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Human lung fibroblast-to-myofibroblast transformation is not driven by an LDH5-dependent metabolic shift towards aerobic glycolysis.

ABSTRACT: BACKGROUND:Idiopathic pulmonary fibrosis (IPF) is a fatal respiratory disease characterized by aberrant fibroblast activation and progressive fibrotic remodelling of the lungs. Though the exact pathophysiological mechanisms of IPF remain unknown, TGF-?1 is thought to act as a main driver of the disease by mediating fibroblast-to-myofibroblast transformation (FMT). Recent reports have indicated that a metabolic shift towards aerobic glycolysis takes place during FMT and that metabolic shifts can directly influence aberrant cell function. This has led to the hypothesis that inhibition of lactate dehydrogenase 5 (LDH5), an enzyme responsible for converting pyruvate into lactate, could constitute a therapeutic concept for IPF. METHODS:In this study, we investigated the potential link between aerobic glycolysis and FMT using a potent LDH5 inhibitor (Compound 408, Genentech). Seahorse analysis was performed to determine the effect of Compound 408 on TGF-?1-driven glycolysis in WI-38 fibroblasts. TGF-?1-mediated FMT was measured by quantifying ?-smooth muscle actin (?-SMA) and fibronectin in primary human lung fibroblasts following treatment with Compound 408. Lactate and pyruvate levels in the cell culture supernatant were assessed by LC-MS/MS. In addition to pharmacological LDH5 inhibition, the effect of siRNA-mediated knockdown of LDHA and LDHB on FMT was examined. RESULTS:We show that treatment of lung fibroblasts with Compound 408 efficiently inhibits LDH5 and attenuates the TGF-?1-mediated metabolic shift towards aerobic glycolysis. Additionally, we demonstrate that LDH5 inhibition has no significant effect on TGF-?1-mediated FMT in primary human lung fibroblasts by analysing ?-SMA fibre formation and fibronectin expression. CONCLUSIONS:Our data strongly suggest that while LDH5 inhibition can prevent metabolic shifts in fibroblasts, it has no influence on FMT and therefore glycolytic dysregulation is unlikely to be the sole driver of FMT.

SUBMITTER: Schruf E

PROVIDER: S-EPMC6507142 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Human lung fibroblast-to-myofibroblast transformation is not driven by an LDH5-dependent metabolic shift towards aerobic glycolysis.

Schruf Eva E Schroeder Victoria V Kuttruff Christian A CA Weigle Sabine S Krell Martin M Benz Maryke M Bretschneider Tom T Holweg Alexander A Schuler Michael M Frick Manfred M Nicklin Paul P Garnett James P JP Sobotta Mirko C MC

Respiratory research 20190509 1

<h4>Background</h4>Idiopathic pulmonary fibrosis (IPF) is a fatal respiratory disease characterized by aberrant fibroblast activation and progressive fibrotic remodelling of the lungs. Though the exact pathophysiological mechanisms of IPF remain unknown, TGF-β1 is thought to act as a main driver of the disease by mediating fibroblast-to-myofibroblast transformation (FMT). Recent reports have indicated that a metabolic shift towards aerobic glycolysis takes place during FMT and that metabolic shi ...[more]

PMID: 31072408

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Project description:The transformation of keratocytes and fibroblasts to myofibroblasts is important to corneal wound healing as well as formation of stromal haze. The purpose of this study was to determine the effect of latrunculin B, an actin cytoskeleton disruptor in conjunction with a fundamental biophysical cue, substrate stiffness, on myofibroblast transformation in vitro and in vivo. Rabbit corneal fibroblasts were cultured on substrates of differing compliance (1.5, 22, and 71?kPa) and tissue culture plastic (TCP; > 1?GPa) in media containing 0 or 10?ng/ml TGF?1 for 72?h. Cells were treated with 0.4??M Lat-B or DMSO for 30?min every 24?h for 72?h. RNA was collected from cells and expression of alpha-smooth muscle actin (?-SMA), keratocan, and ALDH1A1 determined using qPCR; immunocytochemistry was used to assess ?-SMA protein expression. A rabbit phototherapeutic keratectomy (PTK) model was used to assess the impact of 0.1% Lat-B (n?=?3) or 25% DMSO (vehicle control, n?=?3) on corneal wound healing by assessment of epithelial wound size with fluorescein stain and semi-quantitative stromal haze scoring by an observer masked to treatment group as well as Fourier-domain optical coherence tomography (FD-OCT) at set time points. Statistical analysis was completed using one-way or two-way analysis of variance. Treatment with Lat-B versus DMSO resulted in significantly less ?SMA mRNA (P???0.007) for RCF cells grown on 22 and 71?kPa substrates as well as TCP without or with TGF?1, and significantly decreased ?-SMA protein expression in RCFs cultured on the intermediate (22?kPa) stiffness in the absence (P?=?0.028) or presence (P?=?0.018) of TGF?1. Treatment with Lat-B versus DMSO but did not significantly alter expression of keratocan or ALDH1A1 mRNA in RCFs (P?>?0.05) in the absence or presence of TGF?1, but RCFs grown on stiff hydrogels (71?kPa) had significantly more keratocan mRNA expression versus the 22?kPa hydrogel or TCP (P?<?0.001) without TGF?1. Administration of topical Lat-B BID was well tolerated by rabbits post-PTK but did not significantly alter epithelial wound closure, stromal haze score, stromal haze thickness as measured by FD-OCT in comparison to DMSO-treated rabbits. When corneal stromal cells are cultured on substrates possessing biologically relevant substratum stiffnesses, Lat-B modulates mRNA and protein expression of ?-SMA and thus modulates myofibroblast transformation. At a dose and dose-frequency that reduced IOP in human glaucoma patients, Lat-B treatment did not substantially impact corneal epithelial or stromal wound healing in a rabbit PTK model. While a significant impact on wound healing was observed at the concentration and dose frequency reported here was not found, encouraging in vitro data support further investigations of topically applied Lat-B to determine if this compound can reduce stromal fibrosis.

Dataset Information

Human lung fibroblast-to-myofibroblast transformation is not driven by an LDH5-dependent metabolic shift towards aerobic glycolysis.

Publications

Human lung fibroblast-to-myofibroblast transformation is not driven by an LDH5-dependent metabolic shift towards aerobic glycolysis.

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