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1.  Evaluation of bleb characteristics after implantation of the EX-PRESS™ glaucoma filtration device 
Molecular Vision  2012;18:10-13.
Purpose
To compare bleb survival and histology after implantation of the EX-PRESS™ glaucoma filtration device versus silicone tubes in a rabbit model of filtration surgery.
Methods
Glaucoma filtration surgery was performed on one eye each of twelve New Zealand white rabbits. Eyes were randomized to implantation with the EX-PRESS™ filtration device (n=6) or a silicone tube (n=6). Bleb vascularity was evaluated at three and six weeks using a standard scale. At 6 weeks, eyes were enucleated and a histologic analysis was performed. Bleb survival was also recorded for the two groups.
Results
Histologically, a thin capsule consisting of mild fibroblast proliferation associated with intercellular collagen was present around both implants. Both groups demonstrated a mild infiltration of plasma cells and polymorphonuclear leukocytes. Bleb vascularity was similar between both groups at three and six weeks post-operatively. Bleb survival between the two groups was not significantly different.
Conclusions
Similar outcomes were noted after glaucoma filtration surgery using either silicone tubes or the EX-PRESS™ glaucoma filtration device in this rabbit model. Both implants appear to be relatively inert with little difference in biocompatibility and bleb survival.
PMCID: PMC3253066  PMID: 22232564
2.  Effects of bevacizumab on the neovascular membrane of proliferative diabetic retinopathy: reduction of endothelial cells and expressions of VEGF and HIF-1α 
Molecular Vision  2012;18:1-9.
Purpose
Anti-vascular endothelial growth factor (VEGF) agents have recently been used intravitreally during the perioperative period for proliferative diabetic retinopathy (PDR). However, the mechanism of theraputic effects of the agents remains unclear. This study aimed to investigate the effects of intravitreal bevacizumab (IVB) on retinal vascular endothelial cells and expressions of VEGF and hypoxia inducible factor-1α (HIF-1α) in PDR.
Methods
Twenty-four patients with PDR were enrolled and randomized to two groups. Twelve eyes of 12 patients of each group received either an intravitreal injection of 1.25 mg bevacizumab or a sham injection 6 days before vitrectomy. Neovascular membranes (NVMs) were collected during pars plana vitrectomy. The numbers of vascular endothelial cells in the NVMs were counted after staining with hematoxylin and eosin and von Willebrand. The expressions of VEGF and HIF-1α in the NVMs were detected through immunohistochemistry. Ten epiretinal membrane specimens from patients with proliferative vitreoretinopathy (PVR) without IVB treatment were set as an additional control.
Results
The number of vascular endothelial cells in NVMs of the IVB pretreated group was significantly lower than that of the sham group (21.5±3.94 versus 41.33±7.44, p=0.003). The IVB pretreated group also showed significantly lower levels of VEGF and HIF-1α in NVMs than those of the sham group (PHIF-1α=0.02, PVEGF<0.001). A stepwise regression analysis showed that IVB was a significant negative predictor for the numbers of vascular endothelial cells (β=–0.89, p<0.001) and the expressions of VEGF (β=–0.85, p<0.001) and HIF-1α (β=–0.64, p=0.001) in PDR patients. Epiretinal membranes of the PVR group showed negative staining of VEGF and HIF-1α.
Conclusions
Pretreatment with IVB in patients with PDR significantly decreased vascular endothelial cells and expressions of VEGF and HIF-1α, which further supports preoperative use of IVB in such patients.
PMCID: PMC3253067  PMID: 22232563
3.  Chlamydia infection status, genotype, and age-related macular degeneration 
Molecular Vision  2012;18:29-37.
Purpose
To evaluate whether Chlamydia (C.) infections are associated with age-related macular degeneration (AMD) and to assess if this association is influenced by the complement factor H (CFH) Y402H or the high temperature requirement A serine peptidase 1 (HTRA1) rs11200638 risk genotypes.
Methods
One hundred ninety-nine AMD patients with early and late forms of the disease and 100 unaffected controls, at least 50 years old were included in the study. Patients in the AMD and control groups were selected based on known CFH Y402H variant genotype status (one third homozygous CC, one third heterozygous CT, and one third wild-type TT). Plasma from all patients and controls was tested for C. pneumoniae, C. trachomatis, and C. psittaci IgG seropositivity using a micro-immunofluorescent assay to establish previous infection status. Assays were conducted blind to risk genotypes and the results analyzed using univariate and multivariate (logistic regression) analysis.
Results
IgG seropositivity to C. pneumoniae was most prevalent (69.2%, n=207), followed by C. trachomatis (7.4%, n=22) and C. psittaci (3.3%, n=10). No association was found between each of the three Chlamydia species IgG seropositivity and AMD status or severity (early/late). There was also no significant association between Chlamydia species IgG seropositivity and AMD status or severity, in patients carrying at least one CFH Y402H risk allele (C) or HTRA1 rs11200638 risk allele (A), with univariate or logistic regression analysis.
Conclusions
Chlamydia infection status does not appear to be associated with AMD status or severity. The presence of CFH Y402H and HTRA1 rs11200638 risk genotypes does not alter this negative association.
PMCID: PMC3258520  PMID: 22259222
4.  Factors influencing bacterial adhesion to contact lenses 
Molecular Vision  2012;18:14-21.
