Alpha crystallin: the quest for a homogeneous quaternary structure

Alpha A and alpha B crystallins are key members of the small heat-shock protein family. In addition to being a major structural protein of the lens, they are constitutively found in many other cells, where their function is not completely understood. Alpha B crystallin is also known to be over-expressed in many neurological diseases. To date, all efforts to crystallize alpha A or alpha B have failed. Thus, high-resolution data on the tertiary and quaternary structures of alpha crystallin is not available. The main reason for this failure seems to be the polydisperse nature of alpha crystallin. This review deals mainly with the polydisperse properties of alpha crystallin and the influence of post-translational modification, chemical modifications, truncations and mutation on its quaternary structure.     

High glucose‐induced apoptosis in bovine retinal pericytes is associated with transforming growth factor β and βIG‐H3: βIG‐H3 induces apoptosis in retinal pericytes by releasing Arg‐Gly‐Asp peptides

Background: Transforming growth factor β (TGF‐β) plays an important role in diabetic retinopathy. βIG‐H3 is a downstream target molecule of TGF‐β that may participate in the pathogenesis of diabetic retinopathy and in particular in the loss of pericytes during early pathological changes. Methods: We observed bovine retinal pericytes apoptosis and the increased expression of TGF‐β and βIG‐H3 induced by high concentrations of glucose in the cell culture media. An anti‐TGF‐β antibody was used to block glucose‐induced retinal pericytes apoptosis. Retinal pericytes were also transfected with cDNA encodings either wild‐type or mutant βIG‐H3 lacking Arg‐Gly‐Asp (RGD) sequences in order to validate the effects of βIG‐H3 and RGD signalling on retinal pericytes apoptosis. Results: A cell death‐detecting enzyme‐linked immunosorbent assay revealed that 25 mM glucose significantly increased cell death compared with 5.5 mM glucose after 5 or 7 days of exposure (P < 0.01). High glucose significantly increased the TGF‐β levels as compared with 5.5 mM glucose after 5 days, and βIG‐H3 levels after 3, 5 and 7 days of exposure (P < 0.01). TGF‐β increased cell death and βIG‐H3 levels in a dose‐dependent manner, with a maximal effect observed at 1 ng/mL. An anti‐TGF‐β antibody nearly completely blocked high glucose‐induced cell death. Wild‐type βIG‐H3‐transfected cells showed a significant increase in cell death as compared with mutant βIG‐H3‐transfected (Mycb‐c) cells, untransfected or mock‐transfected cells. Conclusion: These results suggest that hyperglycaemia‐induced expression of TGF‐β and βIG‐H3 contributes to accelerated retinal pericytes apoptosis. βIG‐H3 induces pericytes apoptosis through its RGD motif, which may constitute an important pathogenic mechanism leading to pericytes loss in diabetic retinopathy.     

Profile of intraocular tumour necrosis factor‐α and interleukin‐6 in diabetic subjects with different degrees of diabetic retinopathy

