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1.  Molecular genetics of Chinese families with TGFBI corneal dystrophies 
Molecular Vision  2011;17:380-387.
Purpose
To identify clinical features and mutations within the transforming growth factor-beta-induced (TGFBI) gene in three Chinese families with Granular corneal dystrophy, type 1 (GCD1) and Granular corneal dystrophy, type 2 (GCD2).
Methods
Clinical features of GCD1 and GCD2 in three Chinese families were studied with slit-lamp and in vivo laser scanning confocal microscopy (LSCM). Molecular genetic analysis was performed on nine patients and fifteen unaffected individuals from these families. All exons of TGFBI were amplified by polymerase chain reaction (PCR) and sequenced.
Results
Morphological changes in the cornea among affected individuals from three Chinese families examined by in vivo LSCM were almost the same. A heterozygous mutation C>T (R555W) was identified in exon 12 of TGFBI in patients of family A with GCD1. Another heterozygous mutation G>A (R124H) was found in exon 4 of TGFBI in affected members of family B and C with GCD2.
Conclusions
Mutations R555W and R124H in TGFBI were identified in three Chinese families with GCD. Even though there are a variety of mutations in TGFBI of GCD, the different subtypes of GCD (GCD1, GCD2, and GCD3) are in fact the same disorder. Our work supports the hypothesis that corneal dystrophies with the common genetic basis in TGFBI should be grouped together as TGFBI corneal dystrophies.
PMCID: PMC3036564  PMID: 21311742
2.  Retinal whole genome microarray analysis and early morphological changes in the optic nerves of monkeys after an intraorbital nerve irradiated injury 
Molecular Vision  2011;17:2920-2933.
Purpose
To obtain and analyze early retinal changes at the molecular level 24 h after a radiation injury to the ipsilateral intraorbital nerve using gamma knife surgery (GKS), and to examine the morphological changes in bilateral optic nerves.
Methods
Unilateral intraorbital optic nerves of three rhesus macaques were treated by GKS with irradiated doses of 15 Gy, while contralateral optic nerves and retinas served as the control. Gene expression profiles of the control and affected retinas were analyzed with Affymetrix Rhesus Macaque Genome arrays. To verify the results, a quantitative real-time polymerase chain reaction (qRT–PCR) was performed to test the expression patterns of five function-known genes. Morphological changes in the bilateral optic nerves were examined using a transmission electron microscope (TEM) and light microscopy. The glial cell reaction in bilateral optic nerves was studied using immunohistochemistry.
Results
Of the probe sets, 1,597 (representing 1,081 genes) met the criteria for differential expression, of which 82 genes were significantly up-or down-regulated in treated retinas. There was prominent upregulation of genes associated with glial cell activation in the treated retina. Genes related to an early inflammatory reaction and to cell death were also significantly regulated in response to a radiation injury to the intraorbital optic nerve. In contrast, the messenger ribonucleic acid (mRNA) expression levels of retinal ganglion cell (RGC)-specific genes were low. Morphologically, cytoplasmic processes of astrocytes in treated nerves were shorter than those of the control and were not straight, while also being accompanied by decreased GFAP immunostaining. More oligodendrocytes and inflammatory cells were apparent in treated nerves than in the control. In addition, swollen mitochondria and slight chromation condensation could be seen in the glial cells of treated nerves.
Conclusions
We conclude that the current irradiated dose of 15 Gy was sufficient to lead to a radiation injury of the optic nerve and retina. Several transcripts deregulated in retinas after a radiation injury play a key role in radiation-induced neurogenic visual loss, especially for genes associated with RGC, glial cell, and cell death. Glial cells in optic nerves might be the primary target of a radiation injury in the optic nerve.
PMCID: PMC3224835  PMID: 22128239
3.  Down regulation of the PEDF gene in human lens epithelium cells changed the expression of proteins vimentin and αB-crystallin 
Molecular Vision  2010;16:105-112.
Purpose
To study the relationship of pigment epithelium-derived factor (PEDF) expression with the expression of vimentin and αB-crystallin by lens epithelial cells.
Methods
Lens epithelial cells adhering to anterior capsules taken from young donor eyes aged from 20 to 35 years were cultured and passaged. We designed small interfering RNA (siRNA) constructs to specifically downregulate the expression of PEDF by these primary lens epithelial cells. Quantitative PCR was used to confirm the downregulation of PEDF RNA expression following infection of lens epithelial cells. To determine whether altering the expression of PEDF would effect the expression of vimentin or αB-crystallin, we performed western blotting 48 h after expression of the PEDF-directed siRNA.
Results
PEDF RNA expression in the human lens epithelial cells was strongly downregulated by the three separate siRNA constructs. Western blotting revealed that the downregulation of PEDF expression resulted in a concomitant decrease in expression of vimentin and an increase in αB-crystallin protein.
Conclusions
Decreased expression of PEDF in primary human lens epithelial cells resulted in a decrease in the expression of vimentin and the increase of αB-crystallin expression, two proteins critical for maintaining lens clarity.
PMCID: PMC2810874  PMID: 20104255
4.  Novel USH2A compound heterozygous mutations cause RP/USH2 in a Chinese family 
Molecular Vision  2010;16:454-461.
Purpose
To identify the disease-causing gene in a four-generation Chinese family affected with retinitis pigmentosa (RP).
