Regulation of Aqueous Humor Secretion by Melatonin in Porcine Ciliary Epithelium.

Secretion of melatonin, a natural hormone whose receptors are present in the ciliary epithelium, displays diurnal variation in the aqueous humor (AH), potentially contributing to the regulation of intraocular pressure. This study aimed to determine the effects of melatonin on AH secretion in porcine ciliary epithelium. The addition of 100 µM melatonin to both sides of the epithelium significantly increased the short-circuit current (Isc) by ~40%. Stromal administration alone had no effect on the Isc, but aqueous application triggered a 40% increase in Isc, similar to that of bilateral application without additive effect. Pre-treatment with niflumic acid abolished melatonin-induced Isc stimulation. More importantly, melatonin stimulated the fluid secretion across the intact ciliary epithelium by ~80% and elicited a sustained increase (~50-60%) in gap junctional permeability between pigmented ciliary epithelial (PE) cells and non-pigmented ciliary epithelial (NPE) cells. The expression of MT3 receptor was found to be >10-fold higher than that of MT1 and MT2 in porcine ciliary epithelium. Aqueous pre-treatment with MT1/MT2 antagonist luzindole failed to inhibit the melatonin-induced Isc response, while MT3 antagonist prazosin pre-treatment abolished the Isc stimulation. We conclude that melatonin facilitates Cl- and fluid movement from PE to NPE cells, thereby stimulating AH secretion via NPE-cell MT3 receptors.

Mechanistic Effects of Baicalein on Aqueous Humor Drainage and Intraocular Pressure.

Elevated intraocular pressure (IOP) is a major risk factor for glaucoma that results from impeded fluid drainage. The increase in outflow resistance is caused by trabecular meshwork (TM) cell dysfunction and excessive extracellular matrix (ECM) deposition. Baicalein (Ba) is a natural flavonoid and has been shown to regulate cell contraction, fluid secretion, and ECM remodeling in various cell types, suggesting the potential significance of regulating outflow resistance and IOP. We demonstrated that Ba significantly lowered the IOP by about 5 mmHg in living mice. Consistent with that, Ba increased the outflow facility by up to 90% in enucleated mouse eyes. The effects of Ba on cell volume regulation and contractility were examined in primary human TM (hTM) cells. We found that Ba (1-100 µM) had no effect on cell volume under iso-osmotic conditions but inhibited the regulatory volume decrease (RVD) by up to 70% under hypotonic challenge. In addition, Ba relaxed hTM cells via reduced myosin light chain (MLC) phosphorylation. Using iTRAQ-based quantitative proteomics, 47 proteins were significantly regulated in hTM cells after a 3-h Ba treatment. Ba significantly increased the expression of cathepsin B by 1.51-fold and downregulated the expression of D-dopachrome decarboxylase and pre-B-cell leukemia transcription factor-interacting protein 1 with a fold-change of 0.58 and 0.40, respectively. We suggest that a Ba-mediated increase in outflow facility is triggered by cell relaxation via MLC phosphorylation along with inhibiting RVD in hTM cells. The Ba-mediated changes in protein expression support the notion of altered ECM homeostasis, potentially contributing to a reduction of outflow resistance and thereby IOP.

Transcriptional profiling of the chick retina identifies down-regulation of VIP and UTS2B genes during early lens-induced myopia.

Gene expression of the chick retina was examined during the early development of lens-induced myopia (LIM) using whole transcriptome sequencing. Monocular treatment of the right eyes with -10 diopter (D) lenses was performed on newly born chicks for one day (LIM-24) or two days (LIM-48), while the contralateral eyes without lenses served as controls. Myopia development was confirmed by demonstrating significant elongation of the optical axis in lens-treated eyes compared to untreated control eyes. RNA was extracted and RNA-seq was performed using the Illumina HiSeqTM 2000 platform. Data analysis was carried out on a Partek® Flow platform. Using screening criteria of ≥1.30-fold change and a false discovery rate <1%, 11 (five down-regulated and six up-regulated) and 35 differentially expressed genes (six down-regulated and twenty-nine up-regulated) were identified at 24 hours and 48 hours, respectively. Using another cohort for validation, Quantitative PCR confirmed significant changes in the expression of VIP and UTS2B mRNA (P <0.05) after only 24 hours of LIM treatment and numerical changes in the expression for PCGF5 and FOXG1, which were consistent with transcriptome sequencing but did not reach statistical significance. These data suggest that concerted changes of retinal gene expression may be instrumental in the initiation of axial elongation and myopia development.

