Regulation of retinal proteome by topical antiglaucomatous eye drops in an inherited glaucoma rat model

Abstract

Examination of the response of the retinal proteome to elevated intraocular pressure (IOP) and to the pharmacological normalization of IOP is crucial, in order to develop drugs with neuroptorective potential. We used a hereditary rat model of ocular hypertension to lower IOP with travaprost and dorzolamide applied topically on the eye surface, and examine changes of the retinal proteome. Our data demonstrate that elevated IOP causes alterations in the retinal protein profile, in particular in high-mobility-group-protein B1 (HMGB1), calmodulin, heat-shock-protein (HSP) 70 and carbonic anhydrase II expression. The changes of the retinal proteome by dorzolamide or travoprost are different and independent of the IOP lowering effect. This fact suggests that the eye drops exert a direct IOP-independent effect on retinal metabolism. Further investigations are required to elucidate the potential neuroprotective mechanisms signaled through changes of HMGB1, calmodulin, HSP70 and carbonic anhydrase II expression in glaucoma. The data may facilitate development of eye drops that exert neuroprotection through direct pharmacological effect.

Figure 1. IOP readings in an inherited glaucoma rat model.

The IOP was significantly elevated in all glaucoma rats compared to normal control group (red line) (* p<0.05). After treatment with dorzolamide or travoprost the IOP was reduced significantly (* p<0.05), whereas in the glaucoma group, without hypotensive treatment (blue line) the IOP remained elevated.

Figure 2. Peptide mapping of retinal explants obtained from a rat with inherited glaucoma and elevated IOP.

Hypertensive retinal samples showed a marked decrease in calmodulin expression (area marked by a white box and shown in a higher magnification in B)) compared to normotensive retina A). Four weeks treatment with either travaprost C) or dorzolamide D) elevated the calmodulin expression in a similar manner, therefore normalizing its expression.

Figure 3. Peptide mapping of retinal explants obtained from a rat with inherited glaucoma and elevated IOP.

Hypertensive retinal samples showed a marked increase in HMGB-1 expression and slightly increase in CAII expression (area marked by a white box and shown in a higher magnification in B)) compared to normotensive retina A). After 4 weeks anti-glaucomatous topical treatment with travoprost C) and dorzolamide D) HMGB-1 expression is slightly reduced in retinas treated with travaprost C) but not in retinas treated with dorzolamide D), despite of a similar reducing effect on the IOP. The expression of CA II showed nearly constant expression profile independent of treatment or not (A–D).

Figure 4. Peptide mapping of a retinal explants obtained from a rat with inherited glaucoma and elevated IOP.

Hypertensive retinal samples showed a marked decrease in HSP70 expression (area marked by a white box and shown in a higher magnification in B)) compared to normotensive retina A). Four weeks treatment with either travaprost C) or dorzolamide D) slightly reduced the HSP70 expression.

Figure 5. Specific Western blot analysis and the correlated graphs of the relative density of the selected protein normalized to application controls.

A) Western blot showing that HMGB-1 was up-regulated in glaucomatous rats. The up-regulation was significantly reduced in retinas treated with travaprost but not significantly in retinas treated with dorzolamide (A). HSP70 showed a moderate up-regulation in glaucomatous retinas, travaprost (121% ±3) and dorzolamide treated retinas (B). Calmodulin was significantly reduced in glaucomatous retinas while this reduction was prevented in the groups treated with either drugs (C). CAII showed a clear expression in all groups without significant changes (D). Actin or Calnexin was used as a standard control in these probes. Data were presented as relative mean values ± SD. n  = 3 in rat retina. Three independent Western blots were performed. * p<0.05 and ** p<0.01.

Figure 6. Expression of HMGB-1, HSP 70, calmodulin and carbonic anhydrase II in rat and human retinas.

Immunohistochemistry of normal and glaucomatous rat retinas showing up-regulation of HMGB-1 (ab) and the down-regulation of calmodulin (i,j) expression. The immunochemistry staining of HSP 70 and carbonic anhydrase II showed no changes (e,f,m,n) in rat retina. Immunohistochemistry, showing up-regulation of HMGB-1 (c,d) and HSP 70 (g,h) and the down-regulation of calmodulin (k,l) in normal and glaucomatous human retinas.