Glutamine Inhibition Reduces Iatrogenic Laryngotracheal Stenosis.

OBJECTIVE/HYPOTHESIS: Glutamine inhibition has been demonstrated an antifibrotic effect in iatrogenic laryngotracheal stenosis (iLTS) scar fibroblasts in vitro. We hypothesize that broadly active glutamine antagonist, DON will reduce collagen formation and fibrosis-associated gene expression in iLTS mice. STUDY DESIGN: Prospective controlled animal study. METHODS: iLTS in mice were induced by chemomechanical injury of the trachea using a bleomycin-coated wire brush. PBS or DON (1.3 mg/kg) were administered by intraperitoneal injection (i.p.) every other day. Laryngotracheal complexes were harvested at days 7 and 14 after the initiation of DON treatment for the measurement of lamina propria thickness, trichrome stain, immunofluorescence staining of collagen 1, and fibrosis-associated gene expression. RESULTS: The study demonstrated that DON treatment reduced lamina propria thickness (P = .025) and collagen formation in trichrome stain and immunofluorescence staining of collagen 1. In addition, DON decreased fibrosis-associated gene expression in iLTS mice. At day 7, DON inhibited Col1a1 (P < .0001), Col3a1 (P = .0046), Col5a1 (P < .0001), and Tgfß (P = .023) expression. At day 14, DON reduced Co1a1 (P = .0076) and Tgfß (P = .023) expression. CONCLUSIONS: Broadly active glutamine antagonist, DON, significantly reduces fibrosis in iLTS mice. These results suggest that the concept of glutamine inhibition may be a therapeutic option to reduce fibrosis in the laryngotracheal stenosis. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:E2125-E2130, 2021.

M2 Macrophages Promote Collagen Expression and Synthesis in Laryngotracheal Stenosis Fibroblasts.

OBJECTIVE: Macrophages exhibit distinct phenotypes and are dysregulated in a model of iatrogenic laryngotracheal stenosis (iLTS). Increased populations of alternatively activated or M2 macrophages have been demonstrated. However, the role of these macrophages is unknown. The aims of this study are: 1) define the macrophage population in iLTS in the context of classically activated or M1 and M2 macrophage phenotypes, and 2) characterize the effect of monocyte-derived M1 and M2 macrophages on normal airway and LTS-derived fibroblasts (FBs) in vitro. STUDY DESIGN: Comparative analysis; in vitro controlled study. METHODS: Immunohistochemical analysis of human iLTS and control specimens was performed to define the macrophage population. In vitro, M1, and M2 macrophages were polarized using M-CSF + Interferon-gamma and lipopolysaccharide or Interleukin-4, respectively. FBs isolated from laryngotracheal scar (LTS-FBs) and normal tracheal airway (NA-FBs) in eight patients with iLTS were cocultured with polarized macrophages. Fibrosis gene expression, soluble collagen production, and proliferation were assessed. RESULTS: Immunohistochemical analysis revealed increased CD11b + cells (macrophage marker) in laryngotracheal scar specimens (18.3% vs. 8.5%, P = .03) and predominant CD206 (M2) costaining versus CD86 (M1) (51.5% vs. 9.8%, n = 10, P = .001). In vitro, NA-FBs cultured with M2 macrophages demonstrated a 2.41-fold increase in collagen-1 expression (P = .05, n = 8) and an increase in soluble collagen (9.98 vs. 8.875, mean difference: 1.11 95%, confidence interval 0.024-2.192, n = 8, P = .015). CONCLUSION: Increased populations of CD11b cells are present in iLTS specimens and are predominantly CD206+, indicating an M2 phenotype. In vitro, M2 macrophages promoted collagen expression in airway FBs. Targeting macrophages may represent a therapeutic strategy for attenuating fibrosis in iLTS. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E346-E353, 2021.

Inhibition of glutaminase to reverse fibrosis in iatrogenic laryngotracheal stenosis.

OBJECTIVES/HYPOTHESIS: Glutamine metabolism is a critical energy source for iatrogenic laryngotracheal stenosis (iLTS) scar fibroblasts, and glutaminase (GLS) is an essential enzyme converting glutamine to glutamate. We hypothesize that the GLS-specific inhibitor BPTES will block glutaminolysis and reduce iLTS scar fibroblast proliferation, collagen deposition, and fibroblast metabolism in vitro. STUDY DESIGN: Test-tube Lab Research. METHODS: Immunohistochemistry of a cricotracheal resection (n = 1) and a normal airway specimen (n = 1) were assessed for GLS expression. GLS expression was assessed in brush biopsies of subglottic/tracheal fibrosis and normal airway from patients with iLTS (n = 6). Fibroblasts were isolated and cultured from biopsies of subglottic/tracheal fibrosis (n = 6). Fibroblast were treated with BPTES and BPTES + dimethyl α-ketoglutarate (DMK), an analogue of the downstream product of GLS. Fibroblast proliferation, gene expression, protein production, and metabolism were assessed in all treatment conditions and compared to control. RESULTS: GLS was overexpressed in brush biopsies of iLTS scar specimens (P = .029) compared to normal controls. In vitro, BPTES inhibited iLTS scar fibroblast proliferation (P = .007), collagen I (Col I) (P < .0001), collagen III (P = .004), and α-smooth muscle actin (P = .0025) gene expression and protein production (P = .031). Metabolic analysis demonstrated that BPTES reduced glycolytic reserve (P = .007) but had no effects on mitochondrial oxidative phosphorylation. DMK rescued BPTES inhibition of Col I gene expression (P = .0018) and protein production (P = .021). CONCLUSIONS: GLS is overexpressed in iLTS scar. Blockage of GLS with BPTES significantly inhibits iLTS scar fibroblasts proliferation and function, demonstrating a critical role for GLS in iLTS. Targeting GLS to inhibit glutaminolysis may be a successful strategy to reverse scar formation in the airway. LEVEL OF EVIDENCE: NA Laryngoscope, 2020.