Purpose: To assess and correlate the levels of inflammatory mediators in the eyes from non‐diabetic and diabetic subjects without retinopathy (NDR), with non‐proliferative diabetic retinopathy (NPDR) or with proliferative diabetic retinopathy (PDR) to corresponding erum levels. Methods: The levels of interleukin 1β, interleukin‐6 (IL‐6) and tumour necrosis factor‐α (TNF‐α) were analysed by an ELISA‐mimicking technique in the vitreous from 26 diabetic subjects with active PDR and 27 non‐diabetic subjects, or by a multiplex bead assay in the aqueous humour from 35 diabetic subjects with NDR/NPDR and 40 non‐diabetic subjects. Intraocular protein production was estimated in vitreous specimens by calculating a vitreous/serum ratio. Results: In the vitreous, IL‐6 was higher in diabetic [157.5 (25.0–1401.0) pg/ml; median (min–max)] than in non‐diabetic subjects [44.0 (5.0–4425) pg/ml; p = 0.021]. The vitreous/serum ratio was high (55.5:1 and 16:1, respectively), suggesting intraocular production. TNF‐α was lower in diabetic [18.0 (8.0–46.0) pg/ml] than in non‐diabetic subjects [22.0 (13.0–47.0) pg/ml; p = 0.034], but the vitreous/serum ratio was elevated in both groups (2:1 and 3.4:1, respectively). TNF‐α levels were higher in serum from diabetic subjects [9.0 (5.0–53.0) pg/ml versus 6.7 (3.0–11.0) pg/ml; p < 0.001]. Aqueous levels of inflammatory mediators did not differ between diabetic subjects with NDR/NPDR and non‐diabetic subjects despite elevated TNF‐α in serum [27.8 (6.8–153.7) pg/ml versus 16.4 (4.1–42.4) pg/ml; p = 0.021]. Conclusion: Intraocular inflammation seems to be involved in PDR but does not seem to be prominent in early retinopathy stages, i.e. NDR or NPDR. Diabetic subjects have an overall increased inflammatory activity compared to non‐diabetic subjects, as demonstrated by increased serum levels of TNF‐α.     

Semiautomated quantification of β‐zone parapapillary atrophy using blue light fundus autofluorescence

Purpose: To evaluate the reproducibility of measurements of area of β‐zone parapapillary atrophy (β‐PPA) using blue laser fundus autofluorescence (FAF) and confocal scanning laser ophthalmoscopy reflectance (CSLO) measurements and to assess agreement between the two imaging modalities. Methods: Sixty‐five eyes of 45 patients (mean age, 68.2 ± 11.3 years) with established or suspected glaucoma from the Diagnostic Innovations in Glaucoma Study (DIGS) were prospectively included. FAF scans were obtained with the Spectralis HRA+OCT and CSLO reflectance images with the HRTII (both from Heidelberg Engineering, Heidelberg, Germany). Two masked graders independently measured β‐PPA area on 3 consecutive scans using the semi‐automated BluePeak RegionFinder software (BPRF) and on CSLO reflectance images using the optic disc contour line. Reproducibility of β‐PPA area measurements was assessed using intraclass correlation coefficients (ICC). Results: Intragrader reproducibility was 0.997 (95% CI, 0.996–0.998) and 0.995 (95% CI, 0.992–0.996) for grader 1 and 2, respectively, using FAF‐BPRF, and by CSLO, it was 0.991 (95% CI, 0.986–0.994) and 0.988 (95% CI, 0.982–0.992). Intergrader agreement (ICC) was 0.53 (95% CI, 0.331–0.685) for FAF‐BPRF and 0.404 (95% CI, 0.149–0.601) for CSLO (comparison between ICC, p = 0.368). Agreement (ICC) between the two devices was worse for grader 1 (0.356; 95% CI, 0.129–0.549) than grader 2 (0.856; 95% CI, 0.774–0.910) (p < 0.001). Conclusions: Despite excellent intragrader reproducibility for β‐PPA measurements with FAF‐BPRF and CSLO, intergrader reproducibility is low to moderate. Measurements of β‐PPA area obtained with the two instruments are of moderate agreement and, therefore, are not interchangeable.     

NGF and TGF‐β mRNA expression during pregnancy in a rat corneal wound healing model

Background: Growth factors seem to play a major role in corneal wound healing and TGF‐β seems to be associated with abnormal healing after corneal surgical procedures. Few studies have analysed the role of NGF and TGF‐β on corneal wound healing during pregnancy. The aim of the present study was to create an animal model to evaluate the expression of NGF and TGF‐βs during corneal wound healing in two groups: control and pregnant rats. Methods: Corneal mRNA for NGF and the three isoforms of TGF‐β were analysed by RT‐PCR, in a time‐course experiment on different days after epithelial wounding (2, 7, 14 days) in pregnant and control groups Results: The results show high corneal mRNA expression for NGF and TGF‐β1 without any variation throughout the healing process or pregnancy evolution. However, we detected a different expression of corneal mRNAs for TGF‐β2 and TGF‐β3 in the control group. This data was not detected in the pregnant group. Discussion: Our results suggested that pregnancy could have a relevant role on TGF‐β2 and TGF‐β3 mRNA expression during the corneal wound healing process. Additional research should be performed to corroborate these findings.     

