ROCK as a Therapeutic Target of Diabetic Retinopathy

Abstract

The increasing global prevalence of diabetes is a critical problem for public health. In particular, diabetic retinopathy, a prevalent ocular complication of diabetes mellitus, causes severe vision loss in working population. A better understanding of the pathogenesis and the development of new pharmacologic treatments are needed. This paper describes the relevance between Rho/ROCK pathway and the pathogenesis of diabetic retinopathy from its early to late stages. Moreover, the therapeutic potential of ROCK inhibitor in the total management of diabetic retinopathy is discussed.

Figure 1

Rho/ROCK activation in retinal vessels. In immunohistochemical analysis, RhoA (a), ROCK1 (b) and ROCK2 (c) were detected in retinal vessels. Yellow (white arrowhead) indicates double-stained vasculature (magnification: ×400). (d and e) The levels of Rho-GTP were significantly higher in streptozotocin induced-diabetic rat bretinas, compared with those in nondiabetic control, detected by Rho pull-down assay. Average signal intensities are quantified and expressed as percentage of the ratio of control (**P < .01, n = 5 each). Prevention of leukocyte-induced retinal endothelial damage by fasudil. (f–i) In vivo visualization of adhering leukocytes (green, concanavalin A lectin) and injured endothelial cells (red, propidium iodide (PI)) and endothelial nuclei (blue, DAPI) in rat retinas. PI positive cells (white arrowhead) widely coincided with adherent leukocytes (white arrow). The number of PI positive cells per retina was significantly higher in the diabetic animals, compared with the nondiabetic controls. Fasudil caused a significant reduction in the number of PI positive cells in the retinas of the diabetic animals, compared with the vehicle-treated controls (**P < .01, N.S., not significant, n = 5 each).

Figure 2

Impact of fasudil on PDR vitreous-induced collagen gel contraction and MLC phosphorylation. After pretreatment with or without anti-TGF-β mAb or fasudil, hyalocytes were stimulated with vitreous with PDR. (a) In hyalocyte-containing collagen gels, fasudil almost completely suppressed the contraction of collagen gels treated with PDR vitreous. The diameter of the gels was measured and statistically analyzed (*P < .05; **P < .01; NS, not significant, n = 3 each). (b) Western blot analysis was performed to detect phosphorylated MLC (pMLC). Fasudil abolished MLC phosphorylation, induced by PDR vitreous. Lane-loading differences were normalized by MLC. Signal intensities were quantified and expressed as percentages of the pMLC/MLC ratio compared with control (*P < .05; **P < .01, n = 3 each). Experimental PVR in rabbit eyes. (c) Therapeutic potential of fasudil in reducing the progression of experimental PVR. PVR was classified into six stages (0–5). Rhombus, vehicle (n = 5); purple square, fasudil 10 μM (n = 5); trigone, fasudil 30 μM from stage 2 (n = 6); blue square, fasudil 30 μM (n = 5) (*P < .05, **P < .01, not significant versus vehicle). (d) Tractional retinal detachment because of formation and cicatricial contraction of preretinal proliferative membrane was observed by stereomicroscopy in vehicle-treated eyes (stage 5 PVR). (g) In contrast, intravitreal membranes adhered to the retina without causing retinal detachment (arrowhead) in 30 μM fasudil-treated eyes with stage 2 PVR. Micrographs depict α-SMA expression (brown) in preretinal proliferative membrane with stage 5 PVR (e) and stage 2 PVR (h) by immunohistochemical analysis. (Scale bar, 200 μm). (f and i) Magnified images of (e) and (h),respectively, (Scale bar, 10 μm).

Figure 2

Impact of fasudil on PDR vitreous-induced collagen gel contraction and MLC phosphorylation. After pretreatment with or without anti-TGF-β mAb or fasudil, hyalocytes were stimulated with vitreous with PDR. (a) In hyalocyte-containing collagen gels, fasudil almost completely suppressed the contraction of collagen gels treated with PDR vitreous. The diameter of the gels was measured and statistically analyzed (*P < .05; **P < .01; NS, not significant, n = 3 each). (b) Western blot analysis was performed to detect phosphorylated MLC (pMLC). Fasudil abolished MLC phosphorylation, induced by PDR vitreous. Lane-loading differences were normalized by MLC. Signal intensities were quantified and expressed as percentages of the pMLC/MLC ratio compared with control (*P < .05; **P < .01, n = 3 each). Experimental PVR in rabbit eyes. (c) Therapeutic potential of fasudil in reducing the progression of experimental PVR. PVR was classified into six stages (0–5). Rhombus, vehicle (n = 5); purple square, fasudil 10 μM (n = 5); trigone, fasudil 30 μM from stage 2 (n = 6); blue square, fasudil 30 μM (n = 5) (*P < .05, **P < .01, not significant versus vehicle). (d) Tractional retinal detachment because of formation and cicatricial contraction of preretinal proliferative membrane was observed by stereomicroscopy in vehicle-treated eyes (stage 5 PVR). (g) In contrast, intravitreal membranes adhered to the retina without causing retinal detachment (arrowhead) in 30 μM fasudil-treated eyes with stage 2 PVR. Micrographs depict α-SMA expression (brown) in preretinal proliferative membrane with stage 5 PVR (e) and stage 2 PVR (h) by immunohistochemical analysis. (Scale bar, 200 μm). (f and i) Magnified images of (e) and (h),respectively, (Scale bar, 10 μm).

Figure 3

Contractile impacts of ROCK activation in rat retinal vasculature. Intravitreal injections ofllysophosphatidic acid (LPA), Rho activator, were performed into rat's eyes over a period of 1 minute with a 33-gauge needle. The final intraocular concentration of LPA was 20 μM. We monitored the retinal fluorescein with a scanning laser ophthalmoscope ((a) no injection, (b) 5 minutes after injection, (c) 10 minutes after injection). Intravitreal injection of induced sever retinal vessel contraction.

Figure 4

Rho/ROCK pathway and its activation in the development of diabetic retinopathy. RhoA is a small GTP-binding protein, and Rho-kinase (ROCK) is its target protein. Glucose, TNF-α, and TGF-β, elevated in diabetic serum or vitreous, activate Rho/ROCK pathway in endothelial cells or hyalocytes. ROCK activation induces endothelial damage mediated through inactivation of endothelial nitric oxide synthase (eNOS), which has endothelial protective potential. Moreover, ROCK causes firm leukocyte adhesion through the increase of ICAM-1 expression and activation of ezrin, radixin, and moesin (ERM) in endothelial cells. ROCK also has important roles such as leukocyte adhesion, endothelial migration, and contraction of proliferative membrane mediated through myosin light chain (MLC) phosphorylation in diabetic retinopathy. These findings suggest that elevated activity of the Rho/ROCK pathway is involved in the pathogenesis of diabetic microvascular damage, proliferative vitreoretinopathy, and retinal neovascularization.