Late Breaking Abstract - ERBB3 involved in growth factor pathway crosstalk during neonatal lung development in a 3D ex vivo model

Abstract IDH-wildtype GBM is the most common variant of this cancer and occurs in older adults. Unfortunately patients’ tumors are either inherently resistant to standard treatment, which includes radio- and chemo-therapy, or acquire resistance during the therapeutic process. Additionally, although effective in other cancers, targeted therapies have yielded disappointing results in GBM, perhaps because the fully developed disease has significant cellular and molecular heterogeneity, allowing the tumour to adapt to treatments. Better insight into managing GBM might result from a detailed knowledge of its initiating events, which have not yet been elucidated. With this in mind, we developed a mouse model of GBM in which the earliest stages can be studied. This ex vivo model recreates GBM by culturing subventricular zone (SVZ) cells, the putative ‘cell of origin’ of GBM in platelet-derived growth factor A (PDGFA). Under this condition SVZ cells from p53 null mice transform, becoming exogenous growth factor independent and tumorigenic in immune-competent mice. In contrast, wildtype SVZ cells do not proliferate in PDGFA and null cells in EGF/FGF do not transform. To discover why p53 null SVZ cells uniquely transform in PDGFA, we performed array comparative genomic hybridization (aCGH) on cells before and after transformation in PDGFA and whole genome sequencing (WGS) on transformed cells and tumours generated from PDGFA-transformed cells. aCGH and WGS revealed that the genomic landscape of transformed cells displayed a striking similarity to that observed in primary human GBM. Specifically, these studies showed that chromosomal alterations are a hallmark of culturing SVZ cells in PDGFA, an intriguing finding considering GBM is also characterized by a specific landscape of copy number alterations. This model may resemble the pathogenesis of human GBM and be leveraged to investigate the early stages of tumorigenesis, further leading to the development of preventative strategies and novel therapeutics.

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Stretch increases alveolar type 1 cell number in fetal lungs through ROCK-Yap/Taz pathway

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00484.2020 ◽

2021 ◽

Author(s):

Tram Mai Nguyen ◽

Johannes van der Merwe ◽

Linda Elowsson Rendin ◽

Anna-Karin Larsson-Callerfelt ◽

Jan Deprest ◽

...

Keyword(s):

Lung Development ◽

Molecular Mechanisms ◽

Ex Vivo ◽

Fluid Pressure ◽

Fetal Lung ◽

In Vivo Model ◽

Alveolar Type ◽

Ex Vivo Model

Accurate fluid pressure in the fetal lung is critical for its development, especially at the beginning of the saccular stage when alveolar epithelial type 1 (AT1) and type 2 (AT2) cells differentiate from the epithelial progenitors. Despite our growing understanding of the role of physical forces in lung development, the molecular mechanisms that regulate the transduction of mechanical stretch to alveolar differentiation remain elusive. To simulate lung distension, we optimized both an ex vivo model with precision cut lung slices and an in vivo model of fetal tracheal occlusion. Increased mechanical tension showed to improve alveolar maturation and differentiation towards AT1. By manipulating ROCK pathway, we demonstrate that stretch-induced Yap/Taz activation promotes alveolar differentiation towards AT1 phenotype via ROCK activity. Our findings show that balanced ROCK-Yap/Taz signaling is essential to regulate AT1 differentiation in response to mechanical stretching of the fetal lung, which might be helpful in improving lung development and regeneration.

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ID2033 Precision-cut tissue slices as a novel ex-vivo model for evaluating the efficacy of potential drugs

Biomedical Research and Therapy ◽

10.15419/bmrat.v4is.272 ◽

2017 ◽

Vol 4 (S) ◽

pp. 63

Author(s):

Raditya Iswandana ◽

Bao Tung Pham ◽

Theerut Luangmonkon ◽

Henricus A.M. Mutsaers ◽

Peter Olinga

Keyword(s):