Methods
Linkage analysis was performed with a panel of microsatellite markers flanking the candidate genetic loci of RP. These loci included 38 known RP genes. The complete coding region and exon-intron boundaries of Usher syndrome 2A (USH2A) were sequenced with the proband DNA to screen the disease-causing gene mutation. Restriction fragment length polymorphism (RFLP) analysis and direct DNA sequence analysis were done to demonstrate co-segregation of the USH2A mutations with the family disease. One hundred normal controls were used without the mutations.
Results
The disease-causing gene in this Chinese family was linked to the USH2A locus on chromosome 1q41. Direct DNA sequence analysis of USH2A identified two novel mutations in the patients: one missense mutation p.G1734R in exon 26 and a splice site mutation, IVS32+1G>A, which was found in the donor site of intron 32 of USH2A. Neither the p.G1734R nor the IVS32+1G>A mutation was found in the unaffected family members or the 100 normal controls. One patient with a homozygous mutation displayed only RP symptoms until now, while three patients with compound heterozygous mutations in the family of study showed both RP and hearing impairment.
Conclusions
This study identified two novel mutations: p.G1734R and IVS32+1G>A of USH2A in a four-generation Chinese RP family. In this study, the heterozygous mutation and the homozygous mutation in USH2A may cause Usher syndrome Type II or RP, respectively. These two mutations expand the mutant spectrum of USH2A.
PMCID: PMC2842093  PMID: 20309401
5.  Resveratrol protects human lens epithelial cells against H2O2-induced oxidative stress by increasing catalase, SOD-1, and HO-1 expression 
Molecular Vision  2010;16:1467-1474.
Purpose
Oxidative damage induced by H2O2 treatment can irreversibly damage the lens epithelium, resulting in cell death and cataract. Whether the effects of oxidative stress could be attenuated in cultured human lens epithelial cells by incubation with resveratrol (RES) is still unknown. In the present study, we examined the function of resveratrol in protecting human lens epithelial B-3 (HLEB-3) cells against H2O2 induced cell death and cell apoptosis, its role in reducing H2O2 induced intracellular reactive oxygen species (ROS) accumulation, and investigated the mechanism by which resveratrol underlies the effect.
Methods
HLEB-3 cells, a human lens epithelial cell line, were exposed to 100 μM H2O2 with or without RES pre-treatment at different concentrations for different time duration. Cell viabilities were monitored by 4-[3-[4-iodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (WST-1) assay. The apoptosis rate and ROS generation were detected by flow cytometric analysis. Expression levels of superoxide dismutases-1 (SOD-1), catalase, and heme oxygenase-1 (HO-1) proteins were measured by western-blotting analysis. p38 and c-jun N terminal kinase (JNK) activation was also evaluated by western-blotting analysis.
Results
Resveratrol clearly reduced H2O2 induced cell apoptosis and ROS accumulation; protected HLEB-3 cells from H2O2 induced oxidative damage, and increased the expression levels of SOD-1, catalase, and HO-1. Further studies showed that RES also inhibited H2O2 induced p38 and JNK phosphorylation.
Conclusions
These findings suggested that RES protected HLEB-3 cells from H2O2 induced oxidative damage, presumably by inducing three antioxidative enzymes including catalase, SOD-1, and HO-1.
PMCID: PMC2925910  PMID: 20806083
6.  Proteomic analysis of regenerated rabbit lenses reveal crystallin expression characteristic of adult rabbits 
Molecular Vision  2008;14:2404-2412.
Purpose
To explore lens crystallin characteristics and morphology of rabbit regenerated lenses in comparison with wild type natural lenses by means of proteomic analysis and histological assay.
Methods
The lens regeneration model of the New Zealand rabbit was established, and lens regeneration was observed by slit lamp examination and photography. A histological assay was evaluated under light microscopy and transmission electron microscopy (TEM). Protein samples of regenerated lenses were collected from experimental rabbit eyes 2, 4, and 16 weeks after surgery. Two-dimensional gel electrophoresis (2-DE) was performed. Image analyses was done using the ImageMaster 2D Elite 3.01 software package. The protein spots were trypsinized and identified by matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry.
Results
Lens regeneration began in the periphery of the capsule bag about one to two weeks after the surgery and proceeded to regenerate toward the center. The regenerated lens appeared spherical in shape with a fairly translucent cortical structure and a nuclear opacity. Histological findings showed that the remnant lens epithelial cells differentiate at the lens capsule equator and new lens fibers form in a concentric pattern in a manner similar to that observed in natural lenses. However, TEM showed morphological changes in the epithelial cells of the regenerated lenses as compared with natural lenses. 2-D electrophoresis revealed that the patterns of protein spots from regenerated lenses (two weeks, four weeks, and 16 weeks) were analogous to those of 16-week-old natural lenses but were substantially different from those of two-week-old natural lenses, particularly when the two-week-old regenerated lenses were compared with the two-week-old natural lenses.
Conclusions
Proteomic analysis revealed that crystallin expression in regenerated rabbit lenses was analogous to that of natural lenses of adult rabbits but was different from that of very young rabbits (two weeks old), and TEM revealed the presence of morphological changes in the epithelial cells of regenerated lenses. These results suggest that the regrowth of lens materials in the lens capsule after endocapsular phacoemulsification might actually represent the regeneration of “mature” lens substances, which have led us to the conclusion that the regenerative process does not exactly mimic embryonic development.
PMCID: PMC2605429  PMID: 19098996
7.  Differential expression of the catalytic subunits for PP-1 and PP-2A and the regulatory subunits for PP–2A in mouse eye 
Molecular Vision  2008;14:762-773.
