Human pericytes adopt myofibroblast properties in the microenvironment of the IPF lung
Parid Sava, … , Erica L. Herzog, Anjelica L. Gonzalez
Parid Sava, … , Erica L. Herzog, Anjelica L. Gonzalez
Published December 21, 2017
Citation Information: JCI Insight. 2017;2(24):e96352. doi:10.1172/jci.insight.96352.
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Categories: Research Article Cell biology Vascular biology

Human pericytes adopt myofibroblast properties in the microenvironment of the IPF lung

Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown etiology characterized by a compositionally and mechanically altered extracellular matrix. Poor understanding of the origin of α-smooth muscle actin (α-SMA) expressing myofibroblasts has hindered curative therapies. Though proposed as a source of myofibroblasts in mammalian tissues, identification of microvascular pericytes (PC) as contributors to α-SMA–expressing populations in human IPF and the mechanisms driving this accumulation remain unexplored. Here, we demonstrate enhanced detection of α-SMA+ cells coexpressing the PC marker neural/glial antigen 2 in the human IPF lung. Isolated human PC cultured on decellularized IPF lung matrices adopt expression of α-SMA, demonstrating that these cells undergo phenotypic transition in response to direct contact with the extracellular matrix (ECM) of the fibrotic human lung. Using potentially novel human lung–conjugated hydrogels with tunable mechanical properties, we decoupled PC responses to matrix composition and stiffness to show that α-SMA+ PC accumulate in a mechanosensitive manner independent of matrix composition. PC activated with TGF-β1 remodel the normal lung matrix, increasing tissue stiffness to facilitate the emergence of α-SMA+ PC via MKL-1/MTRFA mechanotranduction. Nintedanib, a tyrosine-kinase inhibitor approved for IPF treatment, restores the elastic modulus of fibrotic lung matrices to reverse the α-SMA+ phenotype. This work furthers our understanding of the role that microvascular PC play in the evolution of IPF, describes the creation of an ex vivo platform that advances the study of fibrosis, and presents a potentially novel mode of action for a commonly used antifibrotic therapy that has great relevance for human disease.

Authors

Parid Sava, Anand Ramanathan, Amelia Dobronyi, Xueyan Peng, Huanxing Sun, Adrian Ledesma-Mendoza, Erica L. Herzog, Anjelica L. Gonzalez

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Figure 3

Increased substrate stiffness induces megakaryoblastic leukemia 1–dependent (MKL1-dependent) α-SMA expression.

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Increased substrate stiffness induces megakaryoblastic leukemia 1–depend...
Round glass coverslips were coated with polyacrylamide hydrogels with varying stiffness to evaluate the mechanotransductive effects in pericytes. Elasticity of polyacrylamide hydrogels was tested to generate (A) stress-strain plots and (B) Young’s Moduli calculated at 20% strain, reported as mean ± SEM (one-way ANOVA with Tukey Post-Hoc test, *P < 0.05 versus low-stiffness hydrogel, #P < 0.05 versus medium stiffness hydrogel, n = 9 and 10). Control and IPF lung protein were conjugated to the polymer, and PC were cultured for 7 days. (C) Immunofluorescence images of α-SMA (green) and F-Actin (red) were used to determine (D) cell area ± SEM and (E) shape (aspect ratio ± SEM ) (one-way ANOVA with Tukey Post-Hoc test, *P < 0.05 compared with low-stiffness hydrogel of the same matrix, #P < 0.05 versus medium stiffness hydrogel of the same matrix, n ≥ 10). (F) α-SMA expression was quantified using flow cytometry, presented as mean fold increase over expression of cells on low-stiffness healthy lung ± SEM (one-way ANOVA with Tukey Post-Hoc test *P < 0.05 compared with low-stiffness hydrogel of the same matrix, n = 9). (G) MKL1 translocation was analyzed using immunoblotting and (H) reported as mean ± SEM normalized to lamin A/C (one-way ANOVA with Tukey Post-Hoc test, *P < 0.05 versus low-stiffness hydrogel, n = 5). (I) Immunofluorescence images of α-SMA (green), F-Actin (red), and nuclei (blue) are shown for PC on high-stiffness hydrogels with no treatment (naive) and 0- or 7-day treatment with CCG-1423, an inhibitor of MKL1. (J) α-SMA expression collected cells was quantified using flow cytometry and presented as fold increase in α-SMA ± SEM compared with untreated cells on low-stiffness healthy lung (one-way ANOVA with Tukey Post-Hoc test, *P < 0.05 versus naive PC, n = 4–6).