Rho kinase inhibition by fasudil ameliorates diabetes-induced microvascular damage.
OBJECTIVE
Leukocyte adhesion in retinal microvasuculature substantially contributes to diabetic retinopathy. Involvement of the Rho/Rho kinase (ROCK) pathway in diabetic microvasculopathy and therapeutic potential of fasudil, a selective ROCK inhibitor, are investigated.


METHODS
Localization of RhoA/ROCK and Rho activity were examined in retinal tissues of rats. Impact of intravitreal fasudil administration on retinal endothelial nitric oxide synthase (eNOS) and myosin phosphatase target protein (MYPT)-1 phosphorylation, intercellular adhesion molecule-1 (ICAM-1) expression, leukocyte adhesion, and endothelial damage in rat eyes were investigated. Adhesion of neutrophils from diabetic retinopathy patients or nondiabetic control subjects to cultured microvascular endothelial cells was quantified. The potential of fasudil for endothelial protection was investigated by measuring the number of adherent neutrophils and terminal transferase-mediated dUTP nick-end labeling-positive endothelial cells.


RESULTS
RhoA and ROCK colocalized predominantly in retinal microvessels. Significant Rho activation was observed in retinas of diabetic rats. Intravitreal fasudil significantly increased eNOS phosphorylation, whereas it reduced MYPT-1 phosphorylation, ICAM-1 expression, leukocyte adhesion, and the number of damaged endothelium in retinas of diabetic rats. Neutrophils from diabetic retinopathy patients showed significantly higher adhesion to cultured endothelium and caused endothelial apoptosis, which was significantly reduced by fasudil. Blockade of the Fas-FasL interaction prevented endothelial apoptosis. The protective effect of fasudil on endothelial apoptosis was significantly reversed by Nomega-nitro-l-arginine methyl ester, a NOS inhibitor, whereas neutrophil adhesion remained unaffected.


CONCLUSIONS
The Rho/ROCK pathway plays a critical role in diabetic retinal microvasculopathy. Fasudil protects the vascular endothelium by inhibiting neutrophil adhesion and reducing neutrophil-induced endothelial injury. ROCK inhibition may become a new strategy in the management of diabetic retinopathy, especially in its early stages.
Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Ryoichi
R
Arita
Yasuaki
Y
Hata
Shintaro
S
Nakao
Takeshi
T
Kita
Muneki
M
Miura
Shuhei
S
Kawahara
Souska
S
Zandi
Lama
L
Almulki
Faryan
F
Tayyari
Hiroaki
H
Shimokawa
Ali
A
Hafezi-Moghadam
Tatsuro
T
Ishibashi
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002448:Cell Adhesion / Q000187:drug effects; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism / Q000503:physiopathology*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008297:Male; D008856:Microscopy, Fluorescence; D055806:Microvessels / Q000187:drug effects* / Q000473:pathology; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D047428:Protein Kinase Inhibitors; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D052250:Nitric Oxide Synthase Type III; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; C049347:fasudil