Purpose
Reversible protein phosphorylation is a fundamental regulatory mechanism in all biologic processes. Protein serine/threonine phosphatases-1 (PP-1) and 2A (PP-2A) account for 90% of serine/threonine phosphatase activity in eukaryote cells and play distinct roles in regulating multiple cellular processes and activities. Our previous studies have established the expression patterns of the catalytic subunits for PP-1 (PP-1cs) and PP-2A (PP-2Acs) in bovine and rat lenses. In the present study, we have determined the expression patterns of PP-1cs (PP-1α and PP-1β) and PP-2Acs (PP-2Aα and PP-2Aβ) in the retina and cornea along with the ocular lens of the mouse eye. Moreover, since the function of PP-2A is largely relied on its regulatory subunits, we have also analyzed the expression patterns of the genes encoding the scaffold A subunits of PP-2A, PP2A-Aα and PP2A-Aβ, and the regulatory B family subunits of PP-2A, PP2A-Bα, PP2A-Bβ, and PP2A-Bγ. In addition, we have also demonstrated the differential protections of PP-1 and PP-2A in mouse lens epithelial cell line, αTN4–1, against oxidative stress-induced apoptosis.
Methods
Total RNAs and proteins were extracted from the retina, lens epithelium, lens fiber cells, and cornea of the mouse eye. Reverse transcription polymerase chain reaction (RT–PCR) and real time PCR were used to detect the mRNA expression. Western blot and immunohistochemistry analysis were applied to examine the protein expression and distribution. Stable clones of αTN4–1 cells expressing either PP-1α or PP-2Aα were used to analyze the differential protections against oxidative stress-induced apoptosis.
Results
PP-1 is more abundant than PP-2A in the mouse eye. The catalytic subunits for PP-1 and PP-2A display similar expression patterns in the retina and cornea but much reduced in the lens. The mRNAs for all five isoforms of PP2A-A and PP2A-B subunits are highly expressed in the retina, but only three out of the five mRNAs are expressed in the cornea. In the ocular lens, only PP2A-Aβ and PP2A-Bγ mRNAs are clearly detectable. The A and B subunit proteins of PP-2A are highly expressed in the retina and cornea but are much reduced in the ocular lens. PP2A-Aα/β are differentially distributed in the mouse retina.When transfected into mouse lens epithelial cells, αTN4–1, PP-1α and PP-2Aα display differential protection against oxidative stress-induced apoptosis.
Conclusions
Our results lead to the following conclusions regarding PP-1 and PP-2A in mouse eye: 1) PP1 is a more abundant phosphatase than PP-2A; 2) both PP-1 and PP-2A may play important roles, and the functions of PP-2A appear to be highly regulated by various regulatory subunits; and 3) the genes encoding PP-1α/β, PP-2Aα/β, PP-2A-Aα/β, and PP-2A-B α/β/γ are all differentially expressed.
PMCID: PMC2324119  PMID: 18432318
8.  Developmental expression of three small GTPases in the mouse eye 
Molecular Vision  2007;13:1144-1153.
Purpose
The small GTPases function as "molecular switches" by binding and releasing GTP to mediate downstream signaling effects. The Rho-family of GTPases is central in modulating cell differentiation and cytoskeletal changes. Since eye development requires comprehensive morphogenetic movements and extensive cellular differentiation, we hypothesize that different small GTPases may play important roles during morphogenesis of eye development. To explore this possibility, we examined the expression patterns of three major Rho-GTPases: RhoA, Rac1, and Cdc42 in embryonic, postnatal (one day after birth), and adult (two-month old) mouse eye.
Methods
Various ocular tissues were collected from embryonic, postnatal, and adult C57BL/6 mice. Western blots were conducted using total proteins extracted from cornea, retina, lens epithelial cells, and lens fiber cells of the adult mice or different fractions of rat lenses. Immunohistochemistry (IHC) was performed with 6 μm thick sections cut through the eye ball region of 11.5 pc, 14.5 pc, 17.5 pc, postnatal, and adult mice. Parallel controls were run using the rabbit preimmune and GTPase-specific antibodies blocked with saturating levels of corresponding peptide antigen.
Results
In the embryonic mouse eye, RhoA and Cdc42 expressions were initially detectable in all three compartments at 11.5 pc. However, Rac1 became easily detectable in these compartments at 14.5 pc. Increased levels of RhoA, Rac1, and Cdc42 were detected in the three compartments at 17.5 pc and the strongest signals for RhoA, Rac1, and Cdc42 were observed in the primary lens fiber cells at 17.5 pc. In the postnatal mouse eye, the three small GTPases were significantly expressed in both endothelial and epithelial cells of mouse cornea, epithelial cells of the ocular lens, photoreceptors, horizontal/amacrine/Muller's cells, and some ganglian cells of the retina. Much lower level of expression was observed in the corneal stroma fibroblasts, lens fiber cells, and the inner and outer plexiform layers of the mouse retina. In the adult mouse eye, all three Rho-GTPases were expressed in corneal epithelial cells and retina. However, only RhoA protein was detected in corneal endothelial cells and Rac1 protein detected in the ocular lens.
Conclusions
The strong expression of the three small GTPases in the cornea, lens, and retina of mouse eye at embryonic 17.5 pc and postnatal stage suggests their important functions for the morphogenesis of the different compartments of the mouse eye. Particularly, high levels of expression of RhoA, Rac1, and Cdc42 in embryonic lens fiber cells suggest their involvement in differentiation of primary lens fiber cells. In the adult mouse eye, all three Rho-GTPases seem to be involved in differentiation of corneal epithelial cells and retina, however, RhoA alone may be required for endothelial cell differentiation and Rac1 likely plays an important role in supporting continuous lens growth and maintenance of lens transparency.
