Background Corneal transplant failure with neovascularisation is a leading indication for full-thickness grafts in patients. Lymphangiogenesis is implicated in the pathology of graft failure, and here we systematically evaluate failed human corneal transplants with neovascularisation for the presence of lymphatic vessels.
Methods Nine failed grafts with neovascularisation, based on H&E staining with subsequent immunoperoxidase staining for CD31, a blood vessel marker, were selected. Lymphatics were investigated by immunohistochemical and immunofluorescence approaches using podoplanin as a lymphatic marker. In two of nine cases, fluorescence in situ hybridisation (FISH) was used for detection of lymphatic mRNAs including podoplanin, VEGFR-3 and LYVE-1. All immunofluorescence and FISH samples were compared with positive and negative controls and visualised by confocal microscopy.
Results Corneal neovascularisation was established in all cases by H&E and further confirmed by CD31 immunoreactive profiles. Immunohistochemistry for the podoplanin antibody was positive in all cases and showed morphologies ranging from distinct luminal structures to elongated profiles. Simultaneous immunofluorescence using CD31 and podoplanin showed lymphatic vessels distinct from blood vessels. Podoplanin immunofluorescence was noted in seven of nine cases and revealed clear lumina of varying sizes, in addition to lumen-like and elongated profiles. The presence of lymphatic mRNA was confirmed by FISH studies using a combination of at least two of podoplanin, VEGFR-3 and LYVE-1 mRNAs.
Conclusions The consistent finding of lymphatic vessels in failed grafts with neovascularisation implicates them in the pathogenesis of corneal transplant failure, and points to the lymphatics as a potential new therapeutic target.
- experimental – laboratory
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Contributors MAD, XZ, YG and EG, completed all experimental components and contributed to interpretation of results. MAD, SWSC, YG, YY and NG contributed to experimental design and interpretation of results. MAD, SWSC, YY and NG wrote the paper.
Funding We are grateful to the Harcharan & Prem Singh Foundation and Canadian Institutes of Health Research (MOP119432; YHY, NG).
Competing interests None declared.
Patient consent Not required.
Ethics approval St. Michael's Hospital Research Ethics Board.
Provenance and peer review Not commissioned; externally peer reviewed.
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