Naloxone as mitochondrial phenotype rescuer in a 3D bioprinted LCHADD/VLCADD model
Antonia DegeniD, Michael J. AusserlechneriD, Verena SturmlehneriD, Christian PloneriD, Sara TucciiD, Daniela KaralliD, Hamed Homaei RadiD, Lukas NeumanniD, Judith HagenbuchneriD
Abstract
For patients diagnosed with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) or very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD), fasting episodes or high-energy demands remain life threatening. Due to the low incidence, clinical trials for novel LCHADD/VLCADD therapies are limited, and current mouse models recapitulate human symptoms only partially. Here, we report the use of mitochondrial morphology and 3D bioprinted, vascularized tissue models to establish a robust testing platform for dietary-based and experimental treatment approaches. Using this platform, we demonstrated that mitochondrial morphology is strictly regulated by NOX2-driven ROS formation. Treatment of LCHADD/VLCADD-derived fibroblasts with the NOX2-inhibitor naloxone led to the reassembly of mitochondrial structures controlled by DNM1L/MFN2. Using RNA transcriptomics, we identified a pro-fibrotic phenotype in LCHADD and VLCADD patient cells, which impaired vessel formation in fully 3D bioprinted human tissue equivalents. Metabolic supplementation with dietary approaches, which are used in standard therapy, partially improved vascularization. Naloxone induced the strongest improvement, restoring vessel length and network complexity to those of healthy controls, suggesting increased oxidative stress as main driver. Interestingly, naloxone had no effect on healthy fibroblasts, underscoring its safety. Taken together, these findings suggest the opioid antagonist naloxone as a potential rescue medication during LCHADD/VLCADD-driven metabolic crises.
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Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (22 Sept 2026) · CC BY 4.0
Naloxone, a NOX2 inhibitor, largely restored mitochondrial networks and curbed a pro-fibrotic signature in LCHADD/VLCADD patient fibroblasts, and rescued impaired vessel formation in 3D bioprinted vascularized constructs.
LCHADD and VLCADD trials are limited by low incidence, and mouse models reproduce human disease only partly. This work offers a patient-fibroblast platform, combining mitochondrial readouts with a 3D bioprinted vascularized model, for testing dietary and redox-targeted treatments.
Key results
ShownFour treatments (C7, linoleic acid, glucose, naloxone) each shifted fragmented mitochondria in patient fibroblasts toward connected networks. Naloxone gave the largest drop in glucose consumption and returned elevated ROS to control-comparable levels.
Branch counts rose and fragment counts fell under all treatments (C7 2.5 mmol/L, LA 25 µM, naloxone 1 µM every 12 h). Naloxone reduced glucose consumption most, reaching control level in VLCADD cells (n = 4); ROS fell to control-comparable levels (n = 3-4). · Rescue of mitochondrial network structure and…; Figure legends
ShownNaloxone restored vessel formation in 3D bioprinted constructs containing patient fibroblasts, outperforming glucose supplementation, with no effect in constructs containing healthy fibroblasts.
Patient fibroblasts cut total vessel length by approximately 30-40% and junction density by approximately 30-50% vs healthy controls. Vessel length went from 60-70% to 80-90% of control with glucose, and to healthy-control level with naloxone; junctions 50-70% to 70-80% (glucose) and 90-100% (naloxone). · Development of a 3D bioprinted, vascularized…; Figure legends
ShownUntreated patient fibroblasts carry a large transcriptional shift with a pro-fibrotic, stress-fiber-associated signature. Naloxone altered it extensively in patient cells but not in healthy cells.
Patient vs healthy: 6167 DEGs (FDR < 0.05; 3136 up, 3004 down). Naloxone vs untreated patient: 3695 DEGs (1764 up, 1931 down). Healthy cells with naloxone: no DEGs at FDR < 0.05. Matrix-production genes (SPARC, CCN2, COL11A1, THBS1) decreased; MMP1 and PLAU increased. · Naloxone modulates genes associated with stress…
ShownNaloxone reduced F-actin stress fibers in patient fibroblasts, in line with decreased RHOB and ACTC1 and increased ARHGAP22 expression.
