A high-content, in vitro cardiac fibrosis assay for high-throughput, phenotypic identification of compounds with anti-fibrotic activity

Palano, Giorgia; Jansson, Maria; Backmark, Anna; Martinsson, Stefan; Sabirsh, Alan; Hultenby, Kjell; Åkerblad, Peter; Granberg, Kenneth L.; Jennbacken, Karin; Müllers, Erik; Hansson, Emil M.

doi:10.1016/j.yjmcc.2020.04.002

Cited by 13 publications

(17 citation statements)

References 70 publications

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“…The assay can be run at low costs in 96-well or 384-well format, allowing to robustly assess the values of dose dependency and half-maximal effective concentration (EC50) potency for potential antifibrosis in vitro . Our in vitro assay correctly identified compounds with reported antifibrotic effects in vivo , targeting diverse cellular pathways ( Palano et al, 2020 ), thus, highlighting its utility for high-throughput screening to discover novel compounds and targets for the treatment of cardiac fibrosis. The HCI assay has several advantages, i.e., (1) screening in high-throughput; (2) analyses in microwell format requiring fewer cells and minimizing the amounts of reagents; and (3) quantification of multiparametric readouts on a single-cell basis.…”

Section: High-content Imagingmentioning

confidence: 79%

“…From the perspective of an assay, it is important to understand whether a 3D and/or coculture model has greater physiological relevance, i.e., predictivity, for cardiac fibrosis. To adequately compare models, it is essential to use holistic, integrative readouts, i.e., a better model might show more in vivo -like ( Kong et al, 2014 ) gene expression ( Chothani et al, 2019 ), ( Liu et al, 2017 ) protein expression, ( Gabbiani et al, 1971 ) organoid structure and cellular composition ( Lee et al, 2019 ), or ( Skalli et al, 1989 ) more in vivo -like functional response, that is, higher predictivity for known modulators of cardiac fibrosis ( Palano et al, 2020 ).…”

Section: In Vitro Assays For Cardiac Fibrosismentioning

confidence: 99%

Section: High-content Imagingmentioning

confidence: 99%

“…We recently established cell culture conditions that promote deposition of mature collagen from primary human CFs in vitro ( Palano et al, 2020 ). Based on these conditions, we set up a high-content immunofluorescence assay allowing for high-throughput, phenotypic identification of compounds with antifibrotic activity ( Palano et al, 2020 ). Features extracted from the microscopy images, such as fluorescence intensity, cellular morphology, and staining texture, served as a basis for a linear classifier to classify cells into fibrotic or non-fibrotic phenotype.…”

Section: High-content Imagingmentioning

confidence: 99%

“…Application of HCI and image analysis using ML-based classifiers, as in our phenotypic cardiac fibrosis assay (see section “A Phenotypic High-Throughput in vitro Cardiac Fibrosis Assay”; Palano et al, 2020 ), provides a way forward to develop such an assay toward a coculture and/or 3D model. Having set up a robust biochemical validation and a toolbox of known in vivo modulators of cardiac fibrosis allows for direct comparison and assay optimization toward increased predictivity of in vivo outcomes and, thus, increased physiological relevance.…”

Section: High-content Imagingmentioning

confidence: 99%

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In vitro Assays and Imaging Methods for Drug Discovery for Cardiac Fibrosis

2021

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As a result of stress, injury, or aging, cardiac fibrosis is characterized by excessive deposition of extracellular matrix (ECM) components resulting in pathological remodeling, tissue stiffening, ventricular dilatation, and cardiac dysfunction that contribute to heart failure (HF) and eventually death. Currently, there are no effective therapies specifically targeting cardiac fibrosis, partially due to limited understanding of the pathological mechanisms and the lack of predictive in vitro models for high-throughput screening of antifibrotic compounds. The use of more relevant cell models, three-dimensional (3D) models, and coculture systems, together with high-content imaging (HCI) and machine learning (ML)-based image analysis, is expected to improve predictivity and throughput of in vitro models for cardiac fibrosis. In this review, we present an overview of available in vitro assays for cardiac fibrosis. We highlight the potential of more physiological 3D cardiac organoids and coculture systems and discuss HCI and automated artificial intelligence (AI)-based image analysis as key methods able to capture the complexity of cardiac fibrosis in vitro. As 3D and coculture models will soon be sufficiently mature for application in large-scale preclinical drug discovery, we expect the combination of more relevant models and high-content analysis to greatly increase translation from in vitro to in vivo models and facilitate the discovery of novel targets and drugs against cardiac fibrosis.

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Section: High-content Imagingmentioning

confidence: 79%

Section: In Vitro Assays For Cardiac Fibrosismentioning

confidence: 99%

Section: High-content Imagingmentioning

confidence: 99%

Section: High-content Imagingmentioning

confidence: 99%

Section: High-content Imagingmentioning

confidence: 99%

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In vitro Assays and Imaging Methods for Drug Discovery for Cardiac Fibrosis

2021

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Functional Genomics

Gianni

Escudero‐Ibarz

2022

Genome Editing in Drug Discovery

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Pirfenidone affects human cardiac fibroblast proliferation and cell cycle activity in 2D cultures and engineered connective tissues

Meyer

Santos

Doan

et al. 2023

Naunyn-Schmiedeberg's Arch Pharmacol

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The anti-fibrotic drug pirfenidone (PFD) is currently in clinical testing for the treatment of heart failure with preserved ejection fraction; however, its effects on human cardiac cells have not been fully investigated. Therefore, we aimed to characterize the impact of PFD on human cardiac fibroblasts (CF) in 2D culture as well as in 3D-engineered connective tissues (ECT). We analyzed proliferation by automated cell counting and changes in signaling by immunoblotting. We generated ECT with different geometries to modify the cellular phenotype and investigated the effects of PFD on cell number and viability as well as on cell cycle activity. We further studied its effect on ECT compaction, contraction, stiffening, and strain resistance by ECT imaging, pole deflection analysis, and ultimate tensile testing. Our data demonstrate that PFD inhibits human CF proliferation in a concentration-dependent manner with an IC50 of 0.43 mg/ml and its anti-mitogenic effect was further corroborated by an inhibition of MEK1/2, ERK1/2, and riboprotein S6 (rpS6) phosphorylation. In ECT, a lower cell cycle activity was found in PFD-treated ECT and fewer cells resided in these ECT after 5 days of culture compared to the control. Moreover, ECT compaction as well as ECT contraction was impaired. Consequently, biomechanical analyses demonstrated that PFD reduced the stiffness of ECT. Taken together, our data demonstrate that the anti-fibrotic action of PFD on human CF is based on its anti-mitogenic effect in 2D cultures and ECT.

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A high-content, in vitro cardiac fibrosis assay for high-throughput, phenotypic identification of compounds with anti-fibrotic activity

Cited by 13 publications

References 70 publications

In vitro Assays and Imaging Methods for Drug Discovery for Cardiac Fibrosis

In vitro Assays and Imaging Methods for Drug Discovery for Cardiac Fibrosis

Functional Genomics

Pirfenidone affects human cardiac fibroblast proliferation and cell cycle activity in 2D cultures and engineered connective tissues

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