All-trans retinoic acid increases ARPE-19 cell apoptosis via activation of reactive oxygen species and endoplasmic reticulum stress pathways.

AIM: To explore the apoptosis of ARPE-19 cells after the treatment with different doses of all-trans-retinoic acid (ATRA). METHODS: ARPE-19 cells were used in the in-vitro experiment. Flow cytometry assay was employed to evaluate the level of reactive oxygen species (ROS) and apoptosis. The effects of ATRA (concentrations from 2.5 to 20 µmol/L) on the expression of endoplasmic reticulum stress (ERS) markers in vitro were evaluated by Western blot and real-time quantitative polymerase chain reaction (qRT-PCR) assays. The contribution of ROS and ERS-induced apoptosis in vitro was determined by using N-acetyl-L-cysteine (NAC) and Salubrinal, an antagonist of NAC and ERS, respectively. RESULTS: Flow cytometry showed that ATRA significantly increased ARPE-19 cell apoptosis and ROS levels in each group (F=86.39, P<0.001; F=116.839, P<0.001). Western blot and qRT-PCR revealed that levels of CHOP and BIP were elevated in a concentration-dependent pattern after the cells were incubated with ATRA (2.5-20 µmol/L). The upregulation of VEGF-A and CHOP induced by ATRA could be inhibited by NAC (antioxidant) and Salubrinal (ERS inhibitor) in vitro. CONCLUSION: ATRA induces the apoptosis of ARPE-19 cells via activated ROS and ERS signaling pathways.

Effect of dopamine on bone morphogenesis protein-2 expression in human retinal pigment epithelium.

AIM: To investigate the effect of dopamine on bone morphogenesis protein-2 (BMP-2) expression in retinal pigment epithelium (RPE) cells in vitro. METHODS: ARPE-19 cells as a human RPE cell line were cultured with dopamine for different times (2, 4, 6, 8, 12, 16 and 24h) or with different concentrations (0.1, 1, 2, 5, 10, 20, and 100 µg/mL) in vitro. BMP-2 mRNA expression level in ARPE-19 cells was analyzed with real-time polymerase chain reaction (PCR) analysis and BMP-2 protein level was measured with Western blot analysis. The active form of BMP-2 in the culture medium was measured with enzyme-linked immunosorbent assay (ELISA). RESULTS: The expression level of BMP-2 increased significantly cultured with 20 µg/mL dopamine, at different time points (P<0.05). BMP-2 mRNA level peaked 2h and the protein level peaked at 6 and 8h after treatment. The concentrations of secreted BMP-2 elevated at 12h and peaked at 24h (P<0.05) in a time-dependent manner. Treated with 100 µg/mL dopamine for 6h, the expression levels of BMP-2 mRNA and protein in ARPE-19 cells were enhanced significantly compared to that in the untreated cells (P<0.05). And secreted BMP-2 protein in the cell culture supernatant was also increased (P<0.05). CONCLUSION: Dopamine up-regulate BMP-2 expression in RPE cells, and this may be associated with its inhibitive effect on myopia development.

NF-κB mediates the survival of corneal myofibroblast induced by angiotensin II.

PURPOSE: To investigate the role of angiotensin II (Ang II) in the regulation of corneal myofibroblast apoptosis and the possible signaling pathway. METHODS: Rabbit corneal myofibroblasts were cultured in vitro and the cell phenotype was identified by expression of α-smooth muscle actin (α-SMA) and formation of F-actin. The expression of Ang II type I receptor (AT1R) in keratocytes and corneal myofibroblasts were detected by immunofluorescence staining and Western blot. The effect of Ang II on corneal myofibroblast apoptosis induced by serum starvation and TNFα plus cycloheximide (CHX) was examined by TUNEL, Hoechst 33258 staining, and caspase 3/7 activity assay. The effect of Ang II on nuclear factor-κB (NF-κB)-dependent DNA binding activity and transcriptional activity was studied by electrophoresis mobility shift assay (EMSA) and luciferase reporter assay, respectively. Ang II-induced TGFß1 secretion by corneal myofibroblasts was determined by ELISA. RESULTS: Ang II type I receptor expression was more abundant in corneal myofibroblasts compared with keratocytes. Ang II reduced corneal myofibroblasts apoptotic response to serum starvation or treatment with TNFα plus CHX. This protective effect was attenuated in the presence of AT1R antagonist losartan or NF-κB-specific inhibitor Bay11-7082. Ang II increased NF-κB-dependent DNA-binding activity and transcriptional activity, and also increased TGFß1 production by corneal myofibroblasts. CONCLUSIONS: Ang II induces corneal myofibroblasts resistance to apoptosis via activating NF-κB signaling pathway, and thus should be further investigated as a possible target for therapy of corneal fibrosis.

Regulation of scleral fibroblast differentiation by bone morphogenetic protein-2.

