PGC1α repression in IPF fibroblasts drives a pathologic metabolic, secretory and fibrogenic state

Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal ageing-related disease linked to mitochondrial dysfunction. The present study aimed to determine whether peroxisome proliferator activated receptor gamma co-activator 1-alpha (PPARGC1A, encoding PGC1α), a master regulator of mitochondrial biogenesis, is diminished in IPF and controls pathologic fibroblast activation. Primary human IPF, control lung fibroblasts and fibroblasts sorted from bleomycin-injured mice were used to evaluate the expression and function of PGC1α. In vitro PGC1α manipulation was performed by small interfering RNA knockdown or overexpression. Fibroblast activation was assessed by quantitative PCR, Western blotting, matrix deposition, secreted cytokine array, immunofluorescence and traction force microscopy. Mitochondrial function was assessed by Seahorse analyzer and mitochondria mass and number by flow cytometry, mitochondrial DNA quantification and transmission electron microscopy (TEM). We found that PGC1α levels are stably repressed in IPF fibroblasts. After bleomycin injury in young mice, PGC1α expression drops transiently but then increases prior to fibrosis resolution. In contrast, PGC1α expression fails to recover in aged mice with persistent fibrosis. PGC1α knockdown alone in normal human lung fibroblasts reduces mitochondrial mass and function while enhancing contractile and matrix synthetic fibroblast activation, senescence-related gene expression and soluble profibrotic and prosenescence signalling. Re-expression of PGC1α in IPF fibroblasts ameliorates all of these pathological cellular functions. Pharmacological treatment of IPF fibroblasts with rosiglitazone, but not thyroid hormone, elevated PGC1α expression and attenuated fibroblast activation. The sustained repression of PGC1α and beneficial effects of its rescue in IPF fibroblasts identifies PGC1α as an important regulator of the fibroblast’s pathological state in IPF.

Vascular dysfunction in aged mice contributes to persistent lung fibrosis

“…Numerous studies have shown that bleomycin‐induced lung fibrosis in young mice resolves over time (Tashiro et al, 2017). Using Col1α1‐GFP transgenic mice in combination with fluorescence‐activated cell sorting (FACS) analysis, we have previously shown a transient but reversible expansion of high GFP+ lung fibroblasts from young mice following delivery of a single dose of bleomycin to the lung, whereas fibroblasts isolated from the lungs of aged mice exhibited persistent expansion of the high GFP+ population (Caporarello et al, 2019).…”

“…We have recently demonstrated that lung fibroblast activation following bleomycin challenge is transient in young mice but more persistent in aged ones (Caporarello et al, 2019). Similarly, other groups have shown that fibrosis is persistent in aged mice and resolution is markedly impaired relative to young mice (Hecker et al, 2014; Podolsky et al, 2020; Torres‐Gonzalez et al, 2012; Xu et al, 2009).…”

TGFβ-induced fibroblast activation requires persistent and targeted HDAC-mediated gene repression

“…The broad program of known and putative anti-fibrotic genes repressed by TGFβ and rescued by pracinostat suggests that there may be widespread redundancy in the pathways that maintain fibroblast in an inactive state. To test whether the inhibitory effects of pracinostat on fibroblast activation were specifically dependent on PGC1α, a strong regulator of fibroblast quiescence (Caporarello et al, 2019;Ligresti et al, 2019), we used siRNA to knockdown PGC1α and then measured anti-fibrotic efficacy of pracinostat on ACTA2 expression. Interestingly, pracinostat maintained its inhibitory effect on ACTA2 transcript levels ( Fig.…”

Mitochondrial 8-hydroxy-2′-deoxyguanosine and coronary artery disease in patients with type 2 diabetes mellitus

“…Mitochondrial membrane potential was expressed as the JC-1 fluorescence intensities detected by the Microplate Reader at Ex/Em(green)/Em(red) of 485/538/590 nm. MtDNA 8-OHdG, mtDNA-CN, and mRNA expression of peroxisome proliferator-activated receptor coactivator 1a (PGC-1α, a master gene of mitochondrial biogenesis [35]) gene were determined using qPCR, as previously described [18,36,37]. Culture supernatants of HUVECs were collected to measure the levels of nitric oxide (NO), soluble vascular adhesion molecule 1 (sVCAM-1) and E-selectin using colorimetric kits (for NO) or ELISA (Invitrogen).…”