ALDH3A1: a corneal crystallin with diverse functions

Aldehyde dehydrogenase 3A1 (ALDH3A1) comprises a surprisingly high proportion (5-50% depending on species) of the water-soluble protein of the mammalian cornea, but is present little if at all in the cornea of other species. Mounting experimental evidence demonstrates that this abundant corneal protein plays an important role in the protection of ocular structures against oxidative damage. Corneal ALDH3A1 appears to protect against UV-induced oxidative stress through a variety of biological functions such as the metabolism of toxic aldehydes produced during the peroxidation of cellular lipids, the generation of the antioxidant NADPH, the direct absorption of UV-light, the scavenging of reactive oxygen species (ROS), and the possession of chaperone-like activity. With analogies to the abundant, multifunctional, and taxon-specific lens crystallins, mammalian ALDH3A1 has been considered a corneal crystallin, suggesting that it may contribute to the optical properties of the cornea as well. Recent studies have also revealed a novel role for ALDH3A1 in the regulation of the cell cycle. ALDH3A1-transfected HCE cells have increased population-doubling time, decreased plating efficiency, and reduced DNA synthesis, most likely due to a profound inhibition of cyclins and cyclin-dependent kinases. We have proposed that the ALDH3A1-induced reduction in cell growth may contribute to protection against oxidative stress by extending time for DNA and cell repair. Taken together, the multiple roles of ALDH3A1 against oxidative stress in addition to its contributions to the optical properties of the cornea are consistent with the idea that this specialized protein performs diverse biological functions as do the lens crystallins.     

Anti-inflammatory effects of α-humulene and β-caryophyllene on pterygium fibroblasts

AIMTo investigate the anti-inflammatory effects of the sesquiterpenes α-humulene and β-caryophyllene on pterygium fibroblasts.METHODSPrimary cultures of pterygium fibroblasts were established. Third passage pterygium fibroblasts were exposed to α-humulene and β-caryophyllene separately and together. The cell viability was assessed by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay at 12, 24, 48, and 72h after exposure. The levels of the inflammatory cytokines interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α and IL-10 in the conditioned culture medium were assessed by enzyme-linked immunosorbent assay (ELISA) at 12, 24 and 48h after exposure. Data were statistically analyzed using Friedman repeated measures analysis of variances on ranks.RESULTSThe 25 µmol/L β-caryophyllene induced significant decrease in the IL-6 production by pterygium fibroblasts 48h after the exposure (P=0.041). The levels of IL-1β, IL-8, IL-10, and TNF-α were very low and had no statistically significant variations after exposure to α-humulene, β-caryophyllene, or both compounds together.CONCLUSIONThe exposure to 25 µmol/L of β-caryophyllene significantly reduce the production of IL-6 by pterygium fibroblasts after 48h. This sesquiterpene may be a potential alternative adjuvant agent for the treatment of pterygium.     

Elevated protein carbonyl and HIF‐1α levels in eyes with proliferative diabetic retinopathy