PMCID: PMC2779149  PMID: 17653061
9.  FGFR2 molecular analysis and related clinical findings in one Chinese family with Crouzon Syndrome 
Molecular Vision  2012;18:449-454.
Purpose
The purposed of this study was to investigate the fibroblast growth factor receptor 2 (FGFR2) gene in one Chinese family with Crouzon syndrome and to characterize the related clinical features.
Methods
One family underwent complete ophthalmic examinations, and two patients were diagnosed with Crouzon syndrome. Genomic DNA was extracted from leukocytes of peripheral blood collected from the family and 100 unrelated control subjects from the same population. Exons 8 and 10 of FGFR2 were amplified by polymerase chain reaction (PCR) and directly sequenced. We performed ophthalmic examinations, including best-corrected visual acuity, slit-lamp examination, fundus examination, Pentacam, Goldmann perimetry, and computed tomography (CT) of the skull.
Results
The two patients were affected with shallow orbits and ocular proptosis, accompanied by midface hypoplasia, craniosynostosis, and clinically normal hands and feet. A heterozygous FGFR2 missense mutation c.866A>C (Gln289Pro) in exon 8 was identified in the affected individuals, but not in any of the unaffected family members and the normal controls.
Conclusions
Although FGFR2 mutations and polymorphisms have been reported in various ethnic groups, especially in the area of osteology, we report, for the first time, the identification of one new FGFR2 mutation in Chinese patients with Crouzon syndrome.
PMCID: PMC3283207  PMID: 22355256
10.  Ala344Pro mutation in the FGFR2 gene and related clinical findings in one Chinese family with Crouzon syndrome 
Molecular Vision  2012;18:1278-1282.
Purpose
The purpose of this study was to investigate the fibroblast growth factor receptor 2 (FGFR2) gene in three Chinese patients with Crouzon syndrome and to characterize the related clinical features.
Methods
A single family underwent complete ophthalmic examinations, and three patients were diagnosed with Crouzon syndrome. Genomic DNA was extracted from leukocytes of peripheral blood collected from members of the family as well as from 100 unrelated control subjects from the same population. Exons 8 and 10 of FGFR 2 were amplified by polymerase chain reaction (PCR) and directly sequenced. We performed ophthalmic examinations, including best-corrected visual acuity, slit-lamp examination, fundus examination, Pentacam, Goldmann perimetry, and computed tomography (CT) of the skull.
Results
The three patients were affected with shallow orbits and ocular proptosis, accompanied by mid-face hypoplasia and craniosynostosis, but had clinically normal hands and feet. A heterozygous FGFR2 missense mutation c.1030G>C (Ala344Pro) in exon 10 was identified in the affected individuals, but not in any of the unaffected family members or the normal controls. The mutation we identified has not previously been reported, either in China or abroad.
Conclusions
Although FGFR2 mutations and polymorphisms have been reported in various ethnic groups, especially in the area of osteology, we report, for the first time, the identification of one new FGFR2 gene mutation in Chinese patients with Crouzon syndrome.
PMCID: PMC3365130  PMID: 22665975
11.  Screening of candidate genes for primary open angle glaucoma 
Molecular Vision  2012;18:2119-2126.
Purpose
Primary open-angle glaucoma (POAG) is one of the leading causes of irreversible blindness in the world. To make progress in understanding POAG, it is necessary to identify more POAG-causing genes.
Methods
Using haplotype analysis, we found that mutational region is located on chromosome 2 in two families. Furthermore, we screened 11 candidate genes on chromosome 2 by protein–protein interaction (PPI) analysis, including mutS homolog 6 (MSH6), mutS homolog 2 (MSH2), v-rel reticuloendotheliosis viral oncogene homolog (REL), endothelial PAS domain protein 1 (EPAS1), vaccinia related kinase 2 (VRK2), F-box protein 11 (FBXO11), EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1), reticulon 4 (RTN4), RAB1A, member RAS oncogene family (RAB1A), ARP2 actin-related protein 2 homolog (ACTR2), and calmodulin 2 (phosphorylase kinase, delta; CALM2). These 11 genes are all predicted to be related to trabecular meshwork changes and progressive loss of retinal ganglion cells in POAG patients.
Results
According to our study, FBXO11 and VRK2 may interact with tumor protein p53 to regulate mitochondrial membrane permeability, mitochondrial membrane organization, and apoptosis. MSH2 is responsible for repairing DNA mismatches and RTN4 is for neuronal regeneration. Therefore, they are supposed to play a negative role in cellular process in POAG. CALM2 may be involved in retinal ganglion cell death and oxidative damage to cell communication.
Conclusions
The results demonstrate that the genes above may be associated with pathogenesis of POAG.
PMCID: PMC3413431  PMID: 22876139
12.  The tear film characteristics of spontaneous subconjunctival hemorrhage patients detected by Schirmer test I and tear interferometry 
Molecular Vision  2012;18:1952-1954.
Purpose
To evaluate the tear film characteristics of spontaneous subconjunctival hemorrhage patients by Schirmer test I and tear interferometry.
Methods
Forty-six spontaneous subconjunctival hemorrhage patients and 46 controls were enrolled in the study. Schirmer test I and tear interferometry were performed in all 92 subjects. The results obtained were compared between the two groups.
