Metabolic variations in normal and fibrotic human laryngotracheal-derived fibroblasts: A Warburg-like effect.

OBJECTIVES/HYPOTHESIS: Laryngotracheal stenosis (LTS) is a chronic fibrotic disease characterized by fibroblast proliferation, collagen deposition, and matrix remodeling in the lamina propria of the larynx and/or trachea. Current medical therapies are limited by a poor understanding of the effector cell's (fibroblasts) cellular biology and metabolism. The purpose of this study was to compare cellular proliferation, function, and metabolism between normal and LTS-derived fibroblasts in vitro. We hypothesize that LTS-derived fibroblasts will demonstrate aberrant behavior with faster proliferation, increased collagen production, and altered metabolic allocation compared with normal fibroblasts. STUDY DESIGN: In vitro comparative analysis. METHODS: Human biopsies of normal and iatrogenic LTS tissue (n = 7) were obtained, and fibroblasts were isolated and cultured in vitro. Cellular proliferation, cellular histology, gene expression, and metabolic analyses were performed. Statistical analyses comparing normal and scar-derived fibroblasts were performed. RESULTS: LTS fibroblast proliferation rate, cellular surface area, and collagen-1 expression were increased compared to normal fibroblasts. Cellular metabolic analysis of LTS-derived fibroblasts demonstrated reduced oxidative phosphorylation and increased glycolysis/oxidative phosphorylation ratio compared with normal fibroblasts. CONCLUSIONS: Human iatrogenic LTS-derived fibroblasts demonstrated aberrant behavior when compared with normal fibroblasts. A Warburg-like effect was revealed, suggesting human iatrogenic LTS fibroblasts drive their proliferation with aerobic glycolysis. The distinct metabolism suggests metabolic inhibitors could reduce fibroblast hyperplasia and hypertrophy in LTS and fibrosis in general. LEVEL OF EVIDENCE: NA Laryngoscope, 127:E107-E113, 2017.

Rapamycin inhibits human laryngotracheal stenosis-derived fibroblast proliferation, metabolism, and function in vitro.

OBJECTIVE: To determine if rapamycin inhibits the growth, function, and metabolism of human laryngotracheal stenosis (LTS)-derived fibroblasts. STUDY DESIGN: Controlled in vitro study. SETTING: Tertiary care hospital in a research university. SUBJECTS AND METHODS: Fibroblasts isolated from biopsies of 5 patients with laryngotracheal stenosis were cultured. Cell proliferation, histology, gene expression, and cellular metabolism of LTS-derived fibroblasts were assessed in 4 conditions: (1) fibroblast growth medium, (2) fibroblast growth medium with dimethylsulfoxide (DMSO), (3) fibroblast growth medium with 10(-10) M (low-dose) rapamycin dissolved in DMSO, and (4) fibroblast growth medium with 10(-9) M (high-dose) rapamycin dissolved in DMSO. RESULTS: The LTS fibroblast count and DNA concentration were reduced after treatment with high-dose rapamycin compared to DMSO (P = .0007) and normal (P = .0007) controls. Collagen I expression decreased after treatment with high-dose rapamycin versus control (P = .0051) and DMSO (P = .0093) controls. Maximal respiration decreased to 68.6 pMoles of oxygen/min/10 mg/protein from 96.9 for DMSO (P = .0002) and 97.0 for normal (P = .0022) controls. Adenosine triphosphate (ATP) production decreased to 66.8 pMoles from 88.1 for DMSO (P = .0006) and 83.3 for normal (P = .0003) controls. Basal respiration decreased to 78.6 pMoles from 108 for DMSO (P = .0002) and 101 for normal (P = .0014) controls. CONCLUSIONS: Rapamycin demonstrated an anti-fibroblast effect by significantly reducing the proliferation, metabolism, and collagen deposition of human LTS fibroblast in vitro. Rapamycin significantly decreased oxidative phosphorylation of LTS fibroblasts, suggesting at a potential mechanism for the reduced proliferation and differentiation. Furthermore, rapamycin's anti-fibroblast effects indicate a promising adjuvant therapy for the treatment of laryngotracheal stenosis.

An in situ, in vivo murine model for the study of laryngotracheal stenosis.

IMPORTANCE: Laryngotracheal stenosis (LTS) lacks an ideal animal model to study its unique wound-healing pathophysiology and the effect of interventions. OBJECTIVE: To present an in vivo, in situ mouse model of LTS that can be used to investigate its pathophysiology, mechanisms, and interventions for treatment. DESIGN, SETTING, AND SUBJECTS: Prospective controlled animal study performed at an academic animal research facility on 87 C57BL/6 mice. INTERVENTIONS: Experimental mice (n = 40) underwent bleomycin-coated wire-brush injury to the larynx and trachea, while mechanical injury controls (n = 32) underwent phosphate-buffered saline-coated wire-brush injury. Normal controls (n = 9) underwent no intervention, and mock surgery controls (n = 6) underwent anterior transcervical tracheal exposure only. Laryngotracheal complexes were harvested at days 7, 14, and 21 after injury. At the respective time points, mice in the chemomechanical and mechanical injury groups were killed, and their laryngotracheal complexes were harvested for histologic analysis. Normal and mock surgery controls were killed and then underwent histologic analysis. MAIN OUTCOMES AND MEASURES: The primary outcome measure was lamina propria thickness. RESULTS: The chemomechanical injury group maintained a significant increase in lamina propria thickness through day 21 compared with uninjured controls at day 7 (82.7 vs 41.8 µm; P<.05), day 14 (93.5 vs 26.0 µm; P<.05), and day 21 (91.2 vs 40.8 µm; P<.05). Compared with the mechanical injury group, the chemomechanical injury group demonstrated a significantly increased thickness at 21 days (91.2 vs 33.7 µm; P<.05). CONCLUSIONS AND RELEVANCE: Chemomechanical initiation of fibrosis in situ creates a viable mouse model of LTS that incorporates the physiologic circulatory supply and airflow. This small-animal model may be used to investigate the pathogenesis and inflammatory mechanisms of iatrogenic LTS and test therapeutic interventions to reverse or reduce the development of fibrosis.