Crystallins are regulated biomarkers for monitoring topical therapy of glaucomatous optic neuropathy.

Optic nerve atrophy caused by abnormal intraocular pressure (IOP) remains the most common cause of irreversible loss of vision worldwide. The aim of this study was to determine whether topically applied IOP-lowering eye drugs affect retinal ganglion cells (RGCs) and retinal metabolism in a rat model of optic neuropathy. IOP was elevated through cauterization of episcleral veins, and then lowered either by the daily topical application of timolol, timolol/travoprost, timolol/dorzolamide, or timolol/brimonidine, or surgically with sectorial iridectomy. RGCs were retrogradely labeled 4 days prior to enucleation, and counted. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption ionization mass spectrometry, Western blotting, and immunohistochemistry allowed the identification of IOP-dependent proteomic changes. Genomic changes were scrutinized using microarrays and qRT-PCR. The significant increase in IOP induced by episcleral vein cauterization that persisted until 8 weeks of follow-up in control animals (p<0.05) was effectively lowered by the eye drops (p<0.05). As anticipated, the number of RGCs decreased significantly following 8 weeks of elevated IOP (p<0.05), while treatment with combination compounds markedly improved RGC survival (p<0.05). 2D-PAGE and Western blot analyses revealed an IOP-dependent expression of crystallin cry-βb2. Microarray and qRT-PCR analyses verified the results at the mRNA level. IHC demonstrated that crystallins were expressed mainly in the ganglion cell layer. The data suggest that IOP and either topically applied antiglaucomatous drugs influence crystallin expression within the retina. Neuronal crystallins are thus suitable biomarkers for monitoring the progression of neuropathy and evaluating any neuroprotective effects

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

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

Two-dimensional gelelectrophoresis of retinal proteins.

<p>A Peptide mapping of a retina obtained from a Sprague-Dawley rat with untreated, sustained elevated IOP 8 weeks after IOP induction. Hypertensive retinal samples showed a marked increase in βb2-crystallin (cryβb2) expression (area marked by a black box in <b>A</b>, shown at higher magnification in <b>B1</b> compared to a normotensive sham-treated retina, <b>B2</b>). The 4 weeks of IOP-lowering treatment with topical treatment with Ti (<b>B3,B4</b>), Ti/T (<b>B5</b>), Ti/D (<b>B6</b>), and Ti/B (<b>B7</b>) decreased cryβb2 expression to baseline levels. The proteins identified, which are marked by an arrow in <b>A</b> and <b>B1</b>, are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049730#pone-0049730-t001" target="_blank">Table 1</a> according to the number given.</p

IOP readings in an experimental model of ocular hypertension.

<p>A significant increase in IOP (*<i>p</i><0.05) was observed in eyes after cauterization of three episcleral veins in all groups relative to the normal control group (blue line) after 2 weeks. After the initiation of a medical hypotensive treatment, marked by a black arrow, the IOP was reduced significantly (**<i>p</i><0.05) in all groups, whereas it remained elevated throughout the experimental period in the control group without hypotensive treatment (red line).</p

Immunohistochemical analysis of crystallins.

<p>Staining for cryβb (green) in a normotensive, sham-treated group (<b>A</b>), 7 days after induction of elevated IOP (<b>B</b>), and 28 days after induction of elevated IOP (<b>C</b>) in retinal slices. Cryβb staining revealed a distinctive up-regulation of cryβb after IOP elevation (<b>B</b>, <b>C</b>) relative to normotensive samples. Moreover, cryβb expression increased within the period of exposure to elevated IOP. The signal was more intense after 28 days (<b>C</b>) than after 7 days (<b>B</b>) of IOP elevation. Cryβb2 expression appeared predominantly in the RGC layer. Scale bar: 100 µm.</p

Specific Western blot analysis and the correlated graph of the relative density of selected proteins, including the application controls.

<p>A–G Blots of cryβb2 (<b>A</b>) cryβb3 (<b>C</b>), cryβL (<b>E</b>), and cryβH (<b>G</b>), and their correlated relative densities (<b>B</b>, <b>D</b>, <b>F</b>, and <b>H</b>, respectively), each with the corresponding control with calnexin. Hypertensive samples are demonstrated in <b>A1</b>–<b>D1</b>, normotensive samples in <b>A2</b>–<b>D2</b> and <b>A3</b>–<b>D3</b>, samples following Ti/B treatment in <b>A4</b>–<b>D4</b>, samples following Ti treatment in <b>A5</b>–<b>D5</b>, samples following Ti/Tr treatment in <b>A6</b>–<b>D6</b>, and samples following Ti/D treatment in <b>A7</b>–<b>D7</b>.</p

Specific Western blot analysis of selected proteins with the corresponding application controls.

<p>The graphs next to them illustrate the relative protein densities. <b>A</b>–<b>H</b> Blots of heat-shock protein (HSP)-70 (<b>A</b>), HSP-90 (<b>C</b>), HSP-25 (<b>E</b>), and cryγ (<b>G</b>) and their relative densities (<b>B</b>, <b>D</b>, <b>F</b>, and <b>H</b>, respectively). Hypertensive samples are shown in <b>A1</b>–<b>D1</b>, normotensive samples in <b>A2</b>–<b>D2</b> and <b>A3</b>–<b>D3</b>, samples following Ti/B treatment in <b>A4</b>–<b>D4</b>, samples following Ti therapy in <b>A5</b>–<b>D5</b>, samples following Ti/Tr treatment in <b>A6</b>–<b>D6</b>, and samples following Ti/D treatment in <b>A7</b>–<b>D7</b>.</p

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

<p>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.</p

Retinal proteins identified by two-dimensional gel electrophoresis and subsequent matrix-assisted laser desorption ionization mass spectrometry.

<p>The numbers in column 1 correspond to those given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049730#pone-0049730-g004" target="_blank">Fig. 4</a>. Column 4 lists the molecular mass of the respective protein. HSP = heat-shock protein.</p

Confirmation of crystallin expression at the gene level by microarray analysis and subsequent quantitative real-time polymerase chain reaction.

<p>CryαA, cryαB, cryβb1, cryβb2, cryβb3, and cryβb4 were up-regulated due to elevated IOP at 8 weeks after IOP induction. The change <b>in</b> gene expression is expressed as the change relative to normotensive sham-treated retinas. The alterations in regulation of all six genes were statistically significant (<i>p</i><0.001).</p