Abstract
ObjectivsCytokine-dependent activation of fibroblasts to myofibroblasts, a key event in fibrosis, is accompanied by phenotypic changes with increased secretory and contractile properties dependent on increased energy utilization, yet changes in the energetic profile of these cells are not fully described. We hypothesize that the TGF-β1-mediated transformation of myofibroblasts is associated with an increase in mitochondrial content and function when compared to naive fibroblasts.MethodsCultured NIH/3T3 mouse fibroblasts treated with TGF-β1, a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts (appearance of α-smooth muscle actin [α-SMA] stress fibers) and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols. Expression of mitochondrial-specific proteins was determined using western blotting, and the mitochondrial DNA quantified using Mitochondrial DNA isolation kit.ResultsTreatment with TGF-β1 (5 ng/mL) induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-SMA (6.85 ± 0.27 RU) compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU). TGF-β1 exposure increased the number of mitochondria in the cells, as monitored by membrane potential sensitive dye tetramethylrhodamine, and expression of mitochondria-specific proteins; voltage-dependent anion channels (0.54 ± 0.05 vs. 0.23 ± 0.05 RU) and adenine nucleotide transporter (0.61 ± 0.11 vs. 0.22 ± 0.05 RU), as well as mitochondrial DNA content (530 ± 12 μg DNA/106 cells vs. 307 ± 9 μg DNA/106 cells in control). TGF-β1 treatment was associated with an increase in mitochondrial function with a twofold increase in baseline oxygen consumption rate (2.25 ± 0.03 vs. 1.13 ± 0.1 nmol O2/min/106 cells) and FCCP-induced mitochondrial respiration (2.87 ± 0.03 vs. 1.46 ± 0.15 nmol O2/min/106 cells).ConclusionsTGF-β1 induced differentiation of fibroblasts is accompanied by energetic remodeling of myofibroblasts with an increase in mitochondrial respiration and mitochondrial content.
Highlights
Fibroblasts are the major cells involved in extracellular matrix remodeling and the repair processes following injury through cytokine-dependent transformation into myofibroblasts [1,2,3,4]
Cultured National Institutes of Health (NIH)/3T3 mouse fibroblasts treated with transforming growth factor-β1 (TGF-β1), a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols
Treatment with TGF-β1 (5 ng/mL) induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-smooth muscle actin (α-SMA) (6.85 ± 0.27 RU) compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU)
Summary
Fibroblasts are the major cells involved in extracellular matrix remodeling and the repair processes following injury through cytokine-dependent transformation into myofibroblasts [1,2,3,4]. Differentiation of fibroblasts into myofibroblasts for active repair of damaged tissue is accompanied by major changes in cell phenotype with conversion of non-excitable precursors into excitable myofibroblasts, cells with increased contractility and higher synthetic and secretory capabilities [5,6,7,8,9], processes that increase cellular energy demands [10]. Phenotypic changes with fibroblast differentiation are well characterized, little information is available about mitochondrial remodeling associated with fibroblast differentiation. The aim of this study was to evaluate the changes in mitochondrial content and respiration of fibroblasts treated with vehicle or transforming growth factor-β1 (TGF-β1), a profibrotic cytokine known to activate fibroblasts into myofibroblasts [1,2]
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