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Quantitative and organisational changes in mature extracellular matrix revealed
through high-content imaging of total protein fluorescently stained in situ
#MMPMID28855577
Holdsworth G
; Bon H
; Bergin M
; Qureshi O
; Paveley R
; Atkinson J
; Huang L
; Tewari R
; Twomey B
; Johnson T
Sci Rep
2017[Aug]; 7
(1
): 9963
PMID28855577
show ga
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: TGF?1 delivered a potent pro-fibrotic stimulus,
which was reduced by TGF? 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.
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