 5 and tropoelastin in the RPE/choroid layer in transgenic mice compared to WT. Fibulin 5 is essential for elastogenesis by promoting elastic fiber assembly and maturation. Taken together, our data implicate that HTRA1 overexpression leads to an altered elastogenesis in BM through fibulin 5 cleavage. It highlights the importance of ECM related proteins in the development of AMD and links HTRA1 to other AMD risk genes such as fibulin 5, fibulin 6, ARMS2 and TIMP3.