Targeting Lysosomes to Reverse Hydroquinone-Induced Autophagy Defects and Oxidative Damage in Human Retinal Pigment Epithelial Cells.

In age-related macular degeneration (AMD), hydroquinone (HQ)-induced oxidative damage in retinal pigment epithelium (RPE) is believed to be an early event contributing to dysregulation of inflammatory cytokines and vascular endothelial growth factor (VEGF) homeostasis. However, the roles of antioxidant mechanisms, such as autophagy and the ubiquitin-proteasome system, in modulating HQ-induced oxidative damage in RPE is not well-understood. This study utilized an in-vitro AMD model involving the incubation of human RPE cells (ARPE-19) with HQ. In comparison to hydrogen peroxide (H2O2), HQ induced fewer reactive oxygen species (ROS) but more oxidative damage as characterized by protein carbonyl levels, mitochondrial dysfunction, and the loss of cell viability. HQ blocked the autophagy flux and increased proteasome activity, whereas H2O2 did the opposite. Moreover, the lysosomal membrane-stabilizing protein LAMP2 and cathepsin D levels declined with HQ exposure, suggesting loss of lysosomal membrane integrity and function. Accordingly, HQ induced lysosomal alkalization, thereby compromising the acidic pH needed for optimal lysosomal degradation. Pretreatment with MG132, a proteasome inhibitor and lysosomal stabilizer, upregulated LAMP2 and autophagy and prevented HQ-induced oxidative damage in wildtype RPE cells but not cells transfected with shRNA against ATG5. This study demonstrated that lysosomal dysfunction underlies autophagy defects and oxidative damage induced by HQ in human RPE cells and supports lysosomal stabilization with the proteasome inhibitor MG132 as a potential remedy for oxidative damage in RPE and AMD.

Thrombospondin-1 mediates Rho-kinase inhibitor-induced increase in outflow-facility.

Rho-kinase (ROCK) inhibitors, a novel class of anti-glaucoma agents, act by increasing the aqueous humor outflow through the conventional trabecular meshwork pathway. However, the downstream signaling consequences of the ROCK inhibitor are not completely understood. Our data show that Y39983, a selective ROCK inhibitor, could induce filamentous actin remodeling, reduced cell motility (as measured by cell migration), and transepithelial resistance in primary human TM (hTM) cells. After 2 days Y39983 treatment of hTM cells, a proteomic study identified 20 proteins whose expression was significantly altered. Pathway analysis of those proteins revealed the involvement of the p53 pathway, integrin signaling pathway, and cytoskeletal pathway regulation by Rho GTPase. Thrombospondin-1 (TSP1), a matricellular protein that is increased in glaucoma patients, was downregulated fivefold following Y39983 treatment. More importantly, both TSP1 antagonist leucine-serine-lysine-leucine (LSKL) and small interfering RNA (siRNA) reduced TSP1 gene and protein expressions as well as hTM cell migration. In the presence of Y39983, no further inhibition of cell migration resulted after LSKL and TSP1 siRNA knockdown. Likewise, LSKL triggered a dose-dependent increase in outflow facility in ex vivo mouse eyes, to a similar extent as Y39983 (83.8% increase by Y39983 vs. 71.2% increase by LSKL at 50 µM). There were no additive effects with simultaneous treatment with LSKL and Y39983, supporting the notion that the effects of ROCK inhibition were mediated by TSP1.

Biophysical properties of corneal cells reflect high myopia progression.

Myopia is a common ocular disorder with significant alterations in the anterior ocular structure, including the cornea. The cell biophysical phenotype has been proposed to reflect the state of various diseases. However, the biophysical properties of corneal cells have not been characterized during myopia progression and their relationship with myopia remains unknown. This study characterizes the biophysical properties of corneal cells in normal, myopic, and recovered conditions, using two classical myopia models. Surprisingly, myopic corneal cells considerably reduce F-actin and microtubule content and cellular stiffness and generate elevated traction force compared with control cells. When myopia is restored to the healthy state, these biophysical properties are partially or fully restored to the levels of control cells. Furthermore, the level of chromatin condensation is significantly increased in the nucleus of myopic corneal cells and reduced to a level similar to healthy cells after recovery. These findings demonstrate that the reversible biophysical alterations of corneal cells reflect myopia progression, facilitating the study of the role of corneal cell biophysics in myopia.

