The molecular pathogenesis of Fuchs' endothelial corneal dystrophy.

In this doctoral thesis, we have investigated the pathogenesis of late-onset Fuchs’ endothelial corneal dystrophy (FECD).

FECD is a degenerative disorder of the corneal endothelium (CE). The CE is the inner layer of the cornea (the cornea is the transparent front part of the eye). The cornea does not contain blood vessels, but receives water and nutrients from the anterior eye chamber fluid (a.k.a. aqueous humor) and the tear film. To prevent the cornea from swelling (edema), the CE constantly pumps out excess fluid and waste products, safeguarding the normal corneal thickness and transparency.

FECD is characterized by a loss of CE cells (CECs), resulting in clouded vision, painful ulcerations and even blindness. Usually both eyes are affected. The prevalence of FECD is estimated at 4% of people older than 40. The only curative treatment consists of replacing the diseased CE layer with a healthy donor layer. FECD is currently the most common indication for this kind of surgery in the Western world. However, as an estimated 10 million people worldwide suffer from corneal blindness (awaiting corneal transplantation), and the prevalence of FECD is expected to rise with aging of the population, there is a need to develop alternative therapies. Therefore, we need a better understanding of the underlying molecular mechanisms of FECD. Significant efforts have been made to improve our understanding of the pathophysiology of FECD, but a unifying theory and tangible drug targets are lacking.

This doctoral thesis aimed to gain new insights into the molecular pathogenesis of FECD, and to differentiate between processes that are specific for FECD (primary, causative factors) and processes that are nonspecific (e.g., secondary to corneal edema), to direct the future development of targeted drug therapy.

To this end, we have analyzed the gene expression profile in CECs from patients with late-onset FECD, in comparison with normal controls (normal corneas from donors and eyes with malignant melanoma), and non-FECD edematous (NFE) corneas (including pseudophakic bullous keratopathy and decompensated corneal grafts). To study these gene expression profiles, we used microarray expression analysis, reverse transcriptase quantitative polymerase chain reaction, and RNA-sequencing technology. Subsequently, we have examined interesting findings at the protein level, using immunohistochemistry (IHC) and immunofluorescence on CE whole mount specimens and cross-sections of full-thickness corneas. Finally, we have measured the concentration of molecules of interest in the aqueous humor.

Novel methodological elements in this project are: 1) the use of RNA-sequencing to study the gene expression profile of CECs with FECD, 2) the comparison with a third group of NFE corneas to differentiate between specific and nonspecific findings in FECD, 3) the development of a semi-automated computer algorithm to analyze IHC staining.

The main achievement of this work is the identification of circulating fibrocytes and dendritic derivatives as a consistent finding in CE layers of patients with symptomatic late-onset FECD. This is novel, because, formerly, it was thought that the CE layer from patients with FECD contained only CECs. Circulating fibrocytes are bone marrow-derived, and play a role in normal wound healing, but also in pathogenic fibrosis.

Furthermore, our data indicate that oxidative phosphorylation, mitochondrial dysfunction, decreased unfolded protein response, inhibition of the oxidative stress response, and downregulation of complement system genes play a specific role in the pathogenesis of FECD, whereas other processes, such as premature cellular senescence, extracellular matrix deposition, epithelial-mesenchymal transition, and the newly identified circulating fibrocytes are nonspecific elements (which also play a role in NFE corneas).

Moreover, we have selected a set of 20 genes that are specifically differentially expressed in FECD versus normal and FECD versus NFE, including cochlin (COCH), olfactomedin like 1 (OLFML1) and scm-like with four mbt domains 2 (SFMBT2).

These results will impact the research field, as they lay the foundations for a novel branch of research into the role of circulating fibrocytes in FECD and other CE disorders. Further research is needed to characterize the recruitment, differentiation, and function of circulating fibrocytes in FECD.

Furthermore, patients with FECD might benefit from antifibrotic treatments that are being developed to treat fibrosing disorders of other organs, in which circulating fibrocytes are involved.

Moreover, the new insights into primary and secondary processes in the pathogenesis of FECD, will help direct the future development of non-surgical therapies (for prevention, treatment, and prevention of progression of FECD). In the long run, this should alleviate the burden that FECD imposes on corneal tissue banks, and help reduce corneal blindness worldwide.