Results
The Schirmer test I value of the spontaneous subconjunctival hemorrhage patients was 6.93 (4.72) mm, and that of the controls was 14.70 (3.70) mm. A statistical difference was found between the two groups (independent samples t test, t=-8.79, p<0.001). The mean rank of the tear interferometry patterns of the spontaneous subconjunctival hemorrhage patients was 50.07, and that of the controls was 42.93. No statistical difference was found between the two groups (Mann–Whitney U test, Z=-1.85, p=0.064).
Conclusions
For the spontaneous subconjunctival hemorrhage patients, the Schirmer test I value was lower than that of the controls, whereas the tear interferometry patterns were comparable to that of the controls.
PMCID: PMC3413439  PMID: 22876120
13.  PAX6 analysis of two sporadic patients from southern China with classic aniridia 
Molecular Vision  2012;18:2190-2194.
Purpose
To investigate the paired box 6 (PAX6) gene in two sporadic patients from southern China presenting with classic aniridia.
Methods
The two sporadic patients underwent complete physical and ophthalmic examinations. Genomic DNA was extracted from the leukocytes of the peripheral blood collected from the families of the two sporadic patients and 100 unrelated control subjects from the same population. Exons 4–13 of PAX6 were amplified by polymerase chain reaction (PCR) and sequenced directly. The ophthalmic examinations included best-corrected visual acuity, slit-lamp examination, fundus examination, optical coherence tomography, and Pentacam and Goldmann perimetry.
Results
The two patients were affected with aniridia accompanied by nystagmus. A heterozygous PAX6 frameshift mutation in exon 7, c.375_376delAG (p.Arg125SerfsX7), was identified in sporadic patient 1 and not in any of the unaffected family members and normal controls. One novel mutation in exon 10, c.868_871dupAGTT (p.Phe291X), was detected in sporadic patient 2. The frameshift mutation we identified has not previously been reported either in China or abroad.
Conclusions
Although PAX6 mutations and polymorphisms have been reported in various ethnic groups, we report, for the first time, the identification of one new PAX6 mutation in Chinese aniridia patient.
PMCID: PMC3425573  PMID: 22919266
14.  Characterization and retinal neuron differentiation of WERI-Rb1 cancer stem cells 
Molecular Vision  2012;18:2388-2397.
Purpose
The evidence is increasing that cancer stem cells (CSCs) expressing embryonic and neuronal stem cell markers are present in human retinoblastoma (Rb). This study was conducted to determine whether stem-like cancer cells (SLCCs) in Rb express retinal stem cell–related genes and whether SLCCs can directly differentiate into retinal neurons.
Methods
The cancer stem cell characteristics in WERI-Rb1 cells were determined with Hoechst 33,342 staining, clone formation assay, and CD133 flow cytometry. The expression of embryonic stem cell and retinal stem cell–related genes was analyzed with real-time PCR and immunofluorescence. The SLCCs were induced to differentiate into retinal neurons by the addition of Dickkopf-related protein 1 and Lefty-A.
Results
A small but persistent population of cells excluding Hoechst dye in a verapamil-sensitive manner exhibited a cancer stem cell–like phenotype. The SLCCs displayed highly clonogenic abilities and increased CD133 expression with isolation and expansion in culture in serum-free medium. By comparing the expression of stem cell markers, we found Oct3/4 was more highly expressed in the SLCCs than in human embryonic stem cells. Together with the properties of intrinsic retinal stem cell–related gene expression, we found SLCCs can be induced into neuron-like cells that express glial fibrillary acidic protein and rhodopsin (a photoreceptor cell marker).
Conclusions
These findings provide new insight into cancer stem cells and used a strategy of an artificial change of cancer stem cell fate with transcription factors.
PMCID: PMC3462595  PMID: 23049239
15.  A mouse dry eye model induced by topical administration of benzalkonium chloride 
Molecular Vision  2011;17:257-264.
Purpose
To develop a dry eye model of mouse induced by topical administration of benzalkonium chloride (BAC) and investigate the possible mechanisms.
Methods
BAC at concentration of 0.2% was applied to the mouse ocular surface for 7 days. Phenol red thread tear test, tear break-up time (BUT) test, corneal inflammatory index scoring, fluorescein and rose bengal test were performed to evaluate the toxic effects of BAC on the ocular surface. Global specimens were collected on day (D) 7 and labeled with a series of antibodies including cytokeratin 10 (K10) and mucin 5AC (MUC5AC). Apoptosis of ocular surface epithelium was evaluated by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Histologic analysis and transmission electron microscopy (TEM) were performed on D7.
Results
BAC at a concentration of 0.2% successfully induced a dry eye condition with decreased tear volume and BUTs, increased corneal fluorescein and rose bengal scores. The Inflammatory index was increased in accompanyment with higher tumor necrosis factor-α (TNF-α) expression and more inflammatory infiltration in the cornea. Immunolabeling revealed positive K10 expression in BAC-treated corneal epithelium and fewer MUC5AC-positive cells in the BAC-treated conjunctival fornix. TUNEL assay showed more apoptotic cells in the corneal basal epithelium. TEM showed that the size and intervals of the microvillis were both reduced in the corneal epithelium.
Conclusions
Topical administration of 0.2% BAC in mouse induces changes resembling that of dry eye syndrome in humans, and thus, represents a novel model of dry eye.
PMCID: PMC3030605  PMID: 21283525
16.  Apolipoprotein E gene and age-related macular degeneration in a Chinese population 
Molecular Vision  2011;17:997-1002.
