Under normal conditions, the cornea, being the transparent “windscreen” of the eye, is free of both blood and lymphatic vessels. However, various diseases of the eye, like infections, can interfere with the balance between promoting and inhibiting factors, which leads to ingrowth of blood and lymphatic vessels. The newly formed lymphatic vessels increase the risk of graft rejection after subsequent corneal transplantation. Corneal transplantation is one of the most commonly performed transplantations worldwide, with more than 40,000 surgeries per year in Europe. To date, various anti-hem- and anti-lymphangiogenic treatment strategies have been developed specifically for the corneal vascular endothelial growth factor (VEGF) pathway. Currently, however, no treatment strategies are clinically available to specifically modulate lymphangiogenesis. In this review, we will give an overview about endogenous regulators of hem- and lymphangiogenesis and discuss potential new strategies for targeting pathological lymphangiogenesis. Furthermore, we will review recently identified modulators and demonstrate that the cornea is a suitable model for the identification of novel endogenous modulators of lymphangiogenesis. The identification of novel modulators of lymphangiogenesis and a better understanding of the signaling pathways involved will contribute to the development of new therapeutic targets for the treatment of pathological lymphangiogenesis. This, in turn, will improve graft rejection, not only for the cornea.

Lymphangiogenesis is a key player in several diseases such as tumor metastasis, obesity, and graft rejection. Endogenous regulation of lymphangiogenesis is only partly understood. Here we use the normally avascular cornea as a model to identify endogenous regulators of lymphangiogenesis. Quantitative trait locus analysis of a large low-lymphangiogenic BALB/cN x high-lymphangiogenic C57BL/6 N intercross and prioritization by whole-transcriptome sequencing identify a novel gene responsible for differences in lymphatic vessel architecture on chromosome 17, thecystathionine β-synthase (Cbs). Inhibition of CBS in lymphatic endothelial cells results in reduce proliferation, migration, altered tube-formation, and decrease expression of vascular endothelial growth factor (VEGF) receptor 2 (VEGF-R2) and VEGF-R3, but not their ligands VEGF-C and VEGF-D. Also in vivo inflammation-induced lymphangiogenesis is significantly reduce in C57BL/6 N mice after pharmacological inhibition of CBS. The results confirm CBS as a novel endogenous regulator of lymphangiogenesis acting via VEGF receptor 2 and 3-regulation and open new treatment avenues in diseases associated with pathologic lymphangiogenesis.

Central subendothelial geographic deposits are formed as a fibrillar layer (FL) in advanced Fuchs endothelial corneal dystrophy (FECD). Previous studies demonstrated a significant decrease in corneal endothelial cell (CEC) density and an increase in focal corneal backscatter in the FL area. The present study investigated the association of the FL with edema formation and its localization. Patients (n = 96) presenting for Descemet membrane endothelial keratoplasty (DMEK) for advanced FECD were included. Slit-lamp biomicroscopy with FECD grading was followed by Scheimpflug imaging with en face backscatter analysis and pachymetric analysis. FL dimensions were measured, and correlation with pachymetric values was performed. An FL was detected in 74% of all eyes (n = 71). Pachymetric values in FL-positive versus FL-negative eyes were for corneal thickness at the apex (ACT) 614 ± 52 µm and 575 ± 46 µm (p = 0.001), for peripheral corneal thickness at 1 mm (PCT_1mm) 616 ± 50 µm and 580 ± 44 µm (p = 0.002), for PCT_2mm 625 ± 48 µm and 599 ± 41 µm (p = 0.017), for PCT_3mm 651 ± 46 µm and 635 ± 40 µm (p = 0.128) and for PCT_4mm 695 ± 52 µm and 686 ± 43 µm (p = 0.435), respectively. Correlation analysis indicated a weak correlation for the FL maximum vertical caliper diameter with ACT and PCT_1mm values but no further relevant correlations. In FL-positive eyes, increased focal corneal backscatter and increased corneal thickness showed primarily central and inferotemporal localization. In conclusion, Scheimpflug imaging shows an association of the FL with increased corneal thickness in advanced FECD and shows localization of the FL and increased corneal thickness in the central and inferotemporal region. This may provide important information for progression assessment and therapeutic decision making in FECD patients in the future.

To investigate the genetic cause, clinical characteristics, and potential therapeutic targets of infantile corneal myofibromatosis. Case series with genetic and functional in vitro analyses. Four individuals from 2 unrelated families with clinical signs of corneal myofibromatosis were investigated. Exome-based panel sequencing for platelet-derived growth factor receptor beta gene (PDGFRB) and notch homolog protein 3 gene (NOTCH3) was performed in the respective index patients. One clinically affected member of each family was tested for the pathogenic variant detected in the respective index by Sanger sequencing. Immunohistochemical staining on excised corneal tissue was conducted. Functional analysis of the individual PDGFRB variants was performed in vitro by luciferase reporter assays on transfected porcine aortic endothelial cells using tyrosine kinase inhibitors. Protein expression analysis of mutated PDGFRB was analyzed by Western blot. Sequencing data, immunohistochemical stainings, functional analysis of PDGFRB variants, and protein expression analysis. We identified 2 novel, heterozygous gain-of-function variants in PDGFRB in 4 individuals from 2 unrelated families with corneal myofibromatosis. Immunohistochemistry demonstrated positivity for alpha-smooth muscle actin and β-catenin, a low proliferation rate in Ki-67 (< 5%), marginal positivity for Desmin, and negative staining for Caldesmon and CD34. In all patients, recurrence of disease occurred after corneal surgery. When transfected in cultured cells, the PDGFRB variants conferred a constitutive activity to the receptor in the absence of its ligand and were sensitive to the tyrosine kinase inhibitor imatinib. The variants can both be classified as likely pathogenic regarding the American College of Medical Genetics and Genomics classification criteria. We describe 4 cases of corneal myofibromatosis caused by novel PDGFRB variants with autosomal dominant transmission. Imatinib sensitivity in vitro suggests perspectives for targeted therapy preventing recurrences in the future. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Topical application of vascular endothelial growth factor A (VEGFA) inhibitors including Bevacizumab is used for antiangiogenic therapy at the ocular surface. While clinical studies have suggested that this approach is well-tolerated, the effect of the drug on limbal epithelial stem cells has not been studied. In this study, the effect of Bevacizumab on phenotype and functionality of putative limbal epithelial stem cells (SC) was investigated. The effect of Bevacizumab on human limbal epithelial cells was assessed in terms of metabolic activity and scratch wound closure. The different treatment groups featured no difference in proliferation and colony forming efficiency (CFE) of limbal epithelial cells or their putative SC marker expression. A significant delay in scratch closure of all the Bevacizumab-treated groups was detected at 4 h. RNA and protein quantification indicated a dose-responsive increase of keratin 3. VEGFA RNA expression also increased while VEGFC and D as well as VEGFR1, 2 and 3 were unchanged. This study highlights previously unknown effects of Bevacizumab on cultured putative limbal epithelial SC: a dose-related increase of keratin 3, an increase in VEGFA as well as a delay in scratch wound closure. These in vitro data should be considered when using Bevacizumab in the context of limbal epithelial SC transplantation.