Functional connexin35 increased in the myopic chicken retina.

Our previous research showed that increased phosphorylation of connexin (Cx)36 indicated extended  coupling of AII amacrine cells (ACs) in the rod-dominant mouse myopic retina. This research will determine whether phosphorylation at serine 276 of Cx35-containing gap junctions increased in the myopic chicken, whose retina is cone-dominant. Refractive errors and ocular biometric dimensions of 7-days-old chickens were determined following 12 h and 7 days induction of myopia by a -10D lens. The expression pattern and size of Cx35-positive plaques were examined in the early (12 h) and compensated stages (7 days) of lens-induced myopia (LIM). At the same time, phosphorylation at serine 276 (functional assay) of Cx35 in strata 5 (S5) of the inner plexiform layer was investigated. The axial length of the 7 days LIM eyes was significantly longer than that of non-LIM controls (P < 0.05). Anti-phospho-Ser276 (Ser276-P)-labeled plaques were significantly increased in LIM retinas at both 12 h and 7 days. The density of Ser276-P of Cx35 was observed to increase after 12 h LIM. In the meanwhile, the areas of existing Cx35 plaques did not change. As there was more phosphorylation of connexin35 at Ser276 at both the early and late stages (12 h) and 7 days of LIM chicken retinal activity, the coupling with ACs could be increased in myopia development of the cone-dominated chicken retina.

Autophagy Upregulation by the TFEB Inducer Trehalose Protects against Oxidative Damage and Cell Death Associated with NRF2 Inhibition in Human RPE Cells.

Trehalose is a natural dietary molecule that has shown antiaging and neuroprotective effects in several animal models of neurodegenerative diseases. The role of trehalose in the management of age-related macular degeneration (AMD) is yet to be investigated and whether trehalose could be a remedy for the treatment of diseases linked to oxidative stress and NRF2 dysregulation. Here, we showed that incubation of human retinal pigment epithelial (RPE) cells with trehalose enhanced the mRNA and protein expressions of TFEB, autophagy genes ATG5 and ATG7, as well as protein expressions of macroautophagy markers, LC3B and p62/SQTM1, and the chaperone-mediated autophagy (CMA) receptor LAMP2. Cathepsin D, a hydrolytic lysosomal enzyme, was also increased by trehalose, indicating higher proteolytic activity. Moreover, trehalose upregulated autophagy flux evident by an increase in the endogenous LC3B level, and accumulation of GFP-LC3B puncta and free GFP fragments in GFP-LC3 - expressing cells in the presence of chloroquine. In addition, the mRNA levels of key molecular targets implicated in RPE damage and AMD, such as vascular endothelial growth factor- (VEGF-) A and heat shock protein 27 (HSP27), were downregulated, whereas NRF2 was upregulated by trehalose. Subsequently, we mimicked in vitro AMD conditions using hydroquinone (HQ) as the oxidative insult on RPE cells and evaluated the cytoprotective effect of trehalose compared to vehicle treatment. HQ depleted NRF2, increased oxidative stress, and reduced the viability of cells, while trehalose pretreatment protected against HQ-induced toxicity. The cytoprotection by trehalose was dependent on autophagy but not NRF2 activation, since autophagy inhibition by shRNA knockdown of ATG5 led to a loss of the protective effect. The results support the transcriptional upregulation of TFEB and autophagy by trehalose and its protection against HQ-induced oxidative damage in RPE cells. Further investigation is, therefore, warranted into the therapeutic value of trehalose in alleviating AMD and retinal diseases associated with impaired NRF2 antioxidant defense.

Data on differentially expressed proteins in rock inhibitor-treated human trabecular meshwork cells using SWATH-based proteomics.

