Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Impaired Myofibroblast Dedifferentiation Contributes to Nonresolving Fibrosis in Aging

doi: 10.1165/rcmb.2019-0092OC

Figure Lengend Snippet: Idiopathic pulmonary fibrosis (IPF) lung myofibroblasts demonstrate impaired dedifferentiation capacity. (A–D) Fibroblasts isolated from the lungs of healthy adults and patients with biopsy-proven IPF were evaluated for cellular senescence by qualitative (A) and quantitative (B) measurements of senescence-associated β-gal activity, expression of the senescence markers by Western blotting (C), and densitometric analyses (D). Scale bars: 100 μm. *P < 0.05 and ****P < 0.0001 as compared with adult healthy lungs, using Student’s two-tailed t-test. (E–J) Cells were serum starved for 16 hours, treated with TGF-β (2 ng/ml) for 48 hours (Day 0), and then treated with 0% or 20% FBS for 5 days. Expression of α-SMA, MyoD, and GAPDH was assessed by Western blotting in fibroblasts isolated from adult healthy lungs (E) and from IPF lungs (H). (F–J) α-SMA and MyoD were quantified by densitometric analyses for adult healthy fibroblasts (F and G) and for IPF fibroblasts (I and J). **P < 0.01 and ****P < 0.0001 as compared with Day 0, using Student’s two-tailed t-test. All values represent means ± SEM; n = 3 biological replicates from 3 independent experiments.

Article Snippet: IPF lung fibroblasts were also purchased from Lonza.

Techniques: Isolation, Activity Assay, Expressing, Western Blot, Two Tailed Test

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Impaired Myofibroblast Dedifferentiation Contributes to Nonresolving Fibrosis in Aging

doi: 10.1165/rcmb.2019-0092OC

Figure Lengend Snippet: Impaired dedifferentiation in IPF fibroblasts is associated with apoptosis resistance. Adult healthy and IPF lung fibroblasts were serum starved for 16 hours, treated with TGF-β (2 ng/ml) for 48 hours (Day 0), and then treated with 0% or 20% FBS for 5 days. Cells were treated with vehicle or staurosporine (300 nM) for 8 hours, and Caspase 3 activity was assessed (A). (B–D) Expression of cPARP, cCaspase 3, and GAPDH was assessed by Western blotting (B and C) and densitometric analyses (D). Values represent means ± SEM; n = 3 biological replicates; **P < 0.01 and ***P < 0.001 using Student’s two-tailed t test.

Article Snippet: IPF lung fibroblasts were also purchased from Lonza.

Techniques: Activity Assay, Expressing, Western Blot, Two Tailed Test

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Impaired Myofibroblast Dedifferentiation Contributes to Nonresolving Fibrosis in Aging

doi: 10.1165/rcmb.2019-0092OC

Figure Lengend Snippet: MyoD is upregulated in IPF lung myofibroblasts, and genetic targeting restores apoptosis susceptibility. (A) IPF lung tissue sections were analyzed by immunohistochemistry for expression of MyoD. Scale bars: 100 μm. (B and C) Adult healthy and IPF lung fibroblasts were evaluated for protein expression of MyoD by Western blotting (B) and densitometric analyses (C). (D and E) IPF fibroblasts were transfected with MyoD siRNA. Downregulation of MyoD was confirmed by Western blotting (D) and caspase activity of transfected cells was assessed (E). Values represent means ± SEM; n = 3 biological replicates; **P < 0.01 and ****P < 0.0001 using Student’s two-tailed t test. NT = nontargeting siRNA.

Article Snippet: IPF lung fibroblasts were also purchased from Lonza.

Techniques: Immunohistochemistry, Expressing, Western Blot, Transfection, Activity Assay, Two Tailed Test

Journal: Aging (Albany NY)

Article Title: Modulating in vitro lung fibroblast activation via senolysis of senescent human alveolar epithelial cells

doi: 10.18632/aging.205994

Figure Lengend Snippet: DNA damage is a hallmark of commercial primary IPF fibroblasts in vitro , but not Cdkn2a /p16 ink4a expression or a fibrotic secretome . ( A ) Primary NHLF and IPF cells ( n = 3 different donors, each with 2 technical replicates) were cultured at the same density overnight on standard tissue culture plates in low-serum growth medium and were subsequently fixed. Immunocytochemistry was performed to fluorescently label nuclei (blue) and p21 Waf1/Cip1 (orange). Images were acquired with a 40X water objective using an Operetta High Content Screening instrument and intensity of nuclear p21 Waf1/Cip1 was quantified and calculated as a positive percentage of each population. ( B ) NHLF and IPF cells were treated and imaged as in ( A ), and immunocytochemistry was performed to fluorescently label nuclei (blue) and DNA damage via nuclear γH2A.X (Ser139) foci (orange). The number of nuclear foci per cell was quantified and cells with one or more were reported as positive. ( C ) Following overnight culture in standard tissue culture plates, NHLF and IPF cells were lysed, and qPCR was performed. Expression of senescence and fibrosis-related matrix and secreted factor genes was assessed, and data were normalized to β2m housekeeper expression using the 2 −ΔΔCt method versus NHLF cells. ( D ) Following overnight culture as in ( C ), cell culture supernatants were collected and assayed for determination of the concentration of common fibrosis-related secreted proteins by MSD kits. Statistical analysis was performed using an unpaired t -test ( A , B ) or a two-way ANOVA with a Bonferroni post-test ( C , D ) in GraphPad Prism: * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars represent standard error of the mean (SEM).

Article Snippet: Primary normal human lung fibroblasts (NHLF) were purchased from Lonza and ATCC, and diseased IPF lung fibroblasts were purchased from BioIVT, Lonza, or ATCC.

Techniques: In Vitro, Expressing, Cell Culture, Immunocytochemistry, High Content Screening, Concentration Assay

Journal: Aging (Albany NY)

Article Title: Modulating in vitro lung fibroblast activation via senolysis of senescent human alveolar epithelial cells

doi: 10.18632/aging.205994

Figure Lengend Snippet: Summary of in vitro and ex vivo systems to mimic alveolar epithelial and fibroblast damage and dysfunction in the IPF lung. While IPF fibroblasts and bleomycin treated NHLFs exhibit some hallmarks of senescence, these cells do not appear to demonstrate a phenotype of Cdkn2a /p16 ink4a expression, ECM deposition, or secretion of fibrotic mediators such as TIMP1. Rather, the SASP of senescent, aberrant epithelial cells drives a fibrotic phenotype in NHLFs that is consistent with progressive fibrosis. Development of senolytic agents presents an opportunity for therapeutic impact early in disease pathogenesis and with an orthogonal mechanism than the fibroblast targeting standard of care, Nintedanib. Image created with https://www.biorender.com/ .

Article Snippet: Primary normal human lung fibroblasts (NHLF) were purchased from Lonza and ATCC, and diseased IPF lung fibroblasts were purchased from BioIVT, Lonza, or ATCC.

Techniques: In Vitro, Ex Vivo, Expressing