The process of any contact lens related keratitis generally starts with the adhesion of opportunistic pathogens to contact lens surface. This article focuses on identifying the factors which have been reported to affect bacterial adhesion to contact lenses. Adhesion to lenses differs between various genera/species/strains of bacteria. Pseudomonas aeruginosa, which is the predominant causative organism, adheres in the highest numbers to both hydrogel and silicone hydrogel lenses in vitro. The adhesion of this strain reaches maximum numbers within 1h in most in vitro studies and a biofilm has generally formed within 24 h of cells adhering to the lens surface. Physical and chemical properties of contact lens material affect bacterial adhesion. The water content of hydroxyethylmethacrylate (HEMA)-based lenses and their iconicity affect the ability of bacteria to adhere. The higher hydrophobicity of silicone hydrogel lenses compared to HEMA-based lenses has been implicated in the higher numbers of bacteria that can adhere to their surfaces. Lens wear has different effects on bacterial adhesion, partly due to differences between wearers, responses of bacterial strains and the ability of certain tear film proteins when bound to a lens surface to kill certain types of bacteria.
PMCID: PMC3258521  PMID: 22259220
5.  Genotype-phenotype analysis of F-helix mutations at the kinase domain of TGFBR2, including a type 2 Marfan syndrome familial study 
Molecular Vision  2012;18:55-63.
Purpose
Transforming growth factor beta receptor II (TGFBR2) gene mutations are associated with Marfan syndrome; however, the relationship between the mutations and clinical phenotypes are not clear.
Methods
Genomic DNA from peripheral blood leukocytes of a Chinese proband with Marfan syndrome, five of the proband’s relatives, and 100 unrelated Chinese control subjects were isolated and screened for fibrillin-1 (FBN1) and TGFBR2 gene mutations by direct sequencing, and a genotype-phenotype study was performed following a review of the literature on TGFBR2 mutations in the search area. Also, the structure of TGFBR2 protein before and after gene mutation was analyzed.
Results
The results identified a novel missense TGFBR2 mutation p.V453E (c.1358T>A) in the proband and two relatives that was located in the F-helix in the kinase domain of TGFBR2. No such genetic change was observed in the unrelated controls. No FBN1 mutation was detected in any of the subjects. Genotype-phenotype analyses indicated that F-helix mutations are related to type 2 Marfan syndrome and Loeys-Dietz syndrome, and these mutations can lead to severe cardiovascular (93.8%) and skeletal (81.3%) lesions and minor ocular lesions (25%). Losartan treatment can slow-down the progression of aortic lesions.
Conclusions
The findings extend the mutation spectrum of Marfan syndrome, and that mutations at the F-helix in the kinase domain of TGFBR2 may be associated with the development of severe cardiovascular and skeletal lesions and minor ocular lesions. These findings have implications for genetic testing, diagnosis, and treatment in individuals with transforming growth factor beta (TGF-β) signaling-related disorders.
PMCID: PMC3258522  PMID: 22259224
6.  Effects of a novel DNA methyltransferase inhibitor Zebularine on human lens epithelial cells 
Molecular Vision  2012;18:22-28.
Purpose
Posterior capsular opacification (PCO) is a common long-term complication of modern cataract surgery. We have shown that Zebularine, an inhibitor of DNA methylation, suppresses transforming growth factor-β (TGFβ)-induced lens epithelial cells (LECs)–myofibroblasts transdifferentiation. The purpose of this study is to evaluate the role that Zebularine plays in the inhibition of PCO pathogenesis, including its effect on attachment, migration, and proliferation of LECs in vitro.
Methods
A tetrazolium dye–reduction assay (MTT test) was performed to determine cell proliferation. Cell attachment was assessed by modified MTT test. Migration was determined by the transwell method after incubation of LECs with Zebularine. The effect of Zebularine on DNA-methyltransferase 1 (DNMT1), phospho-p44/42 Map Kinase, and protein kinase B (Akt) were analyzed by western blot.
Results
Zebularine was an effective inhibitor of human LEC proliferation, attachment, and migration in vitro. A Zebularine concentration of 100 μM accounted for the following: inhibition of cell proliferation of 57.2%, reduction in cell attachment to 29.6%, and inhibition of cell migration of 58.9%. All effects were dose dependent. Zebularine treatment resulted in dose-dependent decreases of DNMT1, phosphorylated p44/42 MAP Kinase, and phosphorylated Akt.
Conclusions
Zebularine is capable of inhibiting the crucial cellular events in PCO pathogenesis in vitro. Zebularine acts through the inhibition of DNMT1, and it consequently down regulation of the expression of proliferative and survival genes that relate to pathogenesis of PCO. These findings suggest that Zebularine may become a therapeutic approach for the prevention of PCO.
PMCID: PMC3258523  PMID: 22259221
7.  Regulation of the human tyrosinase gene in retinal pigment epithelium cells: the significance of transcription factor orthodenticle homeobox 2 and its polymorphic binding site 
Molecular Vision  2012;18:38-54.
Purpose
Tyrosinase is the rate-limiting enzyme responsible for melanin biosynthesis in the retinal pigment epithelium (RPE) of the eye. Melanin has an important role in retinal development, function, and protection against light-induced oxidative stress, and melanin levels are associated with age-related macular degeneration (AMD). Because the levels of and protection afforded by melanin seem to decline with increasing age, proper regulation of the human tyrosinase gene (TYR) in the RPE is an important but insufficiently understood process. Our purpose was to obtain detailed information on regulation of the TYR gene promoter in the human RPE and to specify the role of orthodenticle homeobox 2 (OTX2) and microphthalmia-associated transcription factor (MITF).
Methods
We used luciferase reporter constructs to study regulation of the human TYR gene promoter in cultured human RPE cells. We further studied the role of OTX2 and MITF, their binding sites, and endogenous expression by using mutagenesis, electrophoretic mobility shift assay, yeast two-hybrid assay, RNA interference, and gene expression analyses.
