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Rho kinase inhibition by fasudil ameliorates diabetes-induced microvascular damage.

Arita R, Hata Y, Nakao S, Kita T, Miura M, Kawahara S, Zandi S, Almulki L, Tayyari F, Shimokawa H, Hafezi-Moghadam A, Ishibashi T - Diabetes (2008)

Bottom Line: 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.The Rho/ROCK pathway plays a critical role in diabetic retinal microvasculopathy.ROCK inhibition may become a new strategy in the management of diabetic retinopathy, especially in its early stages.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

ABSTRACT

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.

Research design and 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.

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Impact of intravitreal fasudil on retinal ROCK activity. Phosphorylated MYPT-1 (Thr853) and eNOS (Ser1177) were detected in rat retinal preparations by Western blot analysis. Lane loading differences were normalized by reblotting the membranes with an antibody against GAPDH. A: Representative results of phospho-MYPT-1 and GAPDH in rat retinas. B: ROCK activity was expressed as the ratio of phospho-MYPT-1 to GAPDH (**P < 0.01, NS; n = 6 each). C: Representative results of phospho-eNOS and GAPDH. D: Average signal intensities quantified and expressed as percentage of the ratio of control (**P < 0.05, NS; n = 6 each).
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f2: Impact of intravitreal fasudil on retinal ROCK activity. Phosphorylated MYPT-1 (Thr853) and eNOS (Ser1177) were detected in rat retinal preparations by Western blot analysis. Lane loading differences were normalized by reblotting the membranes with an antibody against GAPDH. A: Representative results of phospho-MYPT-1 and GAPDH in rat retinas. B: ROCK activity was expressed as the ratio of phospho-MYPT-1 to GAPDH (**P < 0.01, NS; n = 6 each). C: Representative results of phospho-eNOS and GAPDH. D: Average signal intensities quantified and expressed as percentage of the ratio of control (**P < 0.05, NS; n = 6 each).

Mentions: To examine the efficacy of fasudil for ROCK inhibition in the retina, we quantified the amount of phosphorylated MYPT-1 and eNOS, downstream targets of ROCK. In line with our Rho activity data (Fig. 1D and E), MYPT-1 phosphorylation was markedly increased in the retinas of vehicle-treated diabetic rats, compared with the vehicle-treated nondiabetic controls (2.7-fold, n = 6 each, P < 0.01). In comparison, treatment with fasudil in diabetic rats significantly reduced the increase of phosphorylated MYPT-1 by 72% (n = 6 each, P < 0.01) (Fig. 2B).


Rho kinase inhibition by fasudil ameliorates diabetes-induced microvascular damage.

Arita R, Hata Y, Nakao S, Kita T, Miura M, Kawahara S, Zandi S, Almulki L, Tayyari F, Shimokawa H, Hafezi-Moghadam A, Ishibashi T - Diabetes (2008)

Impact of intravitreal fasudil on retinal ROCK activity. Phosphorylated MYPT-1 (Thr853) and eNOS (Ser1177) were detected in rat retinal preparations by Western blot analysis. Lane loading differences were normalized by reblotting the membranes with an antibody against GAPDH. A: Representative results of phospho-MYPT-1 and GAPDH in rat retinas. B: ROCK activity was expressed as the ratio of phospho-MYPT-1 to GAPDH (**P < 0.01, NS; n = 6 each). C: Representative results of phospho-eNOS and GAPDH. D: Average signal intensities quantified and expressed as percentage of the ratio of control (**P < 0.05, NS; n = 6 each).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2606876&req=5

f2: Impact of intravitreal fasudil on retinal ROCK activity. Phosphorylated MYPT-1 (Thr853) and eNOS (Ser1177) were detected in rat retinal preparations by Western blot analysis. Lane loading differences were normalized by reblotting the membranes with an antibody against GAPDH. A: Representative results of phospho-MYPT-1 and GAPDH in rat retinas. B: ROCK activity was expressed as the ratio of phospho-MYPT-1 to GAPDH (**P < 0.01, NS; n = 6 each). C: Representative results of phospho-eNOS and GAPDH. D: Average signal intensities quantified and expressed as percentage of the ratio of control (**P < 0.05, NS; n = 6 each).
Mentions: To examine the efficacy of fasudil for ROCK inhibition in the retina, we quantified the amount of phosphorylated MYPT-1 and eNOS, downstream targets of ROCK. In line with our Rho activity data (Fig. 1D and E), MYPT-1 phosphorylation was markedly increased in the retinas of vehicle-treated diabetic rats, compared with the vehicle-treated nondiabetic controls (2.7-fold, n = 6 each, P < 0.01). In comparison, treatment with fasudil in diabetic rats significantly reduced the increase of phosphorylated MYPT-1 by 72% (n = 6 each, P < 0.01) (Fig. 2B).

Bottom Line: 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.The Rho/ROCK pathway plays a critical role in diabetic retinal microvasculopathy.ROCK inhibition may become a new strategy in the management of diabetic retinopathy, especially in its early stages.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

ABSTRACT

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.

Research design and 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.

Show MeSH
Related in: MedlinePlus