Rho-associated coiled coil-forming protein kinase (ROCK) inhibitors represent a novel class of anti-glaucoma drugs because of their ocular hypotensive effects. However, the underlying mechanisms responsible for lowering intraocular pressure (IOP) are not completely clear. The protein profile changes in primary human trabecular meshwork (TM) cells after two days treatment with a ROCK inhibitor were studied using label-free SWATH acquisition. These results provided significant data of key protein candidates underlying the effect of ROCK inhibitor. Using the sensitive label-free mass spectrometry approach with data-independent acquisition (SWATH-MS), we established a comprehensive TM proteome library. All raw data generated from IDA and SWATH acquisitions were uploaded and published in the Peptide Atlas public repository (http://www.peptideatlas.org/) for general release (Data ID PASS01254).

Alteration of retinal metabolism and oxidative stress may implicate myopic eye growth: Evidence from discovery and targeted proteomics in an animal model.

Myopia, the most common cause of impaired vision, may induce sight- threatening diseases or ocular complications due to axial elongation. The exact mechanisms underlying myopia development have received much attention and understanding of these is necessary for clinical prevention or therapeutics. In this study, quantitative proteomics using Isotope Coded Protein Label (ICPL) was applied to identify differentially regulated proteins in the retinas of myopic chicks and, from their presence, infer the possible pathogenesis of excessive ocular elongation. Newly hatched white leghorn chicks (n = 15) wore -10D and + 10D lenses bilaterally for 3 and 7 days, respectively, to develop progressive lens-induced myopia (LIM) and hyperopia (LIH). Retinal proteins were quantified with nano-liquid chromatography electrospray ionization coupled with tandem mass spectrometry (nanoLC-ESI-MS/MS). Bioinformatics analysis of differentially regulated proteins revealed that the majority originated from the cytoplasmic region and were related to various metabolic, glycolytic, or oxidative processes. The fold changes of four proteins of interest (vimentin, apolipoprotein A1, interphotoreceptor retinoid binding protein, and glutathione S-transferase) were further confirmed by a novel high-resolution multiple reaction monitoring mass spectrometry (MRM-HR) using a label-free approach. SIGNIFICANCE: Discovery of effective protein biomarkers of myopia has been extensively studied to inhibit myopia progression. This study first applied lens-induced hyperopia and myopia in the same chick to maximize the inter-ocular differences, aiming to discover more protein biomarker candidates. The findings provided new evidence that myopia was metabolism related, accompanied by altered energy generation and oxidative stress at retinal protein levels. The results in the retina were also compared to our previous study in vitreous using ICPL quantitative technology. We have now presented the protein changes in these two adjacent tissues, which may provide extra information of protein changes during ocular growth in myopia.

Data on differentially expressed proteins in retinal emmetropization process in guinea pig using integrated SWATH-based and targeted-based proteomics.

Myopia is generally regarded as a failure of normal emmetropization process, however, its underlying molecular mechanisms are unclear. Retinal protein profile changes using integrated SWATH and MRM-HR MS were studied in guinea pigs at 3- and 21-days of age, where the axial elongation was significantly detected. Differential proteins expressions were identified, and related to pathways which are important in postnatal development in retina, proliferation, breakdown of glycogen-energy and visual phototransduction. These results are significant as key retinal protein players and pathways that underlying emmetropization can be discovered. All raw data generated from IDA and SWATH acquisitions were accepted and published in the Peptide Atlas public repository (http://www.peptideatlas.org/) for general release (Data ID PASS00746). A more comprehensive analysis of this data can be obtained in the article "Integrated SWATH-based and targeted-based proteomics provide insights into the retinal emmetropization process in guinea pig" in Journal of Proteomics (Shan et al., 2018) [1].

Characterization and Regulation of Gap Junctions in Porcine Ciliary Epithelium.

Purpose: Gap junctions provide a conduit between the intracellular fluids of the pigmented (PE) and non-pigmented (NPE) ciliary epithelial cells, and are therefore critical in the secretion of the aqueous humor (AH). However, opinions differ concerning the connexin (Cx) composition of the gap junctions. Therefore, we aimed to characterize the expression of Cx in the porcine ciliary epithelium (CE), a favorable model for humans; and determine the contribution of the highest expressed Cx to AH secretion. Methods: Freshly-harvested porcine CE cells were used. The mRNA and protein expressions of gap junctions were assessed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting (WB), respectively. The relative gene expressions of various Cx were determined by quantitative PCR. The gap junction permeability of isolated PE-NPE cell couplets was evaluated by Lucifer Yellow dye transfer. Results: Using RT-PCR and WB, Cx43, Cx45, Cx47, Cx50, and Cx60 were present in porcine CE, with Cx43 being the most abundant isoform, having over 200-fold higher expression than other Cx. Cx43 was primarily localized in the PE-NPE interface and the basolateral membranes of PE cells. Knockdown of Cx43 by siRNA significantly reduced gene and protein expressions, resulting in reduction of transcellular fluid flow by 90%. Conclusions: Cx43 was found to be the major component of gap junctions in porcine CE. Consistent with results from a bovine model, our results support the important role of Cx43 in mediating AH secretion. This finding may shed light on the development of a novel ocular hypotensive agent.