Results
In the RPE, OTX2 activated the human TYR gene promoter via direct trans-activation of novel OTX2 binding elements. In addition, we found that indirect activation by OTX2 via more proximal MITF binding sites, even in the absence of OTX2 sites, took place. These results are consistent with the physical interaction observed between OTX2 and MITF. Overexpression or knockdown of OTX2 in RPE cells resulted in corresponding changes in tyrosinase mRNA expression. Finally, we found that a single nucleotide polymorphism (SNP rs4547091) at the most proximal OTX2 binding site is associated with altered nuclear protein binding and a remarkable decrease in TYR promoter activity in RPE cells. This single nucleotide polymorphism (SNP) is more common in the European population in which AMD is also more prevalent.
Conclusions
In the RPE, OTX2 activates the human TYR gene promoter by direct DNA binding and by interaction with MITF. Such synergistic interaction highlights the role of OTX2 as a potential coregulator of numerous MITF target genes in the eye. Genetic differences in OTX2 binding sites affect tyrosinase regulation. Collectively, these findings emphasize the role of OTX2 in regulating the human TYR gene, with implications for inter-individual differences in melanin synthesis, retinal development, and function as well as susceptibility to retinal degeneration associated with aging.
PMCID: PMC3258524  PMID: 22259223
8.  Matrix metalloproteinase-2 and -9 activities in the human lens epithelial cells and serum of steroid induced posterior subcapsular cataracts 
Molecular Vision  2012;18:64-73.
Purpose
To evaluate the level of matrix metalloproteinase (MMP)-2 and MMP-9 activities in patients with steroid induced posterior subcapsular cataract (PSC).
Methods
This prospective, observational study comprised of 156 patients having either steroid induced PSC (n=50) or non-steroidal PSC (n=106) were performed to evaluate the level of MMP-2 and MMP-9 activities in the lens epithelial cells (LECs) and the serum. Anterior lens capsules harboring LECs were obtained during phacoemulsification and peripheral blood was collected from patients before administration of anesthesia. Serum was separated by centrifugation at 10,000× g for 15 min at 4 °C. The LECs and serum samples were processed to analyze MMP-2 and MMP-9 activities using succinylated gelatin assay. Quantitative real time-PCR (qRT–PCR) was performed to determine the mRNA levels of MMP-2 and MMP-9 in LECs. The mRNA levels were expressed as a ratio, using the delta-delta method for comparing the relative expression results between cases with steroid induced PSC and cases with non-steroidal PSC. MMP-2 and MMP-9 levels were also compared in the two groups using immunolocalization.
Results
The level of MMP-2 and MMP-9 activity was found to be high in LECs and serum of cases with steroid induced PSC. Further in all steroid induced cases, a 1.4 fold increase was observed in MMP-2 activity in LECs and a 1.4 fold increase in MMP-9 activity in the serum. Both qRT–PCR and immunolocalization showed increased expression of MMP-2 and MMP-9 activity.
Conclusions
MMP-2 and MMP-9 activity in both LECs and serum was significantly higher in cases with steroid induced PSC. The possible use of MMP-9 as a non-invasive biomarker in ascertaining the presence of steroid induced PSC should be evaluated using a larger sample size.
PMCID: PMC3260085  PMID: 22259225
9.  The polyamidoamine-mediated inhibition of bcl-2 by small hairpin RNA to induce apoptosis in human lens epithelial cells 
Molecular Vision  2012;18:74-80.
Purpose
To investigate whether apoptosis of human lens epithelial cells (HLECs) can be induced with the polyamidoamine (PAMAM)-mediated inhibition of bcl-2 (b-cell lymphoma 2) by small hairpin RNA (shRNA).
Methods
HLECs (SRA01/04) were transfected with the fifth generation of PAMAM (PAMAM G5) by bcl-2 shRNA. At 24, 48, and 72 h after transfection, the transfection rate was measured by flow cytometry. The transfection rates mediated by PAMAM and liposome were compared. The bcl-2 mRNA level was detected by real-time PCR. Whole cell protein was extracted and the bcl-2 protein level was detected by western blotting. The percentage of HLECs undergoing apoptosis was measured by Annexin V-FITC/PI staining. The nuclear morphology of HLECs was observed by staining with Hoechst 33258. The expression of cytochrome c and the activity of cleaved caspase-3 were analyzed by western blotting.
Results
At 24, 48, and 72 h after transfection, the rate of transfection of bcl-2 shRNA mediated by PAMAM was higher than in the liposome-mediated group (p<0.05). The mRNA and protein levels of bcl-2 were greatly downregulated. The percentage of HLECs undergoing apoptosis was greatly improved. Hoechst staining showed that bcl-2 shRNA transfected cells had a lower growth status with nuclear fragmentation. The expression of cytochrome c and the activity of cleaved caspase-3 was greatly improved (p<0.05).
Conclusions
PAMAM-mediated bcl-2 shRNA can downregulate the expression of bcl-2 and induce the apoptosis of HLECs by engaging the mitochondrial pathway, including catalytic activation of the caspases.
PMCID: PMC3261082  PMID: 22262940
10.  A novel splicing mutation of the FRMD7 gene in a Chinese family with X-linked congenital nystagmus 
Molecular Vision  2012;18:87-91.
Purpose
To identify a potential pathogenic mutation in a four-generation Chinese family with X-linked congenital nystagmus (XLCN).
Methods
Routine clinical examination and ophthalmic evaluation were performed on normal controls, two patients and two healthy members of the family. Genomic DNA was prepared from the peripheral blood of members of the family and from 50 normal controls. All coding exons and the intronic boundaries of the four-point-one (4.1), ezrin, radixin, moesin (FERM) domain-containing 7 (FRMD7) gene were amplified using polymerase chain reaction (PCR) followed by direct sequencing.
Results
A previously unreported splicing mutation, c.163–1 G→T transversion (c.163–1 G>T), was detected preceding exon3 of FRMD7 in the patients but not in the unaffected family members and 50 unrelated healthy individuals.
