Abstract
Fibrosis is a common driver of end-stage organ failure in most organs. It is characterised by excessive accumulation of extracellular matrix (ECM) proteins. Therapeutic options are limited and novel treatments are urgently required, however current cell-based high-throughput screening (HTS) models to identify molecules affecting ECM accumulation are limited in their relevance or throughput. We report a novel sensitive approach which combines in situ fluorescent staining of accumulated decellularised ECM proteins with automated high-content microscopy. Using this method to measure ECM accumulation in a kidney cell model, we demonstrated good agreement with established radiolabelled amino acid incorporation assays: TGFβ1 delivered a potent pro-fibrotic stimulus, which was reduced by TGFβ antibody or the anti-fibrotic nintedanib. Importantly, our method also provides information about matrix organisation: the extent of ECM accumulation was unaffected by the BMP antagonist Gremlin-1 but a pronounced effect on matrix fibrillar organisation was revealed. This rapid, straightforward endpoint provides quantitative data on ECM accumulation and offers a convenient cross-species readout that does not require antibodies. Our method facilitates discovery of novel pro- and anti-fibrotic agents in 384-well plate format and may be widely applied to in vitro cell-based models in which matrix protein deposition reflects the underlying biology or pathology.
Highlights
The extracellular matrix (ECM) is a complex mix of proteins, proteoglycans and fluid, which was traditionally considered as a relatively inert material that provided a structural framework for cell binding, but is recognised as an important regulator of cellular and tissue function[1,2]
Mature ECM is a highly dynamic structure which is composed of a large number of proteins that are maintained in a homeostatic balance by controlled continuous remodelling, with the synthesis and deposition of ECM components on one side and the activity of clearance enzymes such as matrix metalloproteinases on the other[2]
We demonstrated the ability of TGFβ1 to deliver a potent fibrotic stimulus in vitro using two typical endpoints with different human primary cell types from two organs: renal proximal tubular epithelial cells (RPTEC) and fibroblasts originating from idiopathic pulmonary fibrosis lung (IPF134)
Summary
The extracellular matrix (ECM) is a complex mix of proteins, proteoglycans and fluid, which was traditionally considered as a relatively inert material that provided a structural framework for cell binding, but is recognised as an important regulator of cellular and tissue function[1,2]. The ECM acts as site of growth factor storage and activation, and forms direct interactions with cell-surface molecules such as integrins, thereby delivering extracellular and environmental feedback to the surrounding cells. It actively influences the biology of the cells in its local environment, regulating cellular contraction, migration, proliferation, differentiation and apoptosis. Mature ECM is a highly dynamic structure which is composed of a large number of proteins that are maintained in a homeostatic balance by controlled continuous remodelling, with the synthesis and deposition of ECM components on one side and the activity of clearance enzymes such as matrix metalloproteinases on the other[2]. The resultant decline in the elasticity and function of the affected organ may lead to its failure; fibrosis presents a significant burden of morbidity and mortality[1,3]
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