Purpose
To examine the association between apolipoprotein E (APOE) polymorphisms and age-related macular degeneration (AMD) in a Chinese population.
Methods
The study consisted of 712 subjects, including 201 controls, 363 cases with early AMD, and 148 cases with exudative AMD. Genomic DNA was extracted from venous blood leukocytes. Common allelic variants of APOE (ε2, ε3, and ε4) were analyzed by PCR and direct sequencing.
Results
APOE ε3ε3 was the most frequent genotype, with a frequency of 72.6% in controls, 72.5% in early AMD, and 70.3% in exudative AMD. Frequency of the ε2 allele was 6.7% in controls, 7.4% in early AMD, and 8.8% in exudative AMD. Frequency of the ε4 allele was 8.7% in controls, 7.7% in early AMD, and 7.8% in exudative AMD. No statistically significant difference in APOE genotype and allele frequency distribution was observed among controls, cases with early AMD, and cases with exudative AMD. For ε2 allele carriers, the odds ratio was 1.12 (95% confidence interval [CI], 0.65–1.93) for early AMD and 1.06 (95% CI, 0.53–2.10) for exudative AMD. For ε4 allele carriers, the odds ratio was 1.04 (95% CI, 0.61–1.75) for early AMD and 0.83 (95% CI, 0.42–1.62) for exudative AMD.
Conclusions
Our data provide no evidence to support an association of APOE polymorphisms with early or exudative AMD, suggesting that APOE is less likely to be a major AMD susceptibility gene in the Chinese population.
PMCID: PMC3084239  PMID: 21541275
17.  Characterization of intraocular pressure responses of the Tibetan monkey (Macaca thibetana) 
Molecular Vision  2011;17:1405-1413.
Purpose
To characterize the effects of circadian rhythm, feeding time, age, general anesthesia, and ocular hypotensive compounds on intraocular pressure (IOP) of the Tibetan monkey (Macaca thibetana).
Methods
Tibetan monkeys were trained for IOP measurement with the TonoVet® rebound tonometer without sedation or anesthesia. Their circadian IOP fluctuation was monitored every 3 h. Effects of changing the feeding time, general anesthesia, age (2–3 year-old versus 8–15 year-old animals), and various pharmacological agents, such as travoprost, timolol, naphazoline and spiradoline, on IOP were also evaluated.
Results
After behavioral training, conscious Tibetan monkeys were receptive to IOP measurement. The lowest and highest IOP values in a circadian cycle were recorded at 3:00 AM (19.8±0.4 mmHg, mean±SEM, n=12) and noon (29.3±0.9 mmHg), respectively. Changing the feeding time from 11:30 AM to 12:30 PM lowered the noon IOP to 25.1±1.2 mmHg. General anesthesia lowered IOP in these monkeys, while IOP of young and mature animals were similar. Three hours after topical ocular administration, travoprost reduced IOP by 5.2±0.6 mmHg (n=6, p<0.001), and timolol reduced IOP by 2.8±0.7 mmHg (p<0.05). Naphazoline and spiradoline lowered IOP by 4.8 mmHg and 2.5 mmHg (both p<0.001), respectively, 2 h after drug administration.
Conclusions
The circadian IOP fluctuation in conscious Tibetan monkeys and their responses to travoprost, timolol, and other experimental conditions are similar to other primates. These monkeys appear to be a suitable model for glaucoma research.
PMCID: PMC3108893  PMID: 21654897
18.  Serum levels of Th17-related cytokines in Behcet disease patients after cataract surgery 
Molecular Vision  2011;17:1425-1430.
Purpose
To investigate the profile of T-helper type 17 (Th17) cell–related cytokines (interleukin-23 [IL-23], IL-27, IL-17 and interferon-γ [IFN-γ]) in postoperative inflammation in patients with Behcet disease (BD) after cataract surgery.
Methods
Serum was collected from seven BD patients with complicated cataract, and from nine controls with uncomplicated cataract, before cataract surgery, and again 1, 7, 30, and 90 days after surgery. In addition, aqueous humor was collected at commencement of surgery. The protein levels of IL-23, IL-27, IL-17, and IFN-γ in the serum and in the aqueous humor were measured by an enzyme-linked immunosorbent assay. A laser flare-cell photometer was used to quantify intraocular inflammation.
Results
Serum IL-23, IL-27, and IFN-γ levels were significantly increased after cataract surgery in the BD versus the control patients. In the BD patients, serum levels of IFN-γ and IL-27 correlated strongly with aqueous flare values and cell counts. Remarkably, the levels of serum IL-27 were significantly associated with serum IFN-γ levels in BD patients (r=0.796; p=0.002).
Conclusions
Our data indicates that serum IFN-γ and IL-27 levels are significantly elevated in BD versus control patients and are strongly associated with post-operative intraocular inflammation.
PMCID: PMC3108896  PMID: 21655356
19.  Molecular analysis of the choroideremia gene related clinical findings in two families with choroideremia 
Molecular Vision  2011;17:2564-2569.
Purpose
To investigate the choroideremia (CHM) gene in two families with CHM and to characterize the related clinical features.
Methods
Two families underwent complete ophthalmic examinations and three males were diagnosed with CHM. Genomic DNA was extracted from the leukocytes of peripheral blood collected from the two families and from 100 unrelated control subjects from the same population. Exons 1–15 of CHM were amplified by PCR and directly sequenced. Ophthalmic examinations included best-corrected visual acuity, slit-lamp examination, fundus examination, visual field, optical coherence tomography, electroretinogram, and Pentacam.