Conclusions
We identified a novel mutation (c.163–1 G→T) of FRMD7 in this Chinese family with XLCN. Our finding is the first report related to c.163–1 G→T mutation in FRMD7. The result expands the mutation spectrum of FRMD7 in association with congenital nystagmus.
PMCID: PMC3261083  PMID: 22262942
11.  A new novel mutation in FBN1 causes autosomal dominant Marfan syndrome in a Chinese family 
Molecular Vision  2012;18:81-86.
Purpose
Screening of mutations in the fibrillin-1 (FBN1) gene in a Chinese family with autosomal dominant Marfan syndrome (MFS).
Methods
It has been reported that FBN1 mutations account for approximately 90% of Autosomal Dominant MFS. FBN1 mutations were analyzed in a Chinese family of 36 members including 13 MFS patients. The genomic DNAs from blood leukocytes of the patients and their relatives were isolated and the entire coding region of FBN1 was amplified by PCR. The sequence of FBN1 was dertermined with an ABI 3100 Genetic Analyzer.
Results
A previously unreported the missense mutation G214S (caused by a 640 A→G heterozygous change) in FBN1 was identified in the Chinese family. The mutation was associated with the disease phenotype in patients, but not detected in their relatives or in the 100 normal controls.
Conclusions
This is the first report of molecular characterization of FBN1 in the MFS family of Chinese origin. Our results expand the spectrum of FBN1 mutations causing MFS and further confirm the role of FBN1 in the pathogenesis of MFS. Direct sequencing of the mutation in FBN1 may be used for diagnosis of MFS.
PMCID: PMC3261084  PMID: 22262941
12.  Hypoxia initiates sirtuin1-mediated vascular endothelial growth factor activation in choroidal endothelial cells through hypoxia inducible factor–2α 
Molecular Vision  2012;18:114-120.
Purpose
Hypoxia is a critical pathological factor in a variety of retinal diseases, including age-related macular degeneration. It upregulates angiogenic growth factors and promotes neovascularization. Hypoxia changes the cellular redox state and activates class III histone deacetylase sirtuin1 (SIRT1). Activated SIRT1 signals hypoxia inducible factor (HIF)-2α, which transactivates vascular endothelial growth factor (VEGF) and erythropoietin. In this study, we investigated the role of hypoxia induced SIRT1 in choroidal neovascularization in relation to age-related macular degeneration.
Methods
Choroidal endothelial cells (RF/6A) were maintained in a semiconfluent state and hypoxia was induced by exposing the cells to cobalt chloride for 24 h. Induction of hypoxia was confirmed by flow cytometric analysis and the levels of SIRT1 were noted in a hypoxic condition as well in the cells after blocking SIRT1 activity using sirtinol. The role of SIRT1 in the activation of HIF-2α and nuclear factor–κB (RelA/p65) during hypoxia in the presence or absence of SIRT1 was assessed using immunoblot analysis. VEGF levels were quantified using enzyme-linked immunosorbent assay.
Results
Hypoxic induction was confirmed using flow cytometric analysis, which showed cell cycle arrest starting at a 200 µM concentration of cobalt chloride. Hypoxic treatment (200 µM concentration of cobalt chloride) increased SIRT1 levels to 7.8%, which reduced to control level after its activity was inhibited (p<0.05). Activated SIRT1 mediates HIF-2α and nuclear factor-κB (RelA/p65) expression to 4.5 fold and fivefold, respectively, compared to control, and the levels were suppressed following sirtinol treatment (4.1% and 39.3% respectively; p=0.01). Hypoxic treatment increased VEGF levels by 94.9±19.6 pg/ml compared to control levels (25.58±3.58 pg/ml). These levels decreased to 10.29±0.2 pg/ml after blocking SIRT1 activity using sirtinol, compared to control (p<0.01).
Conclusions
Our study results demonstrate that hypoxia mimetic cobalt chloride induces SIRT1 and augments HIF-2α, which activates and releases VEGF.
PMCID: PMC3265172  PMID: 22275802
13.  Comparison of beneficial factors for corneal wound-healing of rat mesenchymal stem cells and corneal limbal stem cells on the xenogeneic acellular corneal matrix in vitro 
Molecular Vision  2012;18:161-173.
Purpose
This experiment aims to investigate the potential ability of mesenchymal stem cells (MSCs) to produce beneficial factors for corneal recovery when the cells were seeded on a xenogeneic acellular corneal matrix (ACM) in vitro.
Methods
MSCs and corneal limbal stem cells (LSCs) from the corneal limbus region of rats were isolated and cultured. Batch 1 from each type of cell was seeded on a Beagle cornea ACM at a density of 3×103 cells/mm2 for 7 days. The proliferation activity of the cells was quantitatively determined at 1, 3, 5, and 7 days with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Keratin3/12 (CK3/12) growth factors, including vascular endothelial growth factor (VEGF), pigment epithelium-derived factor, epidermal growth factor, and transforming growth factor-beta1, integrin subunits such as α5β1, and α6β1, and elements of the extracellular matrix (ECM) such as fibronectin and laminin were investigated with immunofluorescence staining, reverse transcriptional polymerase chain reaction, and western blot assay, respectively.
Results
The basic expression of growth factors in MSCs was much higher (n=6, p<0.05) than that in the LSCs, including VEGF, epidermal growth factor, and transforming growth factor-beta1. After being seeded on the ACM, those factors in MSCs expressed continuously at a high level, but the seeded corneal epithelium cells presented a downregulated trend in these factors. The expression of VEGF in seeded MSCs decreased, which was similar to the trend for the seeded LSCs (n=6, p<0.05). The expression of keratin3, a sign of mature epithelium cells, was also present in the MSCs after being seeded for 7 days. The expression of pigment epithelium-derived factor by the seeded and normal culture MSCs was equal, while the expression of this factor was not detected in either the seeded or the normal cultured LSCs. There were no significant differences between the integrin subunits (α5, α6, β1) and the extracellular matrix, including fibronectin and laminin, generated by normal cultured or seeded MSCs and LSCs.
