Blindness is a devastating consequence of PVDs such as PDR and PVR. Currently, the progression of these diseases cannot be effectively prevented, and the treatment options are limited to vitreoretinal surgery. An effective pharmacological treatment is thus urgently needed to complement or potentially replace the surgical intervention. In the current study, we show statins’ novel function in inhibiting the Rho/Rho-kinase pathway, the contraction of collagen gel, and the progression of experimental PVR, suggesting the therapeutic potential of these compounds for the treatment of PVDs.
Various cytokines, such as TGF-β, connective tissue growth factor, interleukin-6, and platelet-derived growth factor (PDGF), are overexpressed in the vitreous and membranes associated with PDR and PVR, and they contribute to the pathogenesis of these diseases (15
). Among these cytokines, TGF-β2 induces the transformation of retinal pigment epithelial cells or hyalocytes to myofibroblastic cells (18
) and plays a key role in the formation and contraction of proliferative membranes. In this study, we confirmed the overexpression of TGF-β2 in the vitreous from patients with PVD (PDR and PVR) and showed that TGF-β2 inhibition strongly suppressed the vitreous-induced contraction of the collagen gels. This indicates the possibility that TGF-β2 is the dominant contributor to the contraction of proliferative membrane in the vitreous cavity. Thus, we focused on the role of TGF-β2 to investigate the mechanisms of membrane contraction. TGF-β2 enhanced MLC phosphorylation in hyalocytes that was responsible for the contraction of the hyalocyte-containing collagen gels. We showed that simvastatin suppressed TGF-β2–induced MLC phosphorylation and collagen gel contraction in a dose-dependent fashion by inhibiting GGPP-mediated translocation of Rho to the plasma membrane, whereas no signs of cytotoxicity were apparent.
Differences in the structural characteristics of statin cause different levels of lipophilicity and possibly of efficacies and also cytotoxicity. Pravastatin is strongly hydrophilic, and simvastatin is much more lipophilic than pravastatin (42
). Fluvastatin is also more lipophilic than pravastatin; however, it is less lipophilic than simvastatin (43
). Although our comparison of the inhibitory effects of various statins, simvastatin, fluvastatin, and pravastatin, revealed that simvastatin was more effective in reducing MLC phosphorylation than the other statins under the chosen experimental conditions, the results might vary at other time points or concentrations. Further examination is necessary to determine the order. Additionally, because cerivastatin is more lipophilic than simvastatin (44
), we studied the effects of cerivastatin and simvastatin. Many hyalocytes treated with 5 μmol/l cerivastatin for 24 h shrank and detached from the culture plates, whereas the cells treated with 5 μmol/l simvastatin remained morphologically unchanged. However, at higher concentrations (>20 μmol/l), some hyalocyte shrinkage was observed even with simvastatin (data not shown). Cerivastatin is known to be cytotoxic and induce apoptosis (45
). The morphological changes we observed in cerivastatin-treated hyalocytes in our study suggest that this drug may also be toxic to hyalocytes and promote their apoptosis. Because the lipophilicity of lovastatin is similar to that of simvastatin (42
), we compared their effect and found the inhibitory effect of simvastatin on MLC phosphorylation and collagen gel contraction to be greater than that of 5 μmol/l lovastatin for 24 h (data not shown).
Vitreous samples from patients with PDR and PVR induced MLC phosphorylation and contraction of hyalocyte-containing collagen gels, which were effectively inhibited by simvastatin. It may appear surprising that not only vitreous samples from patients with PVD but also those from non-PVD patients induced MLC phosphorylation and collagen gel contraction. However, this might be explained by the fact that TGF-β2 is expressed in vitreous samples of patients with macular hole at high enough concentrations to induce the observed phenomena. The concentrations of TGF-β2 in vitreous samples from patients with PVD (PDR and PVR) were higher than in those from macular hole patients. Therefore, in a concentration-dependent manner, the inductions might be greater in the vitreous samples from patients with PVD than in those from patients without PVD. The concentrations of TGF-β2 in some vitreous samples from patients with macular hole were higher than those with PDR and PVR. However, the pathology of macular hole does not generally involve proliferative membrane formation and tractional retinal detachment. Occasionally, epiretinal membranes and retinal folds accompany macular hole. Retinal folds are considered to be caused by the contraction of the epiretinal membranes. TGF-β2 concentrations in vitreous samples from patients with macular hole may be high enough to induce contraction of proliferative membranes, however, because the epiretinal membrane in these patients does not extend into the vitreous and is very thin; the expression of TGF-β2 remains inconsequential.
MLC phosphorylation depends on the concentration of TGF-β2 (18
); thus, the eyes with PVR having lower TGF-β2 concentrations might represent a less severe pathology, or remission, compared with those with higher TGF-β2 concentrations. The occurrence of tractional retinal detachment might depend on the presence or absence of proliferative membranes and the concentration of TGF-β2.
Although TGF-β2–stimulated hyalocytes showed no significant change in MLC expression, those stimulated with vitreous samples showed elevated MLC expression that was inhibited by simvastatin treatment. The vitreous includes various cytokines, released from retinal pigment epithelial cells, glial cells, macrophages, and other intravitreal cells (46
). Thus, some cytokines other than TGF-β2 might elevate the expression of MLC. Among the cytokines found in the vitreous, insulin-like growth factor-I (IGF-I), PDGF, and members of the endothelin family have been shown to stimulate extracellular matrix contraction (18
). This study reveals a key role for TGF-β2 in the pathology of PVDs; however, it is possible that other cytokines found in the vitreous might also be involved in MLC phosphorylation and contraction of hyalocyte-containing collagen gels. Because simvastatin almost completely inhibited the phenomena induced by vitreous samples, it might also inhibit the effect of the other cytokines, such as IGF-I, PDGF, and members of the endothelin family and unknowns, which might be exerted through the Rho/Rho-kinase pathway.
Simvastatin also prevented the development of PVR in vivo. Proliferative membranes in simvastatin-injected eyes, even if present, were very thin, whereas the membranes in vehicle injected eyes with PVR in stage 4 or 5 were thick. Thus, simvastatin might have also an inhibitory effect on the formation and growth of proliferative membranes in addition to the cicatricial contraction of proliferative membranes. However, after the end of the simvastatin injections, even in the groups treated with higher concentration of simvastatin (15 μmol/l) the development of PVR was not completely inhibited. This may be due to a short biological half-life time of the compound in the vitreous cavity. Therefore, to sustain a constant level of intravitreal simvastatin concentration, frequent injections or a slow release drug delivery system might be necessary.
Recent findings suggest that statins might have a number of beneficial effects in the eye. Although the applicability of statins on age-related macular degeneration is still under investigation (49
), statins are shown to have protective effects on primary open-angle glaucoma, a major cause of blindness (50
). The regulation of the intraocular pressure (IOP) within a physiological range is of great clinical importance. Statins are reported to downregulate the IOP by increasing aqueous humor outflow via the inhibition of Rho/Rho-kinase pathway in the trabecular meshwork and the ciliary body (50
In conclusion, simvastatin potently inhibits the Rho/Rho-kinase pathway and thus might have therapeutic potential in the prevention of cicatricial contraction of proliferative membranes in vivo. This is the first report demonstrating the beneficial effects of simvastatin in inhibiting the development of PVR. Statins might provide a new strategy for the treatment and prevention of the development of PVDs in humans.