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Effect of Substrate Stiffness on Physicochemical Properties of Normal and Fibrotic Lung Fibroblasts.

ABSTRACT: The presented research aims to verify whether physicochemical properties of lung fibroblasts, modified by substrate stiffness, can be used to discriminate between normal and fibrotic cells from idiopathic pulmonary fibrosis (IPF). The impact of polydimethylsiloxane (PDMS) substrate stiffness on the physicochemical properties of normal (LL24) and IPF-derived lung fibroblasts (LL97A) was examined in detail. The growth and elasticity of cells were assessed using fluorescence microscopy and atomic force microscopy working in force spectroscopy mode, respectively. The number of fibroblasts, as well as their shape and the arrangement, strongly depends on the mechanical properties of the substrate. Moreover, normal fibroblasts remain more rigid as compared to their fibrotic counterparts, which may indicate the impairments of IPF-derived fibroblasts induced by the fibrosis process. The chemical properties of normal and IPF-derived lung fibroblasts inspected using time-of-flight secondary ion mass spectrometry, and analyzed complexly with principal component analysis (PCA), show a significant difference in the distribution of cholesterol and phospholipids. Based on the observed distinctions between healthy and fibrotic cells, the mechanical properties of cells may serve as prospective diagnostic biomarkers enabling fast and reliable identification of idiopathic pulmonary fibrosis (IPF).

SUBMITTER: Raczkowska J

PROVIDER: S-EPMC7600549 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Effect of Substrate Stiffness on Physicochemical Properties of Normal and Fibrotic Lung Fibroblasts.

Raczkowska Joanna J Orzechowska Barbara B Patryas Sabina S Awsiuk Kamil K Kubiak Andrzej A Kinoshita Masaya M Okamoto Masami M Bobrowska Justyna J Stachura Tomasz T Soja Jerzy J Sładek Krzysztof K Lekka Małgorzata M

Materials (Basel, Switzerland) 20201010 20

The presented research aims to verify whether physicochemical properties of lung fibroblasts, modified by substrate stiffness, can be used to discriminate between normal and fibrotic cells from idiopathic pulmonary fibrosis (IPF). The impact of polydimethylsiloxane (PDMS) substrate stiffness on the physicochemical properties of normal (LL24) and IPF-derived lung fibroblasts (LL97A) was examined in detail. The growth and elasticity of cells were assessed using fluorescence microscopy and atomic f ...[more]

PMID: 33050502

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Project description:Pulmonary arterial adventitial fibroblasts (PAAFs) are important regulators of fibrotic vascular remodeling during the progression of pulmonary arterial hypertension (PAH), a disease that currently has no effective anti-fibrotic treatments. We conducted in-vitro experiments in PAAFs cultured on hydrogels attached to custom-made equibiaxial stretchers at 10% stretch and substrate stiffnesses representing the mechanical conditions of mild and severe stages of PAH. The expression of collagens α(1)I and α(1)III and elastin messenger RNAs (Col1a1, Col3a1, Eln) were upregulated by increased stretch and substrate stiffness, while lysyl oxidase-like 1 and α-smooth muscle actin messenger RNAs (Loxl1, Acta2) were only significantly upregulated when the cells were grown on matrices with an elevated stiffness representative of mild PAH but not on a stiffness representative of severe PAH. Fibronectin messenger RNA (Fn1) levels were significantly induced by increased substrate stiffness and transiently upregulated by stretch at 4 h, but was not significantly altered by stretch at 24 h. We modified our published computational network model of the signaling pathways that regulate profibrotic gene expression in PAAFs to allow for differential regulation of mechanically-sensitive nodes by stretch and stiffness. When the model was modified so that stiffness activated integrin β3, the Macrophage Stimulating 1 or 2 (MST1\2) kinases, angiotensin II (Ang II), transforming growth factor-β (TGF-β), and syndecan-4, and stretch-regulated integrin β3, MST1\2, Ang II, and the transient receptor potential (TRP) channel, the model correctly predicted the upregulation of all six genes by increased stiffness and the observed responses to stretch in five out of six genes, although it could not replicate the non-monotonic effects of stiffness on Loxl1 and Acta2 expression. Blocking Ang II Receptor Type 1 (AT1R) with losartan in-vitro uncovered an interaction between the effects of stretch and stiffness and angiotensin-independent activation of Fn1 expression by stretch in PAAFs grown on 3-kPa matrices. This novel combination of in-vitro and in-silico models of PAAF profibrotic cell signaling in response to altered mechanical conditions may help identify regulators of vascular adventitial remodeling due to changes in stretch and matrix stiffness that occur during the progression of PAH in-vivo.

Dataset Information

Effect of Substrate Stiffness on Physicochemical Properties of Normal and Fibrotic Lung Fibroblasts.

Publications

Effect of Substrate Stiffness on Physicochemical Properties of Normal and Fibrotic Lung Fibroblasts.

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