Conclusions
Under the ACM microenvironment, the MSCs presented beneficial factors for corneal recovery comparable to those presented by corneal LSCs. This indicates that MSCs, when combined with an ACM, may compose a competent corneal substitute for healing corneal wounds.
PMCID: PMC3265173  PMID: 22275807
14.  Ursodeoxycholic acid prevents selenite-induced oxidative stress and alleviates cataract formation: In vitro and in vivo studies 
Molecular Vision  2012;18:151-160.
Objective
To evaluate the antioxidative and anticataractogenic potential effect of ursodeoxycholic acid (UDCA) on selenite-induced cataract in vitro and in vivo.
Methods
Enucleated rat lenses were incubated in M199 medium alone (Group I), with 200 μM selenite (Group II), or with 200 μM selenite and 500 μM UDCA (Group III). Selenite was administered on the third day and UDCA treatment was from the second to the fifth day. The development of cataracts was observed under an inverted microscope. Total antioxidative capabilities (T-AOC), mean activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx), glutathione reductase (GR) and glutathione S-transferase (GST), levels of reduced glutathione (GSH), malondialdehyde (MDA), and total sulfhydryl content were analyzed in lenticular samples. In vivo, cataracts were induced in 12-day-old pups by single subcutaneous injections of sodium selenite. The test groups received 180 mg/kg bodyweight/day of UDCA intraperitoneally on postpartum days 11–16 or 0.5% UDCA drops four times daily on postpartum days 11–25.
Results
In vitro, morphological examination of the lenses revealed dense vacuolization and opacification in Group II, minimal vacuolization in 12.5% of Group III, and no opacification in 87.5% of Group III. In Group I, all lenses were clear. UDCA significantly (p<0.05) restored GSH and total sulfhydryl, and decreased MDA levels. T-AOC and the mean activities of the antioxidant enzymes were elevated following treatment with UDCA. In vivo, 0.5% UDCA drops resulted in only 20% nuclear cataract development and 180 mg/kg of UDCA intraperitoneally led to 50% development, compared to 100% in the control group (p<0.05).
Conclusions
UDCA prevents selenite toxicity and cataractogenesis by maintaining antioxidant status and GSH, protecting the sulfhydryl group, and inhibiting lipid peroxidation in lenses.
PMCID: PMC3265174  PMID: 22275806
15.  The association of CD36 variants with polypoidal choroidal vasculopathy compared to typical neovascular age-related macular degeneration 
Molecular Vision  2012;18:121-127.
Purpose
To clarify the association of cluster of differentiation 36 (CD36) variants with polypoidal choroidal vasculopathy (PCV) and compare them with those in typical neovascular age-related macular degeneration (tAMD).
Methods
We included 349 Japanese AMD patients (210 PCV, 139 tAMD) and 198 age-matched controls. Four tag single-nucleotide polymorphisms (SNPs)—rs10499862, rs3173798, rs3211883, and rs3173800—in the CD36 region were genotyped using the TaqMan assay. Allelic and genotypic frequencies of the SNPs were tested.
Results
Although none of the SNPs tested were associated with PCV, the allelic frequencies of rs3173798 and rs3173800 were significantly different between PCV and tAMD patients. Genotype association analysis demonstrated different associations of these two SNPs between PCV and tAMD in the genotype model. Haplotype analysis revealed that the association of the major haplotype (T-T-T-T) at four selected SNPs in CD36 differed significantly between PCV and tAMD patients.
Conclusions
The CD36 region may be associated with the difference in genetic susceptibility for PCV and tAMD.
PMCID: PMC3265175  PMID: 22275803
16.  Accumulation and autofluorescence of phagocytized rod outer segment material in macrophages and microglial cells 
Molecular Vision  2012;18:103-113.
Purpose
To explore the ability of macrophages and microglial cells to phagocytize rod outer segments (ROSs) in a cell culture and characterize the resulting lipofuscin-like autofluorescence (LLAF).
Methods
Either regular or modified ROSs or ROS components (11-cis-retinal, all-trans-retinal, lipids) were fed to macrophages and microglial cells for 4 days. Afterwards, autofluorescence was detected by fluorescence-activated cell sorting (FACS) at two different wavelengths (533 nm and 585 nm), and the cells were imaged by confocal and electron microscopy. Fluorescein isothiocyanate (FITC)-labeled ROSs were added to macrophage and microglial cell cultures for 1–24 h to determine the kinetics of phagocytosis in these cell lines.
Results
Feeding with different ROSs or ROS components led to a significant increase in LLAF in both microglia and macrophages. The 4-hydroxynonenal (HNE)-modified ROSs gave rise to the highest increase in LLAF at both 533 nm and 585 nm. Application of 11-cis-retinal or all-trans-retinal resulted in higher LLAF at 585 nm, compared to application of 9-cis-retinal or liposomes. Fluorescein isothiocyanate-labeled ROSs co-localized well with lysosomes in both types of cells. HNE-modified ROSs were phagocytized more rapidly by both types of cells, compared to unmodified ROSs. Electron microscopy demonstrated inclusion bodies containing whorls of membranes in all types of cells fed with ROSs.
Conclusions
Both macrophages and microglia have the ability to phagocytize ROSs, and this results in increased autofluorescence. Oxidation of ROSs results in faster phagocytosis, higher levels of LLAF, and the appearance of more inclusion bodies inside the cells. Results from the present study suggest that both types of cells accumulate lipofuscin-like material under physiologically relevant conditions. Such accumulation could interfere with their ability to clear cellular debris and could be part of the pathogenetic mechanism for age-related macular degeneration and other lipofuscinopathies.
