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.

Keywords: Cell Biology; Extracellular matrix; Fibrosis; Pericytes; Vascular Biology.

Figure 1. α-SMA⁺ pericyte (PC) expression accumulate in fibrotic foci of idiopathic pulmonary fibrosis (IPF) human lungs.

Control and IPF lung samples were fixed and stained to observe contribution of PC to myofibroblast population in IPF. (A) Immunofluorescence images of human lung tissue from control and IPF patients. Human lungs are costained for α-smooth muscle actin (α-SMA, green) with PC marker NG2 (red) and Dapi (blue). Boxes and arrows represent areas of NG2 and α-SMA dual positive costaining in lung tissue. (B) Image analysis used to quantify the % coexpression ± SEM of α-SMA⁺NG2⁺ cells in control and IPF lung samples. (C) Compared with NHLFs, relative expression of NG2 message is increased in IPF lung fibroblasts (2-tailed Student t test with Bonferroni post-test, ****P < 0.001 compared with control lung, n = 5).

Figure 2. Human idiopathic pulmonary fibrosis (IPF) lung extracellular matrix (ECM) facilitates mechanoresponsive expression of α-SMA.

(A) Seven-day PC cultures on decellularized lung scaffolds were stained for α-SMA and IHC on control and IPF ECM. (B) α-SMA expression presented as mean ± SEM using immunoblotting and normalized to GAPDH (black lines used to designate samples run in the same gel but noncontiguous lanes). (C) MKL1 translocation was examined by extracting nuclear protein for PC cultured on control or IPF lung, was analyzed using immunoblotting, and was presented as mean ± SEM normalized to lamin A/C (2-tailed Student t test with Bonferroni post-test, *P < 0.05 compared with control lung, n = 3 and 4).

Figure 3. Increased substrate stiffness induces megakaryoblastic leukemia 1–dependent (MKL1-dependent) α-SMA expression.

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).

Figure 4. TGF-β1 promotes extracellular matrix (ECM) remodeling and fibrotic foci formation.

(A) PC were cultured on matrices and activated with TGF-β1 for 14 days. Immunofluorescence images of α-SMA (green), vimentin (red), and nuclei (blue) are shown and mean expression ± SEM. (B) α-SMA and vimentin were quantified using flow cytometry (one-way ANOVA with Tukey Post-Hoc test, *P < 0.05 compared with low-stiffness hydrogel of the same activation, ^#P < 0.05 compared with nonactivated condition of the same hydrogel stiffness, n = 3). (C) Immunofluorescence images of collagen IV (green) and fibronectin (red), or collagen I (green) and laminin (red), of fibrotic lesions are shown, and (D) the mean spatial density ± SEM and (E) mean area ± SEM of the lesions were quantified (one-way ANOVA with Tukey Post-Hoc test, *P < 0.05 versus nonactivated PC, n = 6). (F) Immunofluorescence images of α-SMA and vimentin. (G) α-SMA and (H) vimentin expression quantified using flow cytometry (mean fold increase over nonactivated α-SMA or vimentin ± SEM shown) and (I) confirmed using immunoblotting (Student t test with Bonferroni post-test. *P < 0.05 versus nonactivated PC, n ≥ 10).

Figure 5. Effects of TGF-β1 induced lung matrix stiffening and increased α-SMA expression are reversed by nintedanib treatment.

PC were cultured on glass, activated with TGF-β1 for 28 days, and treated with nintedanib for 7 days. (A) Immunofluorescence images of collagen I (green) and fibronectin (red). (B–D) Matrix deposition was quantified using immunoblotting (n = 7), and (E) fibrotic lesions were quantified from images (n ≥ 10) at day 28 (represented as mean ± SEM, Student t test with Bonferroni post-test, *P < 0.05 versus TGF-β1). (F) α-SMA and (G) vimentin expression, quantified using flow cytometry (presented as mean fold increase over protein expression of cells cultured on low-stiffness healthy lung ± SEM) (H) were confirmed using immunoblotting (n ≥ 10). Black lines used to designate samples run in same gel but in noncontiguous lanes. (I) PC were cultured on decellularized control lung with TGF-β1 activation for 28 days and treated with nintedanib for 7 days. Matrix deposition was evaluated by Masson’s trichrome and Picrosirius red staining. (J) Mean Young’s Moduli ± SEM of the decellularized lung were determined using an Instron5848 at 20% strain (Student t test with Bonferroni post-test ,*P < 0.05 versus TGF-β1, n = 7–9). (K) α-SMA expression was evaluated using IHC and (L) spatial density of α-SMA⁺ myofibroblasts was quantified (mean ± SEM) from representative α-SMA IHC images (Student t test with Bonferroni post-test, *P < 0.05 versus TGF-β1, n = 4–6).

Figure 6. Nintedanib induces elevated pericyte (PC) production of metalloproteinases MMP8, MMP9, and MMP13.

(A–E) PC supernatant activated with TGF-β1 ± nintedanib were evaluated using ELISA against (A) MMP2, (B) MMP8, (C) MMP9, (D) MMP13, and (E) MMP10 and data presented as detected MMP concentration (pg/ml) ± SEM (Student t test with Bonferroni post-test; *P < 0.05, **P < 0.01 versus TGF-β1; n = 4, ***P < 0.001). (F and G) Gelatinase zymography was also used to validate activity of (F) MMP2 and (G) MMP9 production by PC ± nintedanib treatment (one-way ANOVA with Tukey post-hoc test, *P < 0.05 versus TGF-β1, n = 6;**P < 0.01).

Figure 7. TGF-β1 induced mechanotransduction pathway of α-SMA⁺ pericyte (PC) accumulation in pulmonary fibrosis.

(A) Pericyte abundant microvasculature reside in the human lung. (B) Production of TGF-β1 by fibroblasts and resident macrophages in the interstitial space results in accumulation of α-SMA⁺ PC in the fibrotic foci. (C) [1] The binding of TGF-β1 to TGF-βR1 and TGF-βR2 on the surface of PC initiates a Smad signaling cascade. [2] Smad2/3 gets phosphorylated and [3] translocates to the cell’s nucleus. [4] The accumulation of P-Smad2/3 in the nucleus leads to increased binding to the Smad binding element (SBE), which results in increased transcription of fibrotic genes. [5] Increased production of matrix proteins, including collagen I, results in an increased ECM stiffness. [6] Focal adhesion and focal adhesion kinases (FAK) binding to the stiffened environment results in [7] increased translocation of megakaryoblastic leukemia 1 (MKL-1), yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), resulting in [8] increased α-SMA expression and myofibroblast transition.