Integrated SWATH-based and targeted-based proteomics provide insights into the retinal emmetropization process in guinea pig.

Myopia is generally regarded as a failure of normal emmetropization process, however, its underlying molecular mechanisms are unclear. To investigate the retinal protein profile changes during emmetropization, we studied differential protein expressions of ocular growth in young guinea pigs at 3 and 21 days old respectively, when significant axial elongation was detected (P < 0.001, n = 10). Independent pooled retinal samples of both eyes were subjected to SWATH mass spectrometry (MS) followed by bioinformatics analysis using cloud-based platforms. A comprehensive retina SWATH ion-library consisting of 3138 (22,871) unique proteins (peptides) at 1% FDR was constructed. 40 proteins were found to be significantly up-regulated and 8 proteins down-regulated during emmetropization (≥log2 of 0.43 with ≥2 peptides matched per protein; P < 0.05). Using pathway analysis, the most significant pathway identifiable was 'phototransduction' (P = 1.412e-4). Expression patterns of 7 proteins identified in this pathway were further validated and confirmed (P < 0.05) with high-resolution Multiple Reaction Monitoring (MRM-HR) MS. Combining discovery and targeted proteomics approaches, this study for the first time comprehensively profiled protein changes in the guinea pig retina during normal emmetropization-associated eye growth. The findings of this study are also relevant to the myopia development, which is the result of failed emmetropization. SIGNIFICANCE: Myopia is considered as a failure of emmetropization. However, the underlying biochemical mechanism of emmetropization, a visually guided process in which eye grows towards the optimal optical state of clear vision during early development, is not well understood. Retina is known as the key tissue to regulate this active eye growth. we studied eye growth of young guinea pigs and harvested their retinal tissues. A comprehensive SWATH ion library with identification of a total 3138 unique proteins were established, in which 48 proteins exhibited significant differential expressions between 3 and 21 days old. After MRM-HR confirmation, 'phototransduction' were found as the most active pathway during emmetropic eye growth. This study is the first in discovering key retinal protein players and pathways which are presumably orchestrated by biological mechanism(s) underlying emmetropization.

New Insight of Common Regulatory Pathways in Human Trabecular Meshwork Cells in Response to Dexamethasone and Prednisolone Using an Integrated Quantitative Proteomics: SWATH and MRM-HR Mass Spectrometry.

The molecular pathophysiology of corticosteroid-induced ocular hypertension (CIH) is not well understood. To determine the biological mechanisms of CIH, this study investigated protein expression profiles of human trabecular meshwork (hTM) cells in response to dexamethasone and prednisolone treatment. Both discovery-based sequential windowed data independent acquisition of the total high-resolution mass spectra (SWATH-MS) and targeted based high resolution multiple reaction monitoring (MRM-HR) confirmation were applied using a hybrid quadrupole-time-of-flight mass spectrometer. A comprehensive list of 1759 proteins (1% FDR) was generated from the hTM. Quantitative proteomics revealed 20 differentially expressed proteins (p-value ≤ 0.05 and fold-change ≥ 1.5 or ≤ 0.67) commonly induced by prednisolone and dexamethasone, both at 300 nM. These included connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1), two proteins previously implicated in ocular hypertension, glaucoma, and the transforming growth factor-ß pathway. Their gene expressions in response to corticosteroids were further confirmed using reverse-transcription polymerase chain reaction. Together with other novel proteins identified in the data sets, additional pathways implicated by these regulated proteins were the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, integrin cell surface interaction, extracellular matrix (ECM) proteoglycans, and ECM-receptor interaction. Our results indicated that an integrated platform of SWATH-MS and MRM-HR allows high throughput identification and confirmation of novel and known corticosteroid-regulated proteins in trabecular meshwork cells, demonstrating the power of this technique in extending the current understanding of the pathogenesis of CIH.

