The use of topical or intraocular therapeutic agents to treat endothelial pathology is an exciting objective that has been the subject of numerous research studies over the past years. Ideally, it would be a question of finding a substance that could induce the regeneration of the endothelial monolayer in case that the cell population undergoes a critical reduction, as in the case of surgical trauma, or reverse the degenerative cellular changes that occur in the dystrophies or in certain infections, situations all that commonly cause dysfunction in the endothelial cell metabolism with the consequent clinical repercussion in the form of edema and loss of corneal transparency. Said substance should also have an adequate safety profile, without local side effects and should not induce uncontrolled cell proliferation that could cause endothelialization of other tissues of the anterior segment such as the angle or the iris, or the formation of tumours¹.

To date there is no commercialized medical treatment that, either by preventing endothelial cell loss or by regenerating the endothelial monolayer, avoids the need for a corneal transplant. Below we detail the main molecules that have been or are subject of study to assess their possible mechanisms of action and indications in the medical treatment of endothelial pathology.

INFLAMMATORY MODULATORS

Several studies have shown how some classic antioxidants can mitigate the loss of endothelial cells under stress conditions, even in cells from patients with Fuchs' dystrophy. Molecules such as coenzyme Q-10², n-acetyl-cysteine³ and sulforaphane⁴ have aroused interest for their protective effect on endothelial cell metabolism. Sulforaphane is a natural isothiocyanate of plant origin that activates an antioxidant metabolic pathway through the nuclear transcription factor Nrf2, which is diminished in the cells of patients with Fuchs' dystrophy. In one study, this molecule was able to reduce cellular apoptosis in 55% of cells not subjected to stress and in 43% of cells subjected to metabolic stress with ter-butyl-hydroperoxide. Another antioxidant agent that is in a phase II clinical trial is RTA 408. It is a synthetic oleanane triterpenoid⁵ that stimulates the synthesis of Nrf2. It is formulated as an ophthalmic suspension and has been shown in preclinical studies to be effective in inducing the expression of antioxidant genes in the human corneal epithelium and endothelium as well as in the reduction of biological markers of postoperative inflammation. These results would open a new way for the use of protective agents that reduce the endothelial cell loss caused by cellular metabolic stress situations of diverse origins.

MAGNETIC CELLULAR ISOLATION

A method has been developed and patented to identify, isolate and enrich human endothelial cells by binding them to reagents or nanoparticles⁶ with positive magnetic affinity⁷. Through this method donor endothelial cells could be obtained, separating them from other possible cells that contaminate the cultures and inject them in the anterior chamber to achieve endothelial cell repopulation. An experimental study⁸ has been published demonstrating its efficacy and safety in the treatment of corneas of rabbits that underwent central endothelial surgical debridement.

FIBROBLASTS GROWTH FACTOR (FGF-1)

FGF-1, also known as acid FGF, is a powerful, broad-spectrum mitogen that promotes repair and regeneration of injured tissues. Its formula obtained by bioengineering (eFGF) has the potential to stimulate the proliferation of endothelial cells, increase their stability and favor their adhesion to the surrounding tissues. Experiments performed in vitro with corneas of rabbits that had been induced cryogenic endothelial damage demonstrated their stimulating effect on endothelial regeneration, compared with the control group. Although no adverse effects have been described in in vivo dosing schedules, hypothetically their angiogenic capacity and the possible elevation of intraocular pressure due to cellular proliferation in the trabecular meshwork would be limiting factors that should be evaluated in future clinical studies.

TRANSPLANTATION OF MESENQUIMAL STEM CELLS

Through the use of mesenchymal stem cells from bone marrow aspirates from healthy donors, a new cell therapy has been developed that is in the clinical trial phase in Japan. Positive results have also been obtained by using a conditioned medium derived from mesenchymal stem cells from the bone marrow (Figure 1). This medium stimulates the proliferation and motility of endothelial cells⁹, maintaining growth inhibition by contact and its endothelial pump functions. Exposure of human endothelial cells in culture to this medium would endow them with endothelial stem cell properties, which would allow the regeneration of the endothelial monolayer.

ROCK-KINASE INHIBITORS

The use of the ROCK kinase enzyme inhibitor Y-27632 (0.4% ripasudil hydrochloride, Glanatec^®)¹⁰ as an agent capable of stimulating the growth and regeneration of the endothelial monolayer has recently been described. Its clinical use in ocular hypertension and glaucoma has been approved in Japan in 2014. This substance is able to promote cell adhesion, inhibit apoptosis and improve endothelial cell proliferation and regeneration. There are contradictory studies¹¹ that show that it has no effect on endothelial proliferative capacity, although it does induce changes in cell shape, increases cell adhesion and improves the cicatricial response ex vivo and in vitro.

The rho-kinases are a family of GTP-ase that are involved in the regulation of the formation and adaptation of the cell cytoskeleton, the generation of focal areas of adhesion and cell motility, and the inhibition of apoptosis and gene transcription. The best known is the Rho-associated spiral-forming protein kinase (ROCK). The ROCK-kinase inhibitor would increase adhesion and cell repair mechanisms although it would not stimulate mitosis or cell proliferation. In recent experimental models¹² a significant improvement has been documented in animals treated with ripasudil eyedrops after surgical endothelial aggression. In the control group, corneal transparency was not achieved at the end of the study, while in the treated group it was achieved in 5 of 6 cases. At present, their combined use with cell transplantation techniques¹³ by injection in the anterior chamber of endothelial cells cultured ex vivo is being evaluated. The introduction of the ROCK inhibitor in the treatment after the cellular injection, would improve the adhesion and cellular repopulation allowing to improve the results of this technique.

At present, the knowledge of the pathophysiological behavior of the human endothelium and its biochemical and biological bases is being significantly deepened. Probably within a reasonable period of time we can assist to the emergence of new medical therapies that, through the modulation and stimulation of cell proliferation and repair mechanisms, succeed in eliminating the need to perform current endothelial cell transplants, obviating the appearance, although every time less frequent, of immunological reactions or the usual accelerated cell loss in the medium term observed with current techniques.