Phalloidin intensity was higher in untreated patient fibroblasts than in healthy controls and decreased significantly after naloxone (n = 3, mean ± SEM). Treated cells showed thinner, more diffuse F-actin bundles. · Naloxone modulates genes associated with stress…; Figure legends
ShownNaloxone increased DNM1L, MFN2 and OXPHOS protein levels in the mitochondria-enriched fraction of patient fibroblasts; at baseline, DNM1L was lower in patient cells than in controls.
Immunofluorescence (at least 10 cells per condition) and fractionation immunoblots (three independent experiments, mean ± SEM) showed higher DNM1L and MFN2 after naloxone 1 µM every 12 h for 72 h. OXPHOS levels, reduced at baseline in all patient lines, rose in the mitochondria-enriched fraction. · Rescue of mitochondrial network structure and…; Figure legends
Methods, data and reproducibility
Patient and healthy fibroblasts, some carrying a mitochondrial GFP reporter, were treated with metabolic or NOX2-targeting compounds. Readouts were mitochondrial morphology, ROS, immunoblots, 3' RNA-seq and phalloidin staining. Fibroblasts were then co-printed with endothelial cells and ASCs in GelMA and vessel networks quantified.
- Skin fibroblasts: one healthy pediatric male donor and patients with LCHADD or VLCADD from three metabolic centers · human · n = healthy donor 1; LCHADD n = 3; VLCADD n = 1
- Adipose-derived stem cells from abdominoplasty donors, plus HDMVEC and HUVEC lines (hTert-EYFP) for bioprinted constructs · human
- 3' RNA-seq of untreated and naloxone-treated fibroblasts (72 h), blocked by cell line · human
- 3D bioprinted vascularized constructs with healthy or patient fibroblasts, with or without naloxone or glucose · human · n = n = 3-5 independent experiments per condition, two constructs each
- GelMA hydrogel rheology at three concentrations · n = at least three independent measurements per concentration
- Methods & tools
- MiMoQuant (YoloX-based mitochondrial quantification), Minimap2 v2.28, Rsubread v2.22.1, edgeR v4.6.3, Camera gene set test, MSigDB Hallmark v2024.1.Hs, GENCODE Release 47 (GRCh38.p14), PromethION 2 Solo (Oxford Nanopore), AngioTool, RegenHu 3D Discovery bioprinter, ElastoSense Bio rheometer, GraphPad Prism v11.0.0
- Reagents & environment
- R 4.5.0 for analysis (R 4.6.0 for plotting), edgeR 4.6.3, Rsubread 2.22.1, Minimap2 2.28, MSigDB Hallmark v2024.1.Hs, GENCODE Release 47, Prism 11.0.0.
- Reproducibility gaps
- No code or data URLs and no RNA-seq accession are given; Table S1 is cited for the healthy-cell DEG result. MiMoQuant is described as in revision. Sequencing was done by a commercial provider (XPseq Analytics).
Limitations (4)
- All work used dermal fibroblasts; the authors note that the pro-fibrotic phenotype may represent a broader mechanism in affected organs, which was not examined here. · stated by the authors
- The glucose dose used reflects emergency treatment during acute crises, not recommended daily carbohydrate intake, so comparisons with naloxone do not mirror routine dietary therapy. · stated by the authors
- Few biological sources: one healthy donor, three LCHADD lines and a single VLCADD line. This limits generalization, especially for VLCADD, and the controls cannot capture inter-individual variation. · noted in this summary
- NOX2-dependence of naloxone was not verified directly in this work, and no in vivo test was done. Glucose is given as 9 g/L in the text but 4.5 g/L in figure legends. · noted in this summary
Who should read:Read this if you work on fatty acid oxidation disorders or mitochondrial redox biology, or want a bioprinted vascularized fibroblast model for testing candidate rescue drugs.
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