224 The lipoxygenase inhibitor ML355 prevents covalent adduction of the corneocyte lipid envelope in a novel preclinical model of congenital ichthyosis

Abstract

Epidermal lipoxygenase (LOX) deficiency is a major cause of autosomal recessive congenital ichthyosis (ARCI), and it could also drive common skin diseases with reduced protein-bound ceramides. In this study, we validated a pharmacological model of epidermal LOX deficiency using the commercially available platelet 12S-LOX inhibitor ML355. ML355 inhibited methyl arachidonate LOX activity in differentiated human foreskin keratinocytes (HFK) lysates in a dose-dependent and noncompetitive manner, with an IC50 of ∼30 μM and >75% inhibition at 100 μM. ML355 did not compromise keratinocyte viability (assessed by trypan blue exclusion in HFKs), but caused dose-dependent barrier dysfunction in human epidermal equivalent (HEE) cultures (assessed by transepithelial electrical resistance, TEER). Protein-bound ceramides and omega-hydroxy fatty acids (analyzed by thin-layer chromatography) were reduced by ML355, while free lipids were relatively preserved. The histopathology and ultrastructure of ML355-treated HEEs were characterized by hyperkeratosis, cytosolic lipid droplet accumulation, and defective lamellar lipid processing, despite normal lamellar body structure and no overt signs of cellular toxicity. Lipid membranes were present surrounding the corneocyte protein envelope in ML355-treated HEEs, but unlike the corneocyte lipid envelopes (CLEs) in control HEEs, these could be removed by organic solvent treatment, indicating that ML355 inhibited covalent adduction of the CLE but not the delivery of CLE lipids. These results demonstrate that ML355 inhibits keratinocyte LOX activity and causes an ichthyosis-like phenotype in HEE cultures. ML355-treated HEEs should be a useful model to test treatments for ARCI and other diseases associated with epidermal LOX deficiency.