Purpose: To evaluate the role of protein carbonyls and hypoxia inducible factor‐1α (HIF‐1α) in diabetic eyes with proliferative diabetic retinopathy (PDR). Methods: Prospective consecutive controlled observational study was performed. Vitreous samples were collected at the start of the 3‐ppp vitrectomy. Protein carbonylation analysis was performed by Western blotting with antibody against 2,4‐Dinitrophenol (anti‐DNP), following derivatization of protein carbonyls with 2,4 Dinitrophenylhydrazine (DNHP). Protein carbonylation was quantified by scanning densitometry analysis and relativized to the total amount of protein into the ponceau staining of membranes. Vitreous HIF‐1 α was determined with ELISA in a subgroup of the samples. Thirty‐one eyes were operated due to PDR (study group). Of the 189 controls, 39 had nonproliferative diabetic retinopathy (non‐PDR), 111 retinal detachment (RD) and 39 macular hole/pucker (MH). Results: Comparison of eyes with PDR with controls revealed that the mean vitreous concentrations of protein carbonyls were significantly higher in the eyes affected with PDR being 242 ± 130 (SD) compared with non‐PDR controls 180 ± 142, nondiabetic eyes affected with RD 175 ± 131 and MH/pucker 140 ± 95 (p = 0.008, one‐way anova ). Mean HIF‐1α values were higher in eyes with PDR compared with controls (RD, MH/pucker); the values being 0.53 ± 0.34 (SEM; n = 4) and 0.13 ± 0.04 (SEM; n = 19), respectively (p = 0.009). Conclusions: Protein carbonyl and HIF‐1 α levels were significantly increased in the vitreous fluid of surgically treated eyes with PDR. Our findings suggest an association between increased intravitreal levels of protein carbonyls and the pathogenesis of PDR.     

The Therapeutic Roles of Recombinant Hsp90α on Cornea Epithelial Injury

PurposeThe purpose of this study was to explore the therapeutic role of heat shock protein 90 (Hsp90) in wound healing of injury cornea epithelium.MethodsThe right eye of C57BL/6N male mice were performed the debridement wounds in the center of the cornea using an algerbrush II blade. The injured area was determined by staining the cornea with fluorescein sodium and measured with image-J. Immunoblotting, ELISA and immunochemistry were used for determining protein expression. The quantitation PCR was performed to measure mRNA expression.ResultsHsp90α is upregulated at both the mRNA and protein levels, and is secreted extracellularly into the corneal stroma and tear film during the healing process after corneal injury in mice. This upregulation is associated with activation of HSF1. Administration of recombinant exogenous Hsp90α (eHsp90α) speeds up wound healing of injured corneal epithelium. The eHsp90α binds to low-density lipoprotein (LDL)-related protein-1 (LRP-1) on the corneal epithelial cells and increases phosphorylation of AKT at S473, which is associated with proliferation and migration corneal epithelial cells in vitro or vivo. Inhibition of AKT by its inhibitor LY294002 abolishes eHsp90α-induced migration and proliferation of corneal epithelial cells.ConclusionsHsp90α is upregulated and secreted after corneal injury and acts to promote the healing process. Recombinant Hsp90α may be a promising therapeutic drug candidate for corneal injury.     

A randomized phase II trial of interferon-α2b versus 5-fluorouracil after trabeculectomy*

Purpose: The aim of the present study was to investigate the safety and potential efficacy of subconjunctival interferon-α2b (IFN-α), either alone or in combination with 5-fluorouracil (5-FU), in reducing the risk of failure of glaucoma surgery. Methods: A prospective, masked randomized phase II study was undertaken in which patients received three subconjunctival injections per week for 3–4 weeks postoperatively. Three treatments were compared: (i) IFN-α (1 × 10 ⁶ IU per dose); (ii) 5-FU (5 mg per dose); and (iii) alternating IFN-α and 5-FU (BOTH). The primary outcome measures were: (i) rate of successful control of intra-ocular pressure without further surgery; and (ii) the incidence of side effects. Results: Fifty-seven patients undergoing glaucoma surgery with an increased risk of failure were evaluated, including 23 patients (40%) undergoing trabeculectomy combined with extracapsular cataract extraction as well as other conventional high-risk groups. With 53 patients (93%) completing 2 years follow up, there was no significant difference in success rates among the three groups. Intra-ocular pressure was controlled without further surgery in 79% of patients (95% confidence interval (CI): 61, 97%) receiving IFN-α, in 89% of patients (76, 100%) receiving 5-FU and in 89% of patients (76, 100%) receiving BOTH. Side effects were similar among the three groups. Conclusions: These results are consistent with a beneficial effect of IFN-α2b given either alone or in combination with 5-FU after glaucoma filtering surgery. However, the lack of a clear and substantial benefit over conventional anti-fibrotic therapy does not support the further clinical evaluation of these treatments.     

