Macromolecular Crowding as a Tool to Screen Anti-fibrotic Drugs: The Scar-in-a-Jar System Revisited

Nataly Puerta Cavanzo,Peter Olinga,Ruud A Bank,Emilia Bigaeva,Miriam Boersema

doi:10.3389/fmed.2020.615774

Nataly Puerta Cavanzo, Peter Olinga + Show 3 more

Open Access

https://doi.org/10.3389/fmed.2020.615774

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Abstract

An unsolved therapeutic problem in fibrosis is the overproduction of collagen. In order to screen the effect of anti-fibrotic drugs on collagen deposition, the Scar-in-a-Jar approach has been introduced about a decade ago. With macromolecular crowding a rapid deposition of collagen is seen, resulting in a substantial decrease in culture time, but the system has never been tested in an adequate way. We therefore have compared six different macromolecular crowders [Ficoll PM 70 (Fc70), Ficoll PM 400 (Fc400), a mixture of Ficoll 70 and 400 (Fc70/400), polyvinylpyrrolidone 40 (PVP40), polyvinylpyrrolidone 360 (PVP360), neutral dextran 670 (ND670), dextran sulfate 500 (DxS500), and carrageenan (CR)] under profibrotic conditions (addition of TGFβ1) with primary human adult dermal fibroblasts in the presence of 0.5 and 10% FBS. We found that (1) collagen deposition and myofibroblast formation was superior with 0.5% FBS, (2) DxS500 and CR results in an aberrant collagen deposition pattern, (3) ND670 does not increase collagen deposition, and (4) CR, DxS500, and Fc40/700 affected important phenotypical properties of the cells when cultured under pro-fibrotic conditions, whereas PVP40 and PVP360 did less or not. Because of viscosity problems with PVP360, we conclude that PVP40 is the most optimal crowder for the screening of anti-fibrotic drugs. Finally, the effect of various concentrations of Imatinib, Galunisertib, Omipalisib or Nintedanib on collagen deposition and myofibroblast formation was tested with PVP40 as the crowder.

Highlights

Most cells are embedded in a tissue-specific micro-environment composed of extracellular matrix (ECM) molecules
As expected, a significant increase is seen in mRNA levels of genes encoding COL1A1, COL5A1, and fibronectin 1 containing extra domain A (FN1EDA), SERPINH1 and PLOD2, alfa-smooth muscle actin (ACTA2) and X-box binding protein 1 (XBP1), whereas a significant decrease was seen in mRNA levels of genes encoding for matrix metalloproteinase 1 (MMP1) and cathepsin K (CTSK)
To test the validity of the Scar-in-a-Jar approach, we investigated the effect of six macromolecular crowders (Fc70/400, polyvinylpyrrolidone 40 (PVP40), polyvinylpyrrolidone 360 (PVP360), neutral dextran 670 (ND670), dextran sulfate 500 (DxS500), and CR) on collagen type I deposition under the same profibrotic conditions at day 4

Summary

Introduction

Most cells are embedded in a tissue-specific micro-environment composed of extracellular matrix (ECM) molecules. One of the structural components of the ECM is collagen. The precursor procollagen needs to be processed into mature collagen to enable the formation of supramolecular structures outside the cell. In culture systems, this processing is time-consuming, and extended culture times are required to obtain a sufficient amount of extracellular matrix. Already in 1986 Bateman et al [1] showed that the addition of neutral polymers in culture medium markedly enhanced the conversion of procollagen into collagen. They showed that the rate-limiting step in culture

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