Sub-Anticoagulant Dose Heparin is a Potential Therapy for Tear Deficient Dry Eye Disease in patients with ocular Graft-Vs-Host Disease (oGVHD).

Abstract

Purpose : We have reported that neutrophil extracellular traps (NETs) can contribute to ocular surface pathology in patients with oGVHD. Our purpose is to investigate whether dismantling of NETs with heparin has therapeutic potential in oGVHD.

Methods : We performed in vitro experiments using cell lines (corneal epithelial cells and conjunctival fibroblast cells) in scratch wound models, and in vivo experiments using murine corneal epitheliopathy models, and compared the effects of NETs with heparinized NETs. Heparin eye drops (100 IU/ml) twice a day were given to a patient to treat severely symptomatic oGVHD.

Results : Incubation of NETs (generated from isolated peripheral blood human neutrophils) with sub-anticoagulant dose of Heparin (100 IU/mL) dismantled NETs as evidenced by the loss of Sytox green stained extracellular DNA strands and reduced levels of histone-associated DNA fragments (which represent intact NETs). Heparin 100 IU/mL dismantles NETs in mucocellular aggregates collected from oGVHD patients. Sub-anticoagulant dose of Heparin (100 IU/mL) did not delay epithelial wound closure whereas anticoagulant doses of heparin (1,000 IU/mL and 10,000 IU/mL) showed significant dose dependent delay in epithelial wound closure. LDH assay was performed using supernatant to determine cytotoxicity. Heparin (100 IU/mL) caused no cytotoxicity as compared to RPMI culture media whereas significant cytotoxicity was observed with heparin 1,000 IU/mL and 10,000 IU/mL. NETs delay epithelial healing, promote fibroblast proliferation and myofibroblast transformation and enhance allogeneic T-cell proliferation. Dismantling with sub-anticoagulant dose heparin abrogates NET-induced epithelial, fibroblast and T-cell affects, Heparin eye drops (100 IU/mL) twice a day were given to a patient with oGVHD who was severely symptomatic despite maximal topical therapy. After one month of heparin therapy, symptoms, as determined by OSDI, reduced from 67.5 to 19.4. Corneal staining decreased from 6/15 to 3/15 and conjunctival staining deceased from 4/6 to 1/6.

Conclusions : Sub-anticoagulant dose heparin (100 IU/mL) eye drop treatment has therapeutic potential in oGVHD. Our data makes a case for controlled clinical trials to investigate the efficacy of heparin eye drops to prevent or treat oGVHD.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.