Rho kinase inhibitor AMA0526 improves surgical outcome in a rabbit model of glaucoma filtration surgery.
OBJECTIVE
First, to elucidate the effect of Rho kinase inhibitor, AMA0526, on Human Tenon Fibroblast (HTF) proliferation and transdifferentiation to myofibroblasts. Second, the effects of ROCK inhibition on the wound healing process and surgical outcome were investigated in a rabbit model of glaucoma filtration surgery.


METHODS
After exposure of HTF to AMA0526 (0.1-25 μM), a water-soluble tetrazolium salt-1 assay and caspase 3/7 activity assay were used to assess its effect on cell proliferation and to elucidate any toxic effects, respectively. Immunohistochemistry of α-smooth muscle actin expression was used to investigate fibroblast-to-myofibroblast differentiation induced by transforming growth factor-beta 1 (TGF-β1) in the presence or absence of the ROCK inhibitor. The effect of topical treatment was studied in a rabbit model of glaucoma filtration surgery. Treatment outcome was studied by performing intraocular pressure (IOP) measurements and clinical investigation of the bleb area and survival. Immunohistological analysis for inflammation (CD45), angiogenesis (CD31), and collagen I was performed at day 8, 14, and 30 after surgery (n=5/time point). Separate control groups treated with vehicle were used as control.


RESULTS
In vitro results showed that AMA0526 dose dependently inhibited proliferation of HTF (P<0.05) without the induction of caspase 3/7 activity. Incubation of HTF with the AMA0526 inhibited TGF-β1 induced fibroblast-to-myofibroblast differentiation. In the rabbit model, topical treatment significantly improved surgical outcome. Compared to vehicle-treated eyes, AMA0526 resulted in increased bleb area (P<0.0001) and prolonged survival (P=0.0025). IOP remained significantly lower throughout the course of the experiment in the AMA0526 group (P<0.0001). Histological evaluation revealed that blebs treated with the ROCK inhibitor were characterized by reduced inflammation, angiogenesis, and collagen deposition at the site of filtration surgery (P<0.05).


CONCLUSIONS
AMA0526 had profound effects on HTF proliferation and myofibroblast transition and improved glaucoma filtration surgery outcome by interfering at different levels of the wound healing process. Therefore, these data indicate that ROCK inhibitors may be considered as more physiological agents which specifically target the wound healing process to improve the outcome of glaucoma surgery.
Laboratory of Ophthalmology, KU Leuven, Leuven, Belgium.
Sarah
S
Van de Velde
Laboratory of Ophthalmology, KU Leuven, Leuven, Belgium.
Tine
T
Van Bergen
Laboratory of Ophthalmology, KU Leuven, Leuven, Belgium; Department of Ophthalmology, University Hospitals Leuven (UZ Leuven), Leuven, Belgium.
Evelien
E
Vandewalle
Amakem Therapeutics, Diepenbeek, Belgium.
Nele
N
Kindt
Amakem Therapeutics, Diepenbeek, Belgium.
Karolien
K
Castermans
Research Group of Neural Circuit Development and Regeneration, KU Leuven, Leuven, Belgium.
Lieve
L
Moons
Laboratory of Ophthalmology, KU Leuven, Leuven, Belgium; Department of Ophthalmology, University Hospitals Leuven (UZ Leuven), Leuven, Belgium. Electronic address: ingeborg.stalmans@uzleuven.be.
Ingeborg
I
Stalmans
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000627:therapeutic use*; D018463:Filtering Surgery / Q000009:adverse effects*; D005901:Glaucoma / Q000188:drug therapy* / Q000601:surgery*; D006801:Humans; D008297:Male; D011183:Postoperative Complications / Q000517:prevention & control; D011817:Rabbits; D018709:Statistics, Nonparametric; D013997:Time Factors; D014746:Vimentin / Q000378:metabolism
D015703:Antigens, CD; D004791:Enzyme Inhibitors; D014746:Vimentin; D020169:Caspases
Fibrosis; Glaucoma filtration surgery; Rho kinase inhibitors; Wound healing