PMCID: PMC3265176  PMID: 22275801
17.  Cloning, characterization, and expression analysis of the pig (Sus scrofa) C1q tumor necrosis factor-related protein-5 gene 
Molecular Vision  2012;18:92-102.
Purpose
Autosomal dominant early-onset long anterior zonules (LAZs) and late-onset retinal degeneration (L-ORD) in humans are associated with the S163R mutation of the complement 1q-tumor necrosis factor related protein-5 (CTRP5) gene. For using the pig as an L-ORD model for the study of pathology, we cloned, characterized, and studied the expression profile of pig CTRP5 (pCTRP5).
Methods
The pCTRP5 was cloned and sequenced from porcine genomic DNA. Bioinformatic analysis was done to evaluate the functional domains present in the pCTRP5 using PROSITE tools. The V5 epitope-tagged constructs of pCTRP5 and the mammalian promoters, elongation factor 1-α (EF) promoter and 579 bp of the putative promoter located upstream to pCTRP5 DNA, were used for in vitro expression analysis. The pCTRP5 expression, protein size, and cellular localization were studied in transiently transfected Cos-7 or ARPE-19 cells by western blot analysis using anti-CTRP5 and anti-V5 epitope antibodies. Expression of pCTRP5 in the pig eye tissues was analyzed by western blot analysis, real-time PCR, and immunohistochemistry.
Results
As predicted, pCTRP5 showed a 92% DNA homology and 98% amino acid homology with human CTRP5 (hCTRP5). Bioinformatic analysis revealed the presence of an alternate in-frame translational start site upstream to the presumed initiator codon. The presence of a putative promoter region upstream to the pCTRP5 was identified. The putative pCTRP5 promoter was found to be functional by western blot analysis. The size of the pCTRP5 protein (pCTRP5) was consistent with its predicted molecular weight, indicating that the potential alternative start site was not used. Western blot and RT–PCR analyses showed that pCTRP5 was predominantly expressed in RPE, a pattern of expression consistent with that found in mouse and human eyes.
Conclusions
The sequence and genomic organization of pCTRP5 was found to be similar to the human homolog. The DNA and protein sequence of pCTRP5 are highly homologous to hCTRP5, indicating that they are highly conserved. A putative promoter region (579 bp) present upstream to pCTRP5 was found to be functional and was able to drive the expression of the pCTRP5 gene cloned downstream. The tissue distribution in the eye and the expression profile of pCTRP5 in transiently transfected cells is consistent with hCTRP5 expression. Immunohistochemistry analysis of the pig retinal sections revealed localization of pCTRP5 to the apical and basolateral regions on the RPE and in the ciliary body. The potential in-frame alternate start site was found to be nonfunctional by western blot analysis of transiently transfected cells. Similarities between human and pig CTRP5 and the presence of an area centralis region in the pig similar to the human macula, together with its large eyeball size, makes the domestic pig a good model for the study of LAZs and L-ORD.
PMCID: PMC3265177  PMID: 22275800
18.  The P2X7 receptor regulates proteoglycan expression in the corneal stroma 
Molecular Vision  2012;18:128-138.
Purpose
Previously, the authors demonstrated that the lack of the P2X7 receptor impairs epithelial wound healing and stromal collagen organization in the cornea. The goal here is to characterize specific effects of the P2X7 receptor on components of the corneal stroma extracellular matrix.
Methods
Unwounded corneas from P2X7 knockout mice (P2X7−/−) and C57BL/6J wild type mice (WT) were fixed and prepared for quantitative and qualitative analysis of protein expression and localization using Real Time PCR and immunohistochemistry. Corneas were stained also with Cuprolinic blue for electron microscopy to quantify proteoglycan sulfation in the stroma.
Results
P2X7−/− mice showed decreased mRNA expression in the major components of the corneal stroma: collagen types I and V and small leucine-rich proteoglycans decorin, keratocan, and lumican. In contrast P2X7−/− mice showed increased mRNA expression in lysyl oxidase and biglycan. Additionally, we observed increases in syndecan 1, perlecan, and type III collagen. There was a loss of perlecan along the basement membrane and enhanced expression throughout the stroma, in contrast with the decreased localization of other proteoglycans throughout the stroma. In the absence of lyase digestion there was a significantly smaller number of proteoglycan units per 100 nm of collagen fibrils in the P2X7−/− compared to WT mice. While digestion was more pronounced in the WT group, double digestion with Keratanase I and Chondroitinase ABC removed 88% of the GAG filaments in the WT, compared to 72% of those in the P2X7−/− mice, indicating that there are more heparan sulfate proteoglycans in the latter.
Conclusions
Our results indicate that loss of P2X7 alters both the expression of proteins and the sulfation of proteoglycans in the corneal stroma.
PMCID: PMC3265178  PMID: 22275804
19.  Interaction between hedgehog signalling and PAX6 dosage mediates maintenance and regeneration of the corneal epithelium 
Molecular Vision  2012;18:139-150.
Purpose
To investigate the roles of intracellular signaling elicited by Hedgehog (Hh) ligands in corneal maintenance and wound healing.
Methods
The expression of Hedgehog pathway components in the cornea was assayed by immunohistochemistry, western blot and reverse-transcription polymerase chain reaction (RT-PCR), in wild-type mice and mice that were heterozygous null for the gene encoding the transcription factor, paired box gene 6 (Pax6).  Corneal epithelial wound healing and cell migration assays were performed after pharmacological upregulation and downregulation of the hedgehog pathway.  Reporter mice, mosaic for expression of the gene encoding β-galactosidase (LacZ), were crossed to Pax6+/- mice, mice heterozygous for the gene encoding GLI-Kruppel family member GLI3, and Pax6+/- Gli3+/- double heterozygotes, to assay patterns of cell migration and corneal epithelial organization in vivo.