Results
The affected men were hemizygous and had night blindness, chorioretinal atrophy spreading from the posterior pole to the mid-periphery, and bareness of the sclera. A novel c.1488delGinsATAAC mutation was detected in CHM in family 1. Another mutation c.1703 C>G (S558X) within exon 14 of CHM was identified in family 2, which caused the serine 558 codon (TCA) to be changed to a stop codon (TGA).
Conclusions
This study identified a novel mutation in CHM associated with CHM and its related clinical features. Our findings expand the genotypic spectrum of CHM mutations associated with CHM and confirm the role of Rab escort protein-1 in the pathogenesis of CHM.
PMCID: PMC3198496  PMID: 22025891
20.  Polymorphisms in the vascular endothelial growth factor gene and the risk of diabetic retinopathy in Chinese patients with type 2 diabetes 
Molecular Vision  2011;17:3088-3096.
Purpose
To investigate whether single nucleotide polymorphisms (SNPs) in the vascular endothelial growth factor (VEGF) gene are associated with diabetic retinopathy (DR) in a cohort of Chinese patients with type 2 diabetes mellitus (T2DM).
Methods
A total of 268 patients with T2DM (129 with DR and 139 without DR) were recruited and enrolled in the study. Patients with T2DM were assigned to a DR group or a diabetic-without-retinopathy group, based on the duration of diabetes and grading of fundus images. Genotypes of eight SNPs in the VEGF gene (rs699947, rs833061, rs13207351, rs2010963, rs833069, rs2146323, rs3025021, and rs3025039) were analyzed using a mass-array genotyping system, and an association study was performed.
Results
After adjusting for covariates, a significant association of DR was observed with the homozygous genotype of the minor allele for promoter SNPs rs699947 (odds ratio (OR)=3.54, 95% confidence interval (CI): 1.12–11.19), rs833061 (OR=3.72, 95% CI: 1.17–11.85) and rs13207351 (OR=3.76, 95% CI: 1.21–11.71). A significant association of DR was also observed with haplotype ACA, as defined by minor alleles of promoter SNPs rs699947, rs833061, and rs13207351 (OR=1.52, 95% CI: 1.03–2.24), and haplotype GAA, as defined by SNPs rs2010963, rs833069, and rs2146323 (OR=1.62, 95% CI: 1.08–2.41).
Conclusions
Our data suggest that polymorphisms in the promoter region of the VEGF gene increase the risk of DR in Chinese patients with T2DM.
PMCID: PMC3233387  PMID: 22162628
21.  PAX6 analysis of one family and one sporadic patient from southern China with classic aniridia 
Molecular Vision  2011;17:3116-3120.
Purpose
To investigate the paired box gene 6 (PAX6) in three patients from southern China presenting with classic aniridia: two patients from two successive generations of one family and one sporadic patient.
Methods
All the available members from two successive generations of one family and one sporadic patient underwent complete physical and ophthalmic examinations. Genomic DNA was extracted from leukocytes of peripheral blood collected from the two generations of family members, the sporadic patient and 100 unrelated control subjects from the same population. Exons 1–13 of the PAX6 gene were amplified by polymerase chain reaction (PCR) and sequenced directly. The ophthalmic examinations included best-corrected visual acuity, slit-lamp examination, fundus examination, optical coherence tomography, and Pentacam and Goldmann perimetry.
Results
The three patients were affected with aniridia accompanied by microcornea, microphthalmia, and nystagmus. A heterozygous PAX6 frameshift mutation, c.891del A(p.Gln297HisfsX68) in exon 10, was identified in the affected individuals and not in any of the unaffected family members, including the unaffected family members of the proband patient’s generation. One novel mutation, c.607C>T(Arg203X) in exon 8, was detected in the unrelated sporadic patient.
Conclusions
Although PAX6 gene mutations and polymorphisms have been reported from various ethnic groups, we report for the first time the identification of two new PAX6 gene mutations in Chinese aniridia patients.
PMCID: PMC3235534  PMID: 22171157
22.  Lutein and zeaxanthin supplementation reduces H2O2-induced oxidative damage in human lens epithelial cells 
Molecular Vision  2011;17:3180-3190.
Purpose
Epidemiological studies suggest that dietary intake of lutein and zeaxanthin is inversely related to the risk for senile cataract. The objectives of this work were to investigate the mechanisms by which these nutrients provide anti-cataract effects. We evaluated their modulation of oxidative damage in human lens epithelial cells (HLEC) and their interaction with intracellular glutathione (GSH).
Methods
Subconfluent HLEC were pre-incubated with or without 5 µM lutein, zeaxanthin, or α-tocopherol for 48 h and then exposed to 100 µM H2O2 for 1 h. Levels of protein carbonyls in the cells were measured by western-blotting analysis following reaction with 2,4-dinitrophenylhydrazine (DNPH). Levels of malondialdehyde (MDA), reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured by an HPLC system. DNA damage was assessed using comet assays. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay.
Results
In the absence of H2O2, HLEC had very low levels of protein carbonyl and MDA. Supplementation with lutein, zeaxanthin, or α-tocopherol to the unstressed HLEC had no detectable effects on levels of oxidized proteins and lipid in the cells. Exposure of HLEC to H2O2 significantly increased levels of oxidized proteins, lipid peroxidation, and DNA damage. Pre-incubation with lutein, zeaxanthin, or α-tocopherol dramatically reduced the levels of H2O2 -induced protein carbonyl, MDA, and DNA damage in HLEC. The protective effects of lutein, zeaxanthin, and α-tocopherol against protein oxidation, lipid peroxidation, and DNA damage were comparable. Supplementation with lutein, zeaxanthin, or α-tocopherol increased GSH levels and GSH:GSSG ratio, particularly in response to oxidative stress. Depletion of GSH resulted in significant increase in susceptibility to H2O2-induced cell death. Supplementation with α-tocopherol, but not lutein or zeaxanthin, can partially restore the resistance of GSH-depleted cells to H2O2.
