title: AMA0428, A Potent Rock Inhibitor, Attenuates Early and Late Experimental Diabetic Retinopathy.
abstract: 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.
  
  
  
  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).
  
  
  
  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.
  
  
  
  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.
authors.affiliation: a Department of Ophthalmology , KU Leuven-University of Leuven , Leuven , Belgium.|b Department of Ophthalmology , University Hospitals Ghent , Ghent , Belgium.
authors.forename: Karolien
authors.initials: K
authors.lastname: Hollanders
authors.affiliation: a Department of Ophthalmology , KU Leuven-University of Leuven , Leuven , Belgium.|c Department of Biology, KU Leuven-University of Leuven , Leuven , Belgium.
authors.forename: Inge Van
authors.initials: IV
authors.lastname: Hove
authors.affiliation: a Department of Ophthalmology , KU Leuven-University of Leuven , Leuven , Belgium.|c Department of Biology, KU Leuven-University of Leuven , Leuven , Belgium.
authors.forename: Jurgen
authors.initials: J
authors.lastname: Sergeys
authors.affiliation: a Department of Ophthalmology , KU Leuven-University of Leuven , Leuven , Belgium.
authors.forename: Tine Van
authors.initials: TV
authors.lastname: Bergen
authors.affiliation: a Department of Ophthalmology , KU Leuven-University of Leuven , Leuven , Belgium.|c Department of Biology, KU Leuven-University of Leuven , Leuven , Belgium.
authors.forename: Evy
authors.initials: E
authors.lastname: Lefevere
authors.affiliation: d Amakem NV , Diepenbeek , Belgium.
authors.forename: Nele
authors.initials: N
authors.lastname: Kindt
authors.affiliation: d Amakem NV , Diepenbeek , Belgium.
authors.forename: Karolien
authors.initials: K
authors.lastname: Castermans
authors.affiliation: a Department of Ophthalmology , KU Leuven-University of Leuven , Leuven , Belgium.|e Department of Ophthalmology , University Hospitals Leuven, KU Leuven-University of Leuven , Leuven , Belgium.
authors.forename: Evelien
authors.initials: E
authors.lastname: Vandewalle
authors.affiliation: f Department of Hepatology , University Hospitals Leuven, KU Leuven-University of Leuven , Leuven , Belgium.
authors.forename: Jos
authors.initials: J
authors.lastname: van Pelt
authors.affiliation: c Department of Biology, KU Leuven-University of Leuven , Leuven , Belgium.
authors.forename: Lieve
authors.initials: L
authors.lastname: Moons
authors.affiliation: a Department of Ophthalmology , KU Leuven-University of Leuven , Leuven , Belgium.|e Department of Ophthalmology , University Hospitals Leuven, KU Leuven-University of Leuven , Leuven , Belgium.
authors.forename: Ingeborg
authors.initials: I
authors.lastname: Stalmans
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental; D003930:Diabetic Retinopathy / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D018450:Disease Progression; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D012160:Retina / Q000473:pathology*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
chemical_list: D004791:Enzyme Inhibitors; D016328:NF-kappa B; D042461:Vascular Endothelial Growth Factor A; D054460:rho-Associated Kinases
keywords: Anti-vascular endothelial growth factor; diabetic retinopathy; oxygen-induced retinopathy model; rho kinase inhibition; streptozotocin-induced retinopathy model