The Effect of CHIR 99021, a Glycogen Synthase Kinase-3β Inhibitor,on Transforming Growth Factor β-Induced Tenon Fibrosis

PurposeThis study investigated the effect of glycogen synthase kinase-3β (GSK-3β) inhibition on the fibrosis of human Tenon''s fibroblasts (HTFs) induced by transforming growth factor-β (TGF-β).MethodsQuantitative real-time PCR and Western blot analyses were performed to determine the expression levels of molecules associated with the fibrosis of HTFs by TGF-β (fibronectin, collagen Iα, and α-smooth muscle actin) and GSK-3β. The levels of phosphorylated Smad2 and Smad3 were also analyzed in the presence of the GSK-3β inhibitor CHIR 99021. The wound healing assay was performed to determine the effect of CHIR 99021 on the migration of HTFs. All experiments were conducted using primary cultured HTFs or human tenon tissues obtained from normal subjects and patients with glaucoma.ResultsTreatment with TGF-β resulted in an increase in the levels of molecules associated with the fibrosis of HTFs. The expression levels of these molecules were higher in the tenon tissues obtained from patients with glaucoma than those from normal subjects. When the HTFs were treated with TGF-β, a significant increase in the active form of GSK-3β (Y216) was observed. A significant decrease in the active form of GSK-3β and molecules associated with fibrosis by TGF-β was noted in HTFs treated with CHIR 99021. CHIR 99021 treatment reduced the phosphorylated Smad2/Smad2 and phosphorylated Smad3/Smad3 ratios in HTFs and attenuated HTF migration.ConclusionsOur results demonstrated the effect of GSK-3β inhibition on the regulation of TGF-β–mediated fibrosis of HTFs, suggesting GSK-3β to be a potential target for maintaining bleb function after glaucoma filtration surgery.     

A Novel Hypoxia-inducible Factor 1α Inhibitor KC7F2 Attenuates Oxygen-induced Retinal Neovascularization

PurposeKC7F2 is a novel molecule compound that can inhibit the translation of hypoxia-inducible factor 1α (HIF1α). It has been reported to exhibit potential antiangiogenic effect. We hypothesized that KC7F2 could inhibit oxygen-induced retinal neovascularization (RNV). The purpose of this study was to investigate this assumption.MethodsOxygen-induced retinopathy (OIR) models in C57BL/6J mice and Sprague-Dawley rats were used for in vivo study. After intraperitoneal injections of KC7F2, RNV was detected by immunofluorescence and hematoxylin and eosin staining. Retinal inflammation was explored by immunofluorescence. EdU incorporation assay, cell counting kit-8 assay, scratch test, transwell assay, and Matrigel assay were used to evaluate the effect of KC7F2 on the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVEC) induced by vascular endothelial growth factor (VEGF) in vitro. Protein expression was examined by Western blot.ResultsKC7F2 treatment (10 mg/kg/d) in OIR mice significantly attenuated pathological neovascularization and decreased the number of preretinal neovascular cell nuclei, without changing the avascular area, which showed the same trends in OIR rats. Consistently, after the KC7F2 intervention (10 µM), cell proliferation was inhibited in VEGF-induced HUVEC, which was in agreement with the trend observed in the retinas of OIR mice. Meanwhile, KC7F2 suppressed VEGF-induced HUVEC migration and tube formation, and decreased the density of leukocytes and microglia colocalizing neovascular areas in the retinas. Moreover, the HIF1α–VEGF pathway activated in retinas of OIR mice and hypoxia-induced HUVEC, was suppressed by KC7F2 treatment.ConclusionsThe current study revealed that KC7F2 was able to inhibit RNV effectively via HIF1α–VEGF pathway, suggesting that it might be an effective drug for RNV treatment.