Conclusions
These data indicate that lutein or zeaxanthin supplementation protects lens protein, lipid, and DNA from oxidative damage and improves intracellular redox status upon oxidative stress. The protective effects are comparable to that of α-tocopherol, except that lutein and zeaxanthin cannot compensate for GSH depletion. The data imply that sufficient intake of lutein and zeaxanthin may reduce the risk for senile cataract via protecting the lens from oxidative damage.
PMCID: PMC3244479  PMID: 22194644
23.  Proteomics analysis of water insoluble-urea soluble crystallins from normal and dexamethasone exposed lens 
Molecular Vision  2011;17:3423-3436.
Purpose
The aim of this study was to identify glucocorticoid induced cataracts (GIC)-specific modified water insoluble-urea soluble (WI-US) crystallins and related changes after rat lens were exposed to dexamethasone (Dex).
Methods
We separated WI-US lens proteins by two-dimensional electrophoresis (2-DE). The crystallins were then analyzed with matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). Protein levels and morphological changes of αA- and αB-crystallins were also determined. Electronic microscope of lens and native-page analysis of crystallins were further determined.
Results
Measured masses, isoelectric points (pIs), and amino acid sequences of all detected crystallins matched previously-reported data. Analysis by 2-DE indicated that αA- and αB-crystallin increased when the lens was viewed under 1 µM and 10 µM Dex, which was identical with the results of western-blot, immuno histochemistry or fluorescence; βB2- and βA3-crystallin increased when lens was viewed under 1 µM Dex and 100 µM Dex. βA1-, βA4-, and βB1-crystallins decreased under 0.1–100 µM Dex. Electronic microscope figures showed the condition of the lens center gradually worsened and cracked between fiber cells that became larger under 1–100 µM Dex. Moreover, αA-crystallins were associated with increased phosphorylation (PI decreased).The newly protein spots: βA2-, βA3-, βB1-, and γs-crystallin appeared under 0.1–100 µM Dex. Native-page showed α-crystallin increased when the lens was exposed to 1 µM Dex; however, β-crystallin did not decrease under 0.1–100 µM Dex. The percentage of α-crystallin gradually decreased, however β-crystallin gradually increased, perhaps because the emergence of newly appeared β-crystallin under Dex.
Conclusions
Our results showed multiple WI-US crystallins may be more vulnerable to glucocorticoid stress because of diminished important roles, which will in turn provide a mechanism for GIC from a proteomics perspective.
PMCID: PMC3249430  PMID: 22219638
24.  AQP1 and SLC4A10 as candidate genes for primary open-angle glaucoma 
Molecular Vision  2010;16:93-97.
Purpose
Recent evidence supports the role of reduced cerebrospinal fluid (CSF) pressure in the pathogenesis of primary open-angle glaucoma (POAG). We investigated the association of variants in two candidate genes that are important in CSF production, aquaporin 1 (AQP1) and solute carrier family 4, sodium bicarbonate transporter, member 10 (SLC4A10), with POAG in the Caucasian population.
Methods
POAG subjects (n=382) met the criteria of glaucomatous optic neuropathy with consistent visual field loss. Intraocular pressure was not used as an inclusion criterion. Control subjects (n=363) did not meet any of the inclusion criteria and had no family history of glaucoma. Eleven tagging single nucleotide polymorphisms (SNPs) for AQP1 and SLC4A10 were genotyped in the POAG and control subjects, using allelic discrimination assays. Genotype frequencies were compared between the POAG and control subjects, using logistic regression adjusted for gender.
Results
There was no statistically significant difference in genotype frequencies between POAG and control subjects for any of the tested SNPs in AQP1 and SLC4A10 (p>0.05).
Conclusions
There was no association between common sequence variants in the AQP1 or SLC4A10 genes and POAG in the Caucasian population. This is the first study to investigate the association between these two candidate genes and increased risk for POAG.
PMCID: PMC2810210  PMID: 20101282
25.  TGFBI gene mutation analysis in a Chinese pedigree of Reis-Bücklers corneal dystrophy 
Molecular Vision  2010;16:556-561.
Purpose
To analyze transforming growth factor beta-induced (TGFBI) gene mutations in a Chinese pedigree with Reis-Bücklers dystrophy (RBCD).
Methods
In a four-generation Chinese family with Reis-Bücklers dystrophy, six members were patients and the rest were unaffected. All members of the family underwent complete ophthalmologic examinations. Exons of TGFBI were amplified by polymerase chain reaction, sequenced, and compared with a reference database. The sequencing results were reconfirmed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).
Results
A single heterozygous C>T (R124C) point mutation was found in exon 4 of TGFBI in all six members of the pedigree affected with RBCD, but not in the unaffected members.
Conclusions
Within this pedigree, RBCD segregates with the R124C variance, which is a known mutation for lattice corneal dystrophy type I. Therefore, along with G623D and R124L, the R124C mutation in TGFBI is also found to be responsible for RBCD.
PMCID: PMC2847680  PMID: 20360992

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