Results
Corneal epithelial wound healing rates increased in response to application of Sonic hedgehog (Shh), but only in mice with wild-type Pax6 dosage.  Downregulation of Hedgehog signalling inhibited corneal epithelial cell proliferation.  Pax6+/- corneal epithelia showed increased proliferation in response to exogenous Shh, but not increased migration. Desert hedgehog (Dhh) was shown to be the major endogenous ligand, with Shh detectable only by RT-PCR and only after epithelial wounding. The activity of phosphatidylinositol-3-OH kinase-γ (PI3Kγ) was not required for the increased migration response in response to Shh.  Nuclear expression of the activator form of the transcription factor Gli3 (which mediates Hh signalling) was reduced in Pax6+/- corneal epithelia. Pax6+/- Gli3+/- double heterozygotes showed highly disrupted patterns of clonal arrangement of cells in the corneal epithelium.
Conclusions
The data show key roles for endogenous Dhh signalling in maintenance and regeneration of the corneal epithelium, demonstrate an interaction between Pax6 and Hh signalling in the corneal epithelium, and show that failure of Hh signalling pathways is a feature of Pax6+/- corneal disease that cannot be remedied pharmacologically by addition of the ligands.
PMCID: PMC3265179  PMID: 22275805
20.  Identification of a novel CRYBB2 missense mutation causing congenital autosomal dominant cataract 
Molecular Vision  2012;18:174-180.
Purpose
To identify the genetic defect in a four-generation Croatian family presenting with autosomal dominant cataract.
Methods
Genome-wide linkage analysis with 250K single nucleotide polymorphism (SNP) arrays was performed using DNA from one unaffected and seven affected individuals. Mutation screening of candidate genes was performed by bidirectional Sanger sequencing.
Results
Evidence for linkage was observed for eight genomic regions. Among these was a locus on chromosome 22 which encompasses the β-crystallin gene cluster. This cluster includes four genes, namely beta-crystallin B1 (CRYBB1), beta-crystallin B2 (CRYBB2), beta-crystallin B3 (CRYBB3), and beta-crystallin A4 (CRYBA4). A novel sequence variant was found in the CRYBB2 gene (p.Arg188His). This variant cosegregated with the disease phenotype in all affected individuals but was not present in the unaffected family members and 100 healthy control subjects.
Conclusions
We report a novel missense mutation, p.Arg188His, in CRYBB2 associated with congenital cataract in a family of Croatian origin. This variant is the most COOH-terminal missense mutation in CRYBB2 that has been identified so far.
PMCID: PMC3272051  PMID: 22312185
21.  Missense mutation outside the forkhead domain of FOXL2 causes a severe form of BPES type II 
Molecular Vision  2012;18:211-218.
Purpose
Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is a developmental disease characterized by a complex eyelid malformation associated or not with premature ovarian failure (POF). BPES is essentially an autosomal dominant disease, due to mutations in the forkhead box L2 (FOXL2) gene, encoding a forkhead transcription factor. More than one hundred unique FOXL2 mutations have been described in BPES in different populations, many of which are missense mutations in the forkhead domain. Here, we report on a very severe form of BPES resulting from a missense mutation outside the forkhead domain.
Methods
A clinical and molecular genetic investigation was performed in affected and unaffected members of an Iranian family with BPES. The FOXL2 coding region was sequenced in an index case. Targeted mutation testing was performed in 8 family members.
Results
We have identified a heterozygous FOXL2 missense mutation c.650C→G (p.Ser217Cys) co-segregating with disease in members of a three-generation family with BPES type II. Only few missense mutations have been reported outside the forkhead domain so far. They were all found in mild BPES, in line with in vitro studies demonstrating mostly normal localization and normal or increased transactivation properties of the mutant proteins. Unlike previous studies, affected members of the family studied here showed a severe BPES phenotype, with bilateral amblyopia due to uncorrected ptosis.
Conclusions
This is the first study demonstrating a severe BPES phenotype resulting from a FOXL2 missense mutation outside the forkhead domain, expanding our knowledge about the phenotypic consequences of missense mutations outside the forkhead domain in BPES.
PMCID: PMC3272052  PMID: 22312189
22.  Non-housekeeping genes expressed in human trabecular meshwork cell cultures 
Molecular Vision  2012;18:241-254.
Purpose
To identify non-housekeeping genes definitively expressed in the human trabecular meshwork (TM).
Methods
Microarray gene expression data on TM cultured cells from four studies were compared. Genes that were queried in at least three studies and assessed to be expressed in at least three studies were considered definitively expressed genes of the human TM. Housekeeping genes were removed from this set of genes. The non-housekeeping TM gene profile was analyzed for pathway enrichment and microRNA targeting, using bioinformatics tools. The results were compared with results of previous non-array based studies.
Results
Nine hundred and sixty-two genes were identified as non-housekeeping TM expressed genes. Analysis of these by Kyoto Encyclopedia of Genes and Genomes led to identification of two enriched biologic pathways that achieved a highly significant Bonferroni p-value (p≤0.01): focal adhesion and extracellular matrix (ECM)-receptor interaction. Many of the genes were previously implicated in TM-related functions and the TM-associated disease glaucoma; however, some are novel. MicroRNAs known to be expressed in the trabecular meshwork were predicted to target some of the genes. Ten genes identified here, ALDH1A1 (aldehyde dehydrogenase 1 family, member A1), CDH11 (cadherin 11, type 2, OB-cadherin), CXCR7 (chemokine (C-X-C motif) receptor 7), CHI3L1 (chitinase 3-like 1), FGF2 (fibroblast growth factor 2), GNG11 (guanine nucleotide binding protein [G protein], gamma 11), IGFBP5 (insulin-like growth factor binding protein 5), PTPRM (protein tyrosine phosphatase, receptor type, M), RGS5 (regulator of G-protein signaling 5), and TUSC3 (tumor suppressor candidate 3), were also reported as TM expressed genes in three earlier non-microarray based studies.
