Inhibition of corneal inflammation by the topical use of Ras farnesyltransferase inhibitors: selective inhibition of macrophage localization.
OBJECTIVE
Ras farnesyltransferase inhibitors are known to block the membrane translocalization of oncogenic Ras protein. They inhibit the cytoplasmic mitogen-activated protein kinase signaling cascade related to Ras protein. Thus far, Ras farnesyltransferase inhibitors have been exclusively regarded with the anticancer drugs. The object of this study was to elucidate the role of Ras farnesyltransferase inhibitors on the corneal opacity induced by an inflammatory stimulus.


METHODS
We used a cauterization-induced corneal inflammation model. The central corneas of BALB/c mice were cauterized with silver nitrate (1 mm in diameter). Ras farnesyltransferase inhibitors, either manumycin or gliotoxin eye drops (each drug dissolved in balanced salt solution [BSS] at concentrations of 1 mM), were topically delivered to the cauterized cornea every 8 hours; BSS eye drops were used as a control. Clinical signs such as corneal edema, opacity, and corneal neovascularization, which are major causes of visual disturbance, were then examined 96 hours after the cauterization. The corneal edema and opacity were clinically scored under a stereoscopic microscope. The corneal neovascularization was evaluated by the length of the blood vessels from the limbus and the sum of extension central angle of vascularized limbus. Furthermore, the corneas were examined histologically, and the phenotypes of the cornea-infiltrating cells were analyzed by flow cytometry.


RESULTS
The control corneas showed prominent edema, neovascularization, and opacity. Histologic analysis revealed corneal epithelial and endothelial cell loss and a large amount of inflammatory cell infiltration into the corneal stroma. Flow cytometric analysis revealed that most of the infiltrating cells were neutrophils and macrophages. In contrast, the degree of corneal edema, neovascularization, and opacity was significantly less in the manumycin- or gliotoxin-treated corneas than in the control corneas. Histologically, the manumycin- and gliotoxin-treated corneas showed minimum edema and good epithelialization. Flow cytometric analysis showed corneal infiltration of macrophages to be selectively and clearly inhibited. Neither manumycin nor gliotoxin produced any side effects in the noncauterized normal cornea either clinically or histologically.


CONCLUSIONS
Ras proteins play an important role in cauterization-induced corneal inflammation and the opacity it induces. Ras farnesyltransferase inhibitors thus have a great potential for improving the treatment of corneal opacity induced by a corneal inflammatory stimulus.
Sonoda
K
K
Department of Ophthalmology, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Sakamoto
T
T
Yoshikawa
H
H
Ashizuka
S
S
Ohshima
Y
Y
Kishihara
K
K
Nomoto
K
K
Ishibashi
T
T
Inomata
H
H
D000287:Administration, Topical; D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors*; D000818:Animals; D002452:Cell Count; D015715:Corneal Edema / Q000209:etiology / Q000473:pathology / Q000517:prevention & control; D016510:Corneal Neovascularization / Q000209:etiology / Q000473:pathology / Q000517:prevention & control; D003318:Corneal Opacity / Q000209:etiology / Q000473:pathology / Q000517:prevention & control; D004728:Endothelium, Corneal / Q000473:pathology; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D019573:Epithelium, Corneal / Q000473:pathology; D005260:Female; D005434:Flow Cytometry; D005912:Gliotoxin / Q000008:administration & dosage / Q000494:pharmacology*; D007634:Keratitis / Q000139:chemically induced / Q000201:enzymology / Q000473:pathology / Q000517:prevention & control*; D008264:Macrophages / Q000187:drug effects* / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D009883:Ophthalmic Solutions; D011090:Polyenes / Q000008:administration & dosage / Q000494:pharmacology*; D053284:Polyunsaturated Alkamides
D004791:Enzyme Inhibitors; D009883:Ophthalmic Solutions; D011090:Polyenes; D053284:Polyunsaturated Alkamides; D005912:Gliotoxin; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; C054474:manumycin