Expression of Lymphangiogenic Markers in Rejected Human Corneal Buttons after Penetrating Keratoplasty

Abstract

Purpose: To investigate the extent and distribution of lymphangiogenesis in the rejected corneal graft, we determined the expression of several lymphangiogenic markers in rejected human corneal buttons.

Material and methods: Thirty-four corneal buttons were obtained from patients who underwent re-keratoplasty for graft rejection after penetrating keratoplasty. All corneas showed signs of rejection, such as, sudden mutton-fat keratic precipitates (KPs) or lines before re-keratoplasty. The corneas were halved, and one half was used for immunostaining and the other half was used for RT-PCR. Expression of vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-D, VEGFR-2, VEGFR-3, LYVE-1 and podoplanin were measured as lymphangiogenic markers. Four non-operated normal corneas were used as controls.

Results: Numerous podoplanin positive cells were found in the anterior and posterior stroma. However, LYVE-1 positive mature lymphatics were found only in herpetic keratitis (HK)-induced graft rejection, and not in pseudophakic bullous keratopathy (PBK). RT-PCR showed that levels of VEGF-A, VEGF-C, VEGFR-2, and VEGFR-3 mRNAs were elevated in rejected corneal buttons versus the non-operated control corneas. Based upon the pre-keratoplasty pathologic conditions, HK cases showed higher levels of VEGF-A and VEGFR-2 than PBK. The mRNA ratios (keratoplastic cornea/normal cornea) for VEGF-A and VEGFR-2 were 8.9 and 5.8, respectively.

Conclusions: The results suggested that the VEGF-A and the VEGFR-2 may be a more important pathway for lymphangiogenesis in rejected corneal grafts than the VEGFR-3. In addition, organized lymphangiogenesis is more prominent in HK than PBK.

Keywords:

• Lymphangiogenesis
• penetrating keratoplasty
• rejection
• VEGF
• VEGFR

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This work was supported by Advanced Science Research Program (grant no.: NRF-2012R1A2A2A02009081) through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology and partially by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant no.: HI13C0055).