Conclusions
A transcriptome consisting of 962 non-housekeeping genes definitively expressed in the human TM was identified. Multiple genes and microRNAs are proposed for further study for a better understanding of TM physiology.
PMCID: PMC3272053  PMID: 22312193
23.  Expression of T-helper-associated cytokines in patients with type 2 diabetes mellitus with retinopathy 
Molecular Vision  2012;18:219-226.
Purpose
Recent studies showed that immunological mechanisms were involved in the pathogenesis of diabetic retinopathy (DR). T-helper (Th) cells play an important role in chronic inflammatory disorders and autoimmune diseases. Whether Th cells participate in the pathogenesis of DR remains unclear.
Methods
To evaluate the role of Th cells in the pathogenesis of type 2 diabetes mellitus with retinopathy, the concentrations of interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 p70, IL-13, IL-17A, IL-22, and tumor necrosis factor (TNF)-α in the serum of 29 patients with type 2 diabetes mellitus and 30 normal controls were measured with FlowCytomix Technology. IL-22 levels in unstimulated and stimulated peripheral blood mononuclear cells (PBMCs) were examined with enzyme-linked immunosorbent assay.
Results
We found that the mean IL-22 serum levels were slightly lower in diabetic patients than in normal controls. The IL-22 level of PBMCs was significantly elevated in patients with proliferative diabetic retinopathy compared with the level in patients with non-proliferative diabetic retinopathy, patients with non-DR, and healthy controls. Additionally, the IL-22 serum and PBMC levels were positively correlated with the duration of diabetes. Serum levels of other associated cytokines showed no significant change in diabetic patients compared to controls.
Conclusions
These results indicate a possible role of Th22 cells in DR, and IL-22 may be involved more in the pathogenesis of proliferative diabetic retinopathy than in other stages of DR.
PMCID: PMC3272054  PMID: 22312190
24.  Coralliform cataract caused by a novel connexin46 (GJA3) mutation in a Chinese family 
Molecular Vision  2012;18:203-210.
Purpose
To identify a novel disease-causing mutation of the GJA3 (gap junction alpha-3 protein) gene in a Chinese family with autosomal dominant congenital cataract (ADCC).
Methods
One family was examined clinically. After informed consent was obtained, genomic DNA was extracted from the venous blood of all participants. Genetic linkage analysis was performed on the known genetic loci for ADCC with a panel of polymorphic markers, and then mutations were screened by direct sequencing. Whenever substitutions were identified in a patient, high-resolution melt curve analysis (HRM) was performed on all available family members and 100 normal controls. Bioinformatics analysis was undergone by the Garnier-Osguthorpe-Robson (GOR) and the PolyPhen (polymorphism phenotyping) programs to predict the effect of variants detected on secondary structure and protein function of the GJA3 protein.
Results
Clinical examination and pedigree analysis revealed one four-generation family with congenital nuclear coralliform cataracts. Significant two-point LOD (linkage odd disequilibrium) score was generated at marker D13S292 (Zmax=2.51, θ=0), and further linkage and haplotype studies confined the disease locus to 13q11–13. Mutations screening of GJA3 in this family revealed an A→T transversion at position 563 (p.N188I) of the cDNA sequence. This novel missense mutation co-segregated with the affected members of the pedigree, but is not present in the unaffected relatives or 100 normal individuals. Secondary structure prediction suggested that the mutant GJA8 188I would replace three turns “T” with three β sheet “E” at amino acid 189–191 and a β sheet “E” with a turn “T” at position 194.
Conclusions
Novel missense mutation in the second extracellular loop (E2) was detected, causing coral-like opacities involving embryonic and fetal nucleus surrounded by blue punctate opacities in the cortical zone of the lens. The results further suggested that the extracellular loop was the mutation hotspot of GJA3.
PMCID: PMC3272055  PMID: 22312188
25.  Age-dependent changes in rat lacrimal gland anti-oxidant and vesicular related protein expression profiles 
Molecular Vision  2012;18:194-202.
Purpose
Anti-oxidation and exocytosis are important for maintaining exocrine tissue homeostasis. During aging, functional and structural alterations occur in the lacrimal gland (LG), including oxidative damage to proteins, lipids, and DNA. The aims of the present study were to determine in the aging LG: a) the effects of aging on LG structure and secretory activity and b) changes in the expression of oxidative stress markers.
Methods
To address these goals, tear secretion composition and corneal impression cytology were compared between male Wistar rats of 2 (control) and 24 (aged) months. LG morphology and the expression levels of vitamin E and malonaldehyde (MDA) were evaluated to determine the anti-oxidant activity and lipid peroxidation, respectively. RT–PCR and western blot analysis were used for the analysis of Ras related in brain GTPase protein (Rab) and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the secretory machinery (i.e.; Rab 3d, Rab 27, vesicle-associated membrane protein-2 (Vamp-2), and syntaxin).
Results
Histological analysis of aged rats revealed a higher frequency of corneal epithelia metaplasia. In the acinar cells, organelles underwent degeneration, and lipofucsin-like material accumulated in the cytoplasm along with declines in the anti-oxidant marker vitamin E. Rab3d and Rab27b mRNA levels fell along with Rab3d protein expression, whereas syntaxin levels increased.
Conclusions
These findings indicate that exocytotic and anti-oxidant mechanisms become impaired with age in the rat LG. In parallel with these structural alterations, functional declines may contribute to the pathophysiology caused by tear film modification in dry eye disease.
PMCID: PMC3272056  PMID: 22312187

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