ROCK as a Therapeutic Target of Diabetic Retinopathy

Figure 2

Impact of fasudil on PDR vitreous-induced collagen gel contraction and MLC phosphorylation. After pretreatment with or without anti-TGF-$\beta$ 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 (${}^{*}P<.05;\mathrm{\hspace{0.17em}\hspace{0.17em}}{}^{**}P<.01$; 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 (${}^{*}P<.05;\mathrm{\hspace{0.17em}\hspace{0.17em}}{}^{**}P<.01,\mathrm{\hspace{0.17em}\hspace{0.17em}}n=3$ 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 $\mu$M ($n=\mathrm{\hspace{0.17em}\hspace{0.17em}}5$); trigone, fasudil 30 $\mu$M from stage 2 ($n=6$); blue square, fasudil 30 $\mu$M ($n=5$) (${}^{*}P<.05,\mathrm{\hspace{0.17em}\hspace{0.17em}}{}^{**}P<.01$, 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 $\mu$M fasudil-treated eyes with stage 2 PVR. Micrographs depict $\alpha$-SMA expression (brown) in preretinal proliferative membrane with stage 5 PVR (e) and stage 2 PVR (h) by immunohistochemical analysis. (Scale bar, 200 $\mu$m). (f and i) Magnified images of (e) and (h),respectively, (Scale bar, 10 $\mu$m).