Growth Factor ◽

Transforming Growth Factor ◽

Ex Vivo ◽

Plasminogen Activator Inhibitor ◽

Plasminogen Activator Inhibitor 1 ◽

Ex Vivo Model ◽

Tissue Slices ◽

Cancer Disease ◽

Fibrotic Process ◽

Pai 1

Background: Recently, we developed a novel model for drug screening by culturing ex-vivo precision-cut tissue slices (PCTS). The tissue slice consists of multiple cell types still in their normal matrix environment and structure provides numerous advantages compare to other models. Our objective was to use this model to investigate the effect of various potential compounds. In this study precision-cut intestinal slices (PCIS) were used to evaluate some transforming growth factor (TGF- β) and platelet-derived growth factor (PDGF) pathway inhibitors. TGF-β and PDGF are key cytokines in fibrotic and cancer diseases and are the main targets for treatment. Methods: Murine PCIS were cultured for 48 h in the presence of profibrotic and/or antifibrotic compounds. The fibrotic process was studied on gene and protein level using a variety of markers including (pro)collagen 1a1 (Col1a1), heat shock protein 47 (Hsp47), fibronectin (Fn2) and plasminogen activator inhibitor-1 (PAI-1). The effects of potential drugs mainly inhibiting the TGFb pathway i.e. valproic acid, tetrandrine, pirfenidone, SB203580 and LY2109761 as well as compounds mainly acting on the PDGF pathway i.e. imatinib, sorafenib and sunitinib were assessed in the model at maximum non-toxic concentrations. Results: Murine PCIS remained viable for 48 h and the onset of fibrosis was observed during culture, as demonstrated by an increased expression of, amongst others, Hsp47, Fn2 and Pai-1. Furthermore, TGFb1 stimulated fibrogenesis while PDGF had no effect. Regarding the tested antifibrotics, pirfenidone, LY2109761 and sunitinib had the most pronounced impact on fibrogenesis, both in the absence and presence of profibrotic factors, as illustrated by reduced levels of Col1a1, Hsp47, Fn2 and Pai-1 following treatment. Moreover, LY2109761 significantly reduced fibronectin protein expression in the presence of TGFb1. Conclusion: PCIS can successfully be used to test drug efficacy. Using the model we demonstrated that tetrandrine, pirfenidone, LY2109761 and sunitinib showed potential antifibrotic effects on a gene level, warranting further evaluation of these compounds for the treatment of fibrosis disease. By using tissue extracted from patient, PCIS could also be a promising model to screen drug for personalized treatment in fibrotic and cancer disease.

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Preclinical evaluation of human secretoglobin 3A2 in mouse models of lung development and fibrosis

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00037.2013 ◽

2014 ◽

Vol 306 (1) ◽

pp. L10-L22 ◽

Cited By ~ 8

Author(s):

Yan Cai ◽

Melissa E. Winn ◽

John K. Zehmer ◽

William K. Gillette ◽

Jacek T. Lubkowski ◽

...

Keyword(s):

Growth Factor ◽

Lung Development ◽

Therapeutic Agent ◽

Ex Vivo ◽

Clinical Settings ◽

Biochemical Measurement ◽

Lung Airways ◽

Further Development ◽

Embryonic Lungs

Secretoglobin (SCGB) 3A2 is a member of the SCGB gene superfamily of small secreted proteins, predominantly expressed in lung airways. We hypothesize that human SCGB3A2 may exhibit anti-inflammatory, growth factor, and antifibrotic activities and be of clinical utility. Recombinant human SCGB3A2 was expressed, purified, and biochemically characterized as a first step to its development as a therapeutic agent in clinical settings. Human SCGB3A2, as well as mouse SCGB3A2, readily formed a dimer in solution and exhibited novel phospholipase A2 inhibitory activity. This is the first demonstration of any quantitative biochemical measurement for the evaluation of SCGB3A2 protein. In the mouse as an experimental animal, human SCGB3A2 exhibited growth factor activity by promoting embryonic lung development in both ex vivo and in vivo systems and antifibrotic activity in the bleomycin-induced lung fibrosis model. The results suggested that human SCGB3A2 can function as a growth factor and an antifibrotic agent in humans. When SCGB3A2 was administered to pregnant female mice through the tail vein, the protein was detected in the dam's serum and lung, as well as the placenta, amniotic fluids, and embryonic lungs at 10 min postadministration, suggesting that SCGB3A2 readily crosses the placenta. The results warrant further development of recombinant SCGB3A2 as a therapeutic agent in treating patients suffering from lung diseases or preterm infants with respiratory distress.