Engineering an immunomodulatory drug-eluting stent to treat laryngotracheal stenosis.

OBJECTIVE: Develop a drug-eluting stent construct with a reliable drug-release profile and adequate mechanically stability for a trial in a small animal model of laryngotracheal stenosis (LTS), a debilitating pathologic narrowing of the airway leading to significant shortness of breath. METHODS: Biodegradable, biocompatible stents containing 1.0% rapamycin made of PLLA-PCL (70% Poly-l-Lactide and 30% Polycaprolactone blend) and 50 : 50 PDLGA (Poly(dl-lactide-co-glycolide)) were compared. Mechanical strength testing and drug elution rates using high performance liquid chromatography analysis (HPLC) was assessed. Next, efficacy of stent elution on LTS derived scar fibroblasts. Finally, stents were placed in situ in an LTS mouse model. RESULTS: The PLLA-PCL stent construct exhibited greater mechanical strength compared to the PDLGA stent over a 4-week period (Young's Modulus (PLLA-PCL) = 13.82; Young's Modulus (PDLGA) = 4.015). Moreover, the PLLA-PCL stent showed a reliable rapamycin release profile for 6 weeks (30% elution for PLLA-PCL stents compared to <1% elution for PDLGA). Collagen 1 (p < 0.05) and fibroblast cell proliferation were decreased in vitro when treated with the rapamycin stent. In vivo, the rapamycin stent reduced lamina propria thickness (p < 0.05) and collagen 1(p < 0.05), collagen 3, TGF-B (p < 0.05) and a-SMA (p < 0.05). CONCLUSIONS: The PLLA-PCL construct demonstrated superior mechanical strength and greater drug elution compared to PDLGA stents. We demonstrated the feasibility of testing this drug-eluting stent in vivo, showing that the rapamycin-eluting stent treats fibrosis. To our knowledge this is the first study to deploy a drug-eluting stent to treat tracheal pathology in an animal model. Optimization of a rapamycin-eluting PLLA-PCL stent for translational investigation will lead to improved treatment strategies of LTS.

Targeting metabolic abnormalities to reverse fibrosis in iatrogenic laryngotracheal stenosis.

OBJECTIVE: Management of laryngotracheal stenosis (LTS) remains primarily surgical, with a critical need to identify targets for adjuvant therapy. Laryngotracheal stenosis scar fibroblasts exhibit a profibrotic phenotype with distinct metabolic shifts, including an increased glycolysis/oxidative phosphorylation ratio. This study examines the effects of the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) on collagen production, gene expression, proliferation, and metabolism of human LTS-derived fibroblasts in vitro. METHOD: Paired normal and scar-derived fibroblasts isolated from subglottic and proximal tracheal tissue in patients with iatrogenic laryngotracheal stenosis (iLTS) were cultured. Proliferation rate, gene expression, protein production, and cellular metabolism were assessed in two conditions: 1) fibroblast growth medium, and 2) fibroblast growth medium with 1 × 10-4 M DON. RESULTS: DON treatment reduced cellular proliferation rate (n = 7, P = 0.0150). Expression of genes collagen 1 and collagen 3 both were reduced (n = 7, P = 0.0102, 0.0143, respectively). Soluble collagen production decreased (n = 7, P = 0.0056). As measured by the rate of extracellular acidification, glycolysis and glycolytic capacity decreased (n = 7, P = 0.0082, 0.0003, respectively). adenosine triphosphate (ATP) production and basal respiration decreased (n = 7, P = 0.0045, 0.0258, respectively), determined by measuring the cellular rate of oxygen consumption. CONCLUSION: The glutamine antagonist DON reverses profibrotic changes by inhibiting both glycolysis and oxidative phosphorylation in iLTS scar fibroblasts. In contrast to untreated iLTS scar fibroblasts, collagen gene expression, protein production, metabolic rate, and proliferation were significantly reduced. These results suggest DON and/or its derivatives as strong candidates for adjuvant therapy in the management of iatrogenic laryngotracheal stenosis. Enzymes involved in glutamine metabolism inhibited by DON offer targets for future investigation. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E59-E67, 2018.

Relation of depression diagnoses to 2-year outcomes in cocaine-dependent patients in a randomized continuing care study.

This study examined the relation between depression diagnoses and outcomes in 132 cocaine-dependent patients who were randomized to relapse prevention (RP) or standard 12-step focused group continuing care and followed for 2 years. Depressed patients attended more treatment sessions and had more cocaine-free urines during treatment than participants without depression, but they drank alcohol more frequently before treatment and during the 18-month posttreatment follow-up. Cocaine outcomes in depressed patients deteriorated to a greater degree after treatment than did cocaine outcomes in patients without depression, particularly in patients in RP who had a current depressive disorder at baseline. The best alcohol outcomes were obtained in nondepressed patients who received RP. The results suggest that extended continuing care treatment may be warranted for cocaine-dependent patients with co-occurring depressive disorders.