AMA0428, A Potent Rock Inhibitor, Attenuates Early and Late Experimental Diabetic Retinopathy

ABSTRACT

Purpose: Diabetic retinopathy (DR) is characterized by an early stage of inflammation and vessel leakage, and an advanced vasoproliferative stage. Also, neurodegeneration might play an important role in disease pathogenesis. The aim of this study was to investigate the effect of the Rho kinase (ROCK) inhibitor, AMA0428, on these processes.

Methods: The response to ROCK inhibition by AMA0428 (1 µg) was studied in vivo using the murine model for streptozotocin (STZ)-induced diabetes, focusing on early non-proliferative DR features and the oxygen-induced retinopathy (OIR) model to investigate proliferative DR. Intravitreal (IVT) administration of AMA0428 was compared with murine anti-VEGF-R2 antibody (DC101, 6.2 µg) and placebo (H₂O/PEG; 1C8). Outcome was assessed by analyzing leukostasis using fluorescein isothiocyanate coupled concanavalin A (FITC-ConA) and vessel leakage (bovine serum albumin conjugated with fluorescein isothiocyanate; FITC-BSA)/neovascularization and neurodegeneration by immunohistological approaches (hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase–mediated biotinylated UTP nick end labeling (TUNEL), Brn3a). ELISA and Western blotting were employed to unravel the consequences of ROCK inhibition (1 µM AMA0428) on myosin phosphatase target protein (MYPT)-1 phosphorylation, endothelial nitric oxide synthase (eNOS) phosphorylation, and vascular endothelial growth factor (VEGF) levels in retinas of diabetic mice, on NF-κβ activity and ICAM-1 expression in endothelial cells (ECs).

Results: In vivo, AMA0428 significantly reduced vessel leakage and neovascularization, respectively, in the STZ and OIR model, comparable to DC101 therapy. Additionally, the ROCK inhibitor decreased neurodegeneration in both models and inhibited leukostasis by 30% (p < 0.05) in the STZ model (p < 0.05), while DC101 had no positive effect on the outcome of these latter processes. ROCK activity was upregulated in the diabetic retina and AMA0428 administration resulted in decreased phospho-MYPT-1, enhanced phospho-eNOS, and reduced VEGF levels. In vitro, AMA0428 interfered with NF-κβ activity, thereby inhibiting ICAM-1 expression in ECs.

Conclusions: Targeting ROCK with AMA0428 effectively attenuated outcome in an early DR model (STZ) and a late vasoproliferative retinopathy model (OIR). These findings make AMA0428 a promising candidate with an additional anti-inflammatory and neuroprotective benefit for DR patients, as compared with anti-VEGF treatment.

KEYWORDS:

• Anti-vascular endothelial growth factor
• diabetic retinopathy
• oxygen-induced retinopathy model
• rho kinase inhibition
• streptozotocin-induced retinopathy model

Acknowledgments

The authors thank Sofie Beckers, Ann Verbeek, Lut Noterdaeme, Lieve Geenen, and Martine Leijssen for their technical support. We thank Prof. Dr. Frederik Nevens, Prof. Dr. Schalk van der Merwe, Ingrid Vander Elst, and Petra Windmolders for their assistance in the integration of western blots. We are grateful to Prof. Dr. Jaan Toelen from the pediatric department for the use of the hyperoxygenation chamber. AMA0428 was kindly provided by Amakem Therapeutics; DC101 and 1C8 were kindly provided by Thrombogenics NV.

Declaration of interest

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

Funding

Karolien Hollanders was financially supported by Amakem Therapeutics; Inge Van Hove was financially supported by Amakem Therapeutics via IWT Innovation fellowship; and Tine Van Bergen was financially supported by ThromboGenics NV via IWT Innovation fellowship. Nele Kindt and Karolien Castermans are employees at Amakem Therapeutics. This study was supported by a grant from the “Funds for Research in Ophthalmology” (FRO) and by the “Fund John W. Mouton Pro Retina,” which is managed by the King Baudouin Foundation for medical scientific research. Ingeborg Stalmans holds a “Chair in Ophthalmology Translational Research,” which is supported by an unrestricted grant form Amakem Therapeutics. The PhD project from Karolien Hollanders, in which this study is performed, is supported with this chair.