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Keratinocyte growth factor attenuates hydrostatic pulmonary edema in an isolated, perfused rat lung model

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.280.3.h1311 ◽

2001 ◽

Vol 280 (3) ◽

pp. H1311-H1317 ◽

Cited By ~ 3

Author(s):

David A. Welsh ◽

Benoit P. H. Guery ◽

Bennett P. Deboisblanc ◽

Elizabeth P. Dobard ◽

Colette Creusy ◽

...

Keyword(s):

Growth Factor ◽

Pulmonary Edema ◽

Ex Vivo ◽

Keratinocyte Growth Factor ◽

Lung Model ◽

Rat Lung ◽

Ex Vivo Model ◽

Lung Weight ◽

Edema Formation ◽

Alveolar Epithelial

Hydrostatic pulmonary edema is a common complication of congestive heart failure, resulting in substantial morbidity and mortality. Keratinocyte growth factor (KGF) is a mitogen for type II alveolar epithelial and microvascular cells. We utilized the isolated perfused rat lung model to produce hydrostatic pulmonary edema by varying the left atrial and pulmonary capillary pressure. Pretreatment with KGF attenuated hydrostatic edema formation. This was demonstrated by lower wet-to-dry lung weight ratios, histological evidence of less alveolar edema formation, and reduced alveolar accumulation of intravascularly administered FITC-labeled large-molecular-weight dextran in rats pretreated with KGF. Thus KGF attenuates injury in this ex vivo model of hydrostatic pulmonary edema via mechanisms that prevent increases in alveolar-capillary permeability.

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An ex vivo model to induce early fibrosis-like changes in human precision-cut lung slices

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00084.2017 ◽

2017 ◽

Vol 312 (6) ◽

pp. L896-L902 ◽

Cited By ~ 49

Author(s):

Hani N. Alsafadi ◽

Claudia A. Staab-Weijnitz ◽

Mareike Lehmann ◽

Michael Lindner ◽

Britta Peschel ◽

...

Keyword(s):

Growth Factor ◽

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Ex Vivo ◽

Disease Onset ◽

Water Soluble ◽

High Morbidity ◽

Ex Vivo Model ◽

Early Fibrosis ◽

Precision Cut Lung Slices

Idiopathic pulmonary fibrosis (IPF) is a devastating chronic interstitial lung disease (ILD) characterized by lung tissue scarring and high morbidity. Lung epithelial injury, myofibroblast activation, and deranged repair are believed to be key processes involved in disease onset and progression, but the exact molecular mechanisms behind IPF remain unclear. Several drugs have been shown to slow disease progression, but treatments that halt or reverse IPF progression have not been identified. Ex vivo models of human lung have been proposed for drug discovery, one of which is precision-cut lung slices (PCLS). Although PCLS production from IPF explants is possible, IPF explants are rare and typically represent end-stage disease. Here we present a novel model of early fibrosis-like changes in human PCLS derived from patients without ILD/IPF using a combination of profibrotic growth factors and signaling molecules (transforming growth factor-β, tumor necrosis factor-α, platelet-derived growth factor-AB, and lysophosphatidic acid). Fibrotic-like changes of PCLS were qualitatively analyzed by histology and immunofluorescence and quantitatively by water-soluble tetrazolium-1, RT-qPCR, Western blot analysis, and ELISA. PCLS remained viable after 5 days of treatment, and fibrotic gene expression ( FN1, SERPINE1, COL1A1, CTGF, MMP7, and ACTA2) increased as early as 24 h of treatment, with increases in protein levels at 48 h and increased deposition of extracellular matrix. Alveolar epithelium reprogramming was evident by decreases in surfactant protein C and loss of HOPX. In summary, using human-derived PCLS, we established a novel ex vivo model that displays characteristics of early fibrosis and could be used to evaluate novel therapies and study early-stage IPF pathomechanisms.

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