Developmental Reprogramming in Mesenchymal Stromal Cells of Human Subjects with Idiopathic Pulmonary Fibrosis - PubMed (original) (raw)

Ashish Kurundkar 1, Sunad Rangarajan 1, Morgan Locy 1, Karen Bernard 1, Nirmal S Sharma 1, Naomi J Logsdon 1, Hui Liu 1, David K Crossman 2, Jeffrey C Horowitz 3, Stijn De Langhe 4, Victor J Thannickal 1

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Developmental Reprogramming in Mesenchymal Stromal Cells of Human Subjects with Idiopathic Pulmonary Fibrosis

Diptiman Chanda et al. Sci Rep. 2016.

Abstract

Cellular plasticity and de-differentiation are hallmarks of tissue/organ regenerative capacity in diverse species. Despite a more restricted capacity for regeneration, humans with age-related chronic diseases, such as cancer and fibrosis, show evidence of a recapitulation of developmental gene programs. We have previously identified a resident population of mesenchymal stromal cells (MSCs) in the terminal airways-alveoli by bronchoalveolar lavage (BAL) of human adult lungs. In this study, we characterized MSCs from BAL of patients with stable and progressive idiopathic pulmonary fibrosis (IPF), defined as <5% and ≥10% decline, respectively, in forced vital capacity over the preceding 6-month period. Gene expression profiles of MSCs from IPF subjects with progressive disease were enriched for genes regulating lung development. Most notably, genes regulating early tissue patterning and branching morphogenesis were differentially regulated. Network interactive modeling of a set of these genes indicated central roles for TGF-β and SHH signaling. Importantly, fibroblast growth factor-10 (FGF-10) was markedly suppressed in IPF subjects with progressive disease, and both TGF-β1 and SHH signaling were identified as critical mediators of this effect in MSCs. These findings support the concept of developmental gene re-activation in IPF, and FGF-10 deficiency as a potentially critical factor in disease progression.

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Figures

Figure 1

Figure 1. Heat map analysis of lung developmental genes.

Heat map representing color coded expression levels of differentially expressed genes in progressive IPF compared to stable IPF (n = 3 in each group; p < 0.005). Up-regulated genes were shown in shades of red whereas down-regulated genes were shown in shades of green.

Figure 2

Figure 2. Validation of lung developmental genes of interest.

Total RNA was isolated from MSCs from stable IPF (n = 7) and progressive IPF (n = 8), and subjected to real-time PCR analysis for FGF-10, BMP-4, Meox2 and HoxA2. Data were normalized to 18 S rRNA and represented graphically as fold change compared to stable IPF (s-IPF).

Figure 3

Figure 3. Gene interaction network of organismal development-related genes.

(A) Ingenuity Pathway Analysis (IPA) was used to generate gene interaction network in progressive IPF. This network contains four developmental genes of interest (FGF-10, BMP-4, Meox2, HOxA2; Table 1). Transcriptional information was projected onto the interaction map such that up-regulated genes are depicted in shades of red and down-regulated genes are in shades of green. (B) Shortest path gene interaction network of growth factors (FGF-10 and BMP-4) and transcription regulators (Meox2 and HoxA2). IPA was used to generate this network using their Path Explorer filter which calculates the shortest path between these genes. Blue lines mark the genes found in the canonical transforming growth factor-β (TGF-β) and sonic hedgehog (SHH) signaling; whereas, orange lines indicate genes found to be involved in lung development.

Figure 4

Figure 4. Effects of TGF-β1, SHH and SAG on BAL-derived MSCs.

(A,B) Myofibroblast differentiation. MSCs were isolated from surveillance bronchoscopies and BAL from lung transplant recipients without bronchiolitis obliterans or infection. MSCs were seeded in 6-well tissue culture plates and serum deprived for 24 h followed by either TGF-β1 treatment (2.5 ng/ml) or SHH (0, 50, 100, 500 ng/ml) for 48 h. Cell lysates were prepared in RIPA buffer and subjected to SDS-PAGE and western blot analysis for α-SMA; GAPDH antibody was used as loading control. Densitometry was performed to quantitate the ratio of α-SMA and GAPDH and plotted graphically; bar graphs represent mean ± SEM, n = 3; *p < 0.05, compared to vehicle treated control. Full-length western blots are presented in Supplementary Figure S6. (C–F) RNA expression of developmental genes of interest. Total RNA was isolated from MSCs 48 h post-treatment with either TGF-β1 or SHH or combination and subjected to real-time PCR analysis. Data were normalized to 18 S rRNA and relative mRNA expressions are represented graphically as fold change compared to control. Data represents mean ± SEM; n = 3 (each analyzed in triplicate); *p < 0.01; **p < 0.001; ***p < 0.0001. Individual colored marker represents average relative mRNA expression of single lung transplant recipient. (G) The smoothened agonist, SAG, downregulates FGF-10. MSCs were treated with vehicle and SAG (100 ng/ml) for 48 h; total RNA was extracted and subjected to real-time PCR analysis. Data were normalized to 18 S rRNA and relative mRNA expressions represented graphically as fold change compared to control. Data represents mean ± SEM; n = 3, *p < 0.01.

Figure 5

Figure 5. Immunohistochemical localization of α-SMA and FGF-10 in IPF and normal lungs.

Six micron (6 μ) sections were obtained from normal (n = 4, failed donor lung) and IPF (n = 4; explant during lung transplantation). Immunohistochemical staining showing expression of the myofibroblast marker, α-SMA, in fibroblastic foci of IPF lung tissues; note that α-SMA staining in normal lung is restricted to large airways and blood vessels (arrows). FGF-10 immunostaining was not detected in the fibroblastic foci although expression of this growth factor was observed in interstitial mesenchymal cells in regions with less fibrotic remodeling (arrows). Higher magnification images of specific regions (boxes) are shown in middle panels.

References

    1. Duffield J. S., Lupher M., Thannickal V. J. & Wynn T. A. Host responses in tissue repair and fibrosis. Annu Rev Pathol. 8, 241–276 (2013). -PMC -PubMed
    1. Thannickal V. J., Toews G. B., White E. S., Lynch J. P. 3rd & Martinez F. J. Mechanisms of pulmonary fibrosis. Annu Rev Med. 55, 395–417 (2004). -PubMed
    1. Selman M., Pardo A. & Kaminski N. Idiopathic pulmonary fibrosis: aberrant recapitulation of developmental programs? PLoS Med. 5, e62 (2008). -PMC -PubMed
    1. Hardie W. D., Glasser S. W. & Hagood J. S. Emerging concepts in the pathogenesis of lung fibrosis. Am J Pathol. 175, 3–16 (2009). -PMC -PubMed
    1. Collard H. R. et al. Changes in clinical and physiologic variables predict survival in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 168, 538–542 (2003). -PubMed

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