Region-specific differential corneal and scleral mRNA expressions of MMP2, TIMP2, and TGFB2 in highly myopic-astigmatic chicks.

Myopia and astigmatism, two common refractive errors frequently co-exist, are affecting vision at all working distances in the affected populations worldwide. Eyeballs having these refractive errors are known to exhibit abnormal eye shape at the anterior and posterior eye segments, but whether the outer coats of these abnormal eyeballs, cornea anteriorly and sclera posteriorly, are regulated by region-specific molecular mechanism remains unclear. Here we presented the changes in mRNA expression levels of three genes (MMP2, TIMP2, and TGFB2), all known to participate in extracellular matrix organization, at five regions of the cornea and sclera in chickens developing high myopia and astigmatism induced by form deprivation. We found that, compared to normal chicks, the highly myopic-astigmatic chicks had significantly higher expression of all three genes in the superior sclera (Mann-Whitney tests, all p ≤ 0.05), as well as higher TIMP2 expression in the central cornea and nasal sclera (Mann-Whitney tests, both p ≤ 0.05). Strikingly, the superior scleral region stood out as showing the strongest and most widespread correlations between mRNA expression and biometry parameters including axial and astigmatic components (r = + 0.52~ + 0.85, all p < 0.05). These results imply that local molecular mechanism may manipulate the eye shape remodeling across the globe during refractive-error development.

Inhibition of Dexamethasone-induced Fatty Liver Development by Reducing miR-17-5p Levels.

Steatosis is a pivotal event in the initiation and progression of nonalcoholic fatty liver disease (NAFLD) which can be driven by peroxisome proliferator-activated receptor-α (PPAR-α) dysregulation. Through examining the effect of PPAR-α on fatty liver development, we found that PPAR-α is a target of miR-17-5p. Transgenic mice expressing miR-17 developed fatty liver and produced higher levels of triglyceride and cholesterol but lower levels of PPAR-α. Ectopic expression of miR-17 enhanced cellular steatosis. Gain-of-function and loss-of-function experiments confirmed PPAR-α as a target of miR-17-5p. On the other hand, PPAR-α bound to the promoter of miR-17 and promoted its expression. The feed-back loop between miR-17-5p and PPAR-α played a key role in the induction of steatosis and fatty liver development. Mice with high levels of miR-17-5p were sensitive to Dexamethasone-induced fatty liver formation. Inhibition of miR-17-5p suppressed this process and enhanced PPAR-α expression in mice treated with Dexamethasone. Clofibrate, Ciprofibrate, and WY-14643: three agents used for treatment of metabolic disorders, were found to promote PPAR-α expression while decreasing miR-17-5p levels and inhibiting steatosis. Our studies show that miR-17-5p inhibitor and agents used in metabolic disorders may be applied in combination with Dexamethasone in the treatment of anti-inflammation, immunosuppression, and cancer patients.

Glutathione attenuates nitric oxide-induced retinal lipid and protein changes.

BACKGROUND: Elevated levels of nitric oxide (NO(•) ), a pro-oxidant that has been associated with numerous retinal diseases, have been implicated in experimental glaucoma models. This study investigated the oxidative effects of sodium nitroprusside (SNP), a nitric oxide donor, on the retinal lipids and proteins and evaluated the potential protective effects of glutathione (GSH). METHODS: Porcine retinal homogenates were incubated with 0, 1, 2, 3, 4 and 5 µm SNP. Malondialdehyde (MDA) levels were assayed spectrophotometrically to quantify lipid peroxidation. Differential protein expressions of 3 µm SNP-treated retinal homogenates were compared with controls after the conduction of two-dimensional gel electrophoresis. Mass spectrometric data was used to identify proteins in NCBInr database. Furthermore, GSH was co-incubated with 3 µm SNP-treated retinal homogenates. MDA levels and protein expressions were compared with SNP-treated controls. RESULTS: SNP significantly increased retinal-MDA levels (p = 0.0002). 2-D gel electrophoresis images displayed a significant change in 13 protein spot expressions (p < 0.05). GSH suppressed SNP-induced MDA elevation (p < 0.0001) and selected protein changes (p < 0.05). SNP down-regulated paraoxonase/arylesterase 2 precursor (PON2), ß-actin and ß-tubulin; however, these effects were prevented by a co-incubation with GSH, as confirmed by Western blots. CONCLUSIONS: Nitric oxide induced lipid and protein changes in retinal tissues. The effects were partially reversed by co-incubation with GSH. Data from this study suggests that nitric oxide-induced retinal oxidative stress induces specific molecular changes. This may enable us to better understand the pathogenesis of glaucoma. Further studies are indicated to explore potential pharmacological applications of GSH in nitric oxide-related retinal diseases.

