Local Rho kinase inhibition, an innovative treatment strategy in the fight against blinding retinopathies

Age-related macular degeneration (AMD), retinopathy of prematurity (ROP) and diabetic retinopathy (DR) are common eye diseases that are the most frequent causes of vision loss due to the formation of blood vessels (angiogenesis), inflammation and scarring (fibrosis). Moreover, they are associated with loss of neuroretinal cells. The available therapies are not effective enough, and carry the risk of life- and vision-threatening complications. We therefore aimed at developing new therapies for these retinopathies. The Rho family of small GTPases, with its downstream effectors ROCK 1 andnbsp;2, controls a diverse array of cellular processes, such as gene expression, proliferation, survival, migration and permeability. Therefore, Rho and ROCK respond to cell surface receptors for various cytokines, adhesion molecules and different growth factors, such as vascular endothelial growth factor (VEGF). Pharmacological inhibition of ROCK strongly disrupts VEGF-mediated angiogenesis, suggesting that it may be a new way to treat angiogenesis-related disorders, such as proliferative retinopathies. Moreover, a potential advantage of ROCK inhibitors, compared to anti-VEGF therapy, would be the additional effect on inflammation and fibrosis, which has already been shown in different models. Indeed, ROCK is a key mediator in several inflammatory processes, such as transcription of inflammatory cytokines through NF-κβ activation and anchoring of leukocytes. Once scarring has developed at the retina, no therapy can restore vision in an eye. Therefore, if ROCK inhibition might be able to prevent scar formation, vision might even be improved. Recent studies revealed that ROCK is also associated with neurodegeneration and suggest that ROCK inhibition might protect against neuronal damage.nbsp;This project was designed to investigate a locally acting ROCK inhibitor, AMA0428 (developed by Amakem Therapeutics), for AMD, ROP and DR. ROCK inhibitors have anti-angiogenic, -inflammatory, -fibrotic and neuroprotective capacities, and might therefore offer a superior strategy in the treatment of these retinopathies. Due to its local mode of action, AMA0428 might have an additional therapeutic benefit compared to anti-VEGF therapy, minimizing systemic side effects. Within a first objective of this thesis, we demonstrated the in vitro effect of the ROCK inhibitor, AMA0428, on cell apoptosis, proliferation, migration, cell morphology and cell surface proteins in human endothelial cells (HEC) and human pericytes (HP). A caspase 3/7 apoptosis assay showed no toxic effects of the compound up to 10µM for all cell types. Proliferation was assessed using a WST-1 assay and showed a decrease in HEC proliferation after treatment with AMA0428, but no clear effect wasnbsp;on cell proliferation of HP, neither stimulation nor inhibition. To investigate the effect on migration, we used the scratchnbsp;and the under-agarose recruitment assay. After treatment with AMA0428, HEC showed a dose-dependent decrease in migration, whereas migration and recruitment of HP was stimulated after exposure to AMA0428. This effect was confirmed by immuonostaining for actin, vinculin and phosphorylated myosin light chain, which showed a migratory phenotype when pericytes were exposed to the ROCK inhibitor. Additionally, ELISA experiments revealed thatkappa;β activity in HEC was impaired after AMA0428 treatment. This result was validated by Western blot, where AMA0428 exposure inhibited ICAM-1 expression in HEC, which is coordinated via the NF-beta; pathway.These in vitro data point to a possible attenuating effect of AMA0428 on neovascularization and inflammatory changes in vivo. Indeed targeting ROCK inhibits HEC proliferation and migration; and might promote maturation of blood vessels by stimulating pericyte recruitment. Additionally, the Rho/ROCK pathway is involved in NF- beta; activity and ICAM-1 expression on vascular EC, which is important in leukocyte adhesion and thus inflammation. In the second approach taken within this PhD project, we explored the in vivo effect of ROCK inhibition by AMA0428 on inflammation, neovascularization and fibrosis in AMD, ROP and DR via three different mouse models: the laser-induced choroidal neovascularization (CNV) model, the oxygen-induced retinopathy (OIR) model and the streptozotocin (STZ)-induced diabetic model. Before exploring the effect of AMA0428, the models were optimized and validated using murine and human anti-VEGF. Next, the potential side effects of intravitreally administered AMA0428 was investigated, but no significant toxicity to the neurosensory retina and its blood vessels was observed in mice. In the CNV model, ROCK activity and VEGF and PlGF levels were significantly increased in choroid and retina compared to naïve mice. However, after AMA0428 injections all factors were reduced, strengthening the hypothesis that ROCK inhibition may be a new way to address the underlying pathophysiology.The inflammatory process has been found to play a crucial role in various retinopathies. In the current study, an anti-inflammatory effect was demonstrated after intravitreal administration of AMA0428 in the laser-induced CNV model as well as in the STZ-induced diabetes model, whereas anti-VEGF therapy did not affect the inflammatory process in either model. Intravitreal administration with AMA0428 also impaired neovascularization/vessel leakage consistently in all three models, similar to anti-VEGF injections. Moreover, a combination therapy of AMA0428 and anti-VEGF allowed reducing the dose of anti-VEGF and thus might lower the side effects, while maintaining the efficacy of the full dose of anti-VEGF therapy. These data implicate that ROCK inhibition might be a new approach as alternative or additive to anti-VEGF in the treatment of retinopathies, with an improved safety profile. Besides inflammation and neovascularization, there is an important fibrotic response in most retinopathies that might cause severe visual impairment due to tractional retinal detachment. We demonstrated that injections with AMA0428 effectively reduced fibrosis in the CNV model, whereas anti-VEGF had no clear effect.Overall, intravitreal administration of AMA0428 consistently induced a decrease in inflammation, neovascularization, vessel leakage and fibrosis in three independent mouse models for neovascular AMD, ROP and DR. In the third scope of this thesis, we studied the neuroprotective potential of AMA0428. Recent studies indicate that patients with ROP or DR might suffer vision loss due to neuronal degeneration. ROCK inhibitors might have neuroprotective capacities by inhibiting apoptosis and promoting regeneration of neuronal cells. In the current study, AMA0428 indeed significantly reduced apoptosis of retinal neurons in the OIR and the STZ model, pointing to a possible neuroprotective feature of AMA0428 treatment. Finally, we investigated the effect of AMA0428 administration on gene expression in the STZ model. The expression of genes involving eye development, like crystallins, which stimulate axonnbsp;was up-regulated in the retina after AMA0428 treatment. This might suggest that AMA0428 treatment induces the expression of specific genes that could help in restoring the damage caused by diabetes. The expression of cell signaling genes was down-regulated, suggesting a potential restore mechanism by AMA0428 on diabetes-inflicted damage to cell communication.nbsp;In summary, this project elucidated the therapeutic potential of ROCK inhibition in various retinopathies, such as neovascular age-related macular degeneration, retinopathy of prematurity and diabetic retinopathy. The used locally acting ROCK inhibitor, AMA0428, was able to attenuate inflammation, neovascularization, vessel leakage, fibrosis and neurodegeneration in three different mouse models. This pleiotropic working mechanism ofnbsp;Rho/ROCK pathway, make ROCK inhibitors anbsp;and innovative treatment strategy that may radically change the quality of life for many patients that are suffering from these retinopathies, and whose vision is at risk.

Publication year:2015

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