Cyclic Adenosine Monophosphate Activates Retinal Apolipoprotein A1 Expression and Inhibits Myopic Eye Growth.

PURPOSE: Apolipoprotein A1 (ApoA1) has been shown to inhibit myopia development in chicks, but the underlying biological mechanism remains unknown. Because ApoA1 interacts with cyclic adenosine monophosphate (cAMP) in many cellular systems, we examined whether this interaction is important in myopia development. METHODS: The nonmetabolizable cAMP analogue 8-Bromo-cAMP (8-Br-cAMP) was administered intravitreally to the right eyes of 8-day old chicks for 4 consecutive days. Control eyes received vehicle. Chicks in group 1 received 8-Br-cAMP (0.1 mM or 1 mM) and were fitted with -10 diopter (D) lenses on both eyes, whereas chicks in group 2 (0.1 mM 8-Br-cAMP) wore plano lenses over both eyes. The levels of retinal cAMP and ApoA1 were examined in another two groups of chicks wearing -10 D (group 3) and +10 D lenses (group 4) over their right eyes for 3 days, respectively (plano over left eyes). RESULTS: The 8-Br-cAMP significantly inhibited development of lens-induced myopia (group 1: 0.1 mM versus vehicle: +1.71 ± 1.22 D versus -8.00 ± 2.19 D; 1 mM versus vehicle: +1.38 ± 1.34 D versus -9.96 ± 1.14 D, mean ± SEM, P < 0.01 for both); 1 mM, but not 0.1 mM 8-Br-cAMP increased expression of retinal ApoA1 levels in right eyes (P < 0.01). The 8-Br-cAMP had minimal effect on normal eye growth. Both retinal cAMP and ApoA1 levels were significantly increased only in hyperopic eyes (group 4). CONCLUSIONS: The 8-Br-cAMP robustly inhibited development of lens-induced myopia. The increase in retinal ApoA1 observed in cAMP-treated and hyperopic eyes suggested a possible interplay between ApoA1 and cAMP in regulating eye growth.

miR-17 targets tissue inhibitor of metalloproteinase 1 and 2 to modulate cardiac matrix remodeling.

We aimed to investigate the role of miR-17 in cardiac matrix remodeling following myocardial infarction (MI). Using real-time PCR, we quantified endogenous miR-17 in infarcted mouse hearts. Compared with related microRNAs, miR-17 was up-regulated most dramatically: 3.7-fold and 2.4-fold in the infarct region 3 and 7 d post-MI, respectively, and 2.4-fold in the border zone at d 3 compared to sham control (P<0.01). Chimeric luciferase reporter constructs were cloned for miR-17 target validation. miR-17 targeted the 3'-UTR of TIMP2 and the protein coding region of TIMP1. The miR-17 mimic decreased TIMP2 (P<0.01) and TIMP1 (P<0.05) protein expression compared with the scrambled control. Inhibition of endogenous miR-17 by in vivo antagomir delivery enhanced TIMP2 (P<0.01) and TIMP1 (P<0.05) protein expression compared to the mismatch group, decreased MMP9 activity (P<0.05), reduced infarct size as early as 7 d post-MI (P<0.05), and improved cardiac function (fractional shortening and fractional area contraction, P<0.05) at d 21 and 28 post-MI. Transgenic mice overexpressing miR-17 in the heart confirmed the deleterious role of miR-17 in matrix modulation. Our study suggests that miR-17 participates in the regulation of cardiac matrix remodeling and provides a novel therapeutic approach using miR-17 inhibitors to prevent remodeling and heart failure after MI.