Bone morphogenesis proteins (BMPs) are multi-functional growth factors. They are expressed in retina, retinal pigment epithelium (RPE) and sclera and serve as a regulator in the growth and development of the eye. This article reviewed the chondrogenic potency of the sclera, biochemical and pathological changes of myopic scleral tissue and the differentiation of chondrogenesis by BMP-2. We proposed the hypothesis that BMP-2 can regulate differentiate of scleral fibroblasts and affect the development of myopia.

Efficacy of progressive addition lenses in the treatment of ametropia after the single eye's IOL implantation.

PURPOSE: To investigate the efficacy of progressive addition lenses on the treatment of ametropia and loss of accomodation after the single eye's IOL implantation. METHODS: Eighty four patients undergoing IOL implantation in single eyes were prescribed with progressive addition lenses for ametropia correction and regularly followed up to observe subsequent correction effect. RESULTS: Among these 84 patients, 72 could comfortably adapt to the use of progressive addition lenses to improve visual acuity and accomondation, while the remaining 12 patients failed to accomodate the usage of progressive addition lenses. CONCLUSION: Wearing progressive addition lenses acts as a relatively feasible approach to improve visual acuity and alleviate disorders of accomodation for patients who underwent IOL implantation in single eyes. The patients should be prescribed with progressive lenses under professional instructions and guidance.

Effects of 7-methylxanthine on form-deprivation myopia in pigmented rabbits.

AIM: To determine the effect of 7-methylxanthine (7-MX) on the posterior sclera of form-deprivation myopia (FDM) in pigmented rabbits. METHODS: Sixteen pigmented rabbits were monocularly deprived (MD) by suturing the right eyelids after natural eye opening (ten-day old) for a period of 30 days. Two groups of pigmented rabbits were fed either 7-MX (30 mg per kg body weight; n=8) or vehicle control (saline equal volume with 7-MX; n=8). Ocular refractions, axial lengths and body weights were measured at the start and the end of the experiment 30 days later. Electron microscopy was used to measure and determine the collagen fibril diameters in the posterior pole of sclera. RESULTS: In vehicle control MD pigmented rabbits, 30 days of MD produced -1.10D±0.78D of myopia and the axial length increased 0.51mm±0.09mm. In MD pigmented rabbits fed with 7-MX, 30 days of MD induced only -0.21D±0.11D of myopia and the axial length increased 0.07mm±0.10mm. There was significant change in axial length of vehicle control MD pigmented rabbits (13.11mm±0.19mm versus 12.60mm±0.06mm; P=0.03). The changes in refraction and axial length of two MD groups' contralateral eyes during the 30 days were not significantly different (2.75D±0.27D versus 2.75D±0.35D, P>0.05; 12.60mm±0.06mm versus 12.45mm±0.14mm, P>0.05). The weights of the two groups pigmented rabbits had no significant changes (187g±22.1g versus 189g±19.3g, P>0.05). The diameter of scleral collagen fibers increased in both eyes of 7-MX treated pigmented rabbits. There was significant difference in collagen fibril diameters of inner layer (111.34nm±28.30nm versus 94.80nm±27.52nm, P=0.002) and outer layer (167.92nm±55.82 nm versus 144.04 nm±47.02nm, P=0.016) in the posterior sclera between the myopic eyes of vehicle control MD group and contralateral eyes of 7-MX treated MD group. CONCLUSION: 7-MX appears to prevent FDM in pigmented rabbits by remodeling the posterior sclera.

Characteristics of the macula in amblyopic eyes by optical coherence tomography.

AIM: To test the hypothesis that amblyopic neuroretina may have an altered thickness when compared to the normal. METHODS: Twenty-five amblyopic, young patients between the ages of 7 and 11 years old were studied. The interested neuroretina areas are defined into 10 sub-regions according to superior-inferior, nasal-temoral, and peri-para axis, which cross the fovela structure. The thicknesses of ten, defined macular regions were separately measured by optical coherence tomography (OCT) and analyzed by t-test. RESULTS: The average thickness of neuroretina in the exact foveola of the amblyopic eyes is larger than that of normal eyes (P<0.05), but the other nine regions have no significant difference. Interestingly, in both the normal and amblyopic eyes, the temporal area looks thinner than other quadrants (P<0.05). CONCLUSION: Thickness alteration may be associated with amblyopic disorders in young patients. Studying a larger volume of subjects of similar age is required to confirm this observation.

Distribution of bone morphogenetic protein receptors in human scleral fibroblasts cultured in vitro and human sclera.

AIM: To investigate the distribution of bone morphogenetic protein receptors (BMPRs) in human scleral fibroblsasts (HSFs) and in human sclera. METHODS: Primary HSFs were cultured in vitro. The mRNA levels of BMP-2 and BMPRs in HSFs were assayed by reverse transcription-polymerase chain reaction (RT-PCR). The protein distributions of BMP-2 and BMPRs in HSFs were further detected by immunocytofluorescence and western blot. Their protein expression was also detected in frozen human posterior scleral sections by immunohistofluorescence. RESULTS: BMP-2 and BMPRs were expressed in both HSFs and human sclera not only at mRNA level but also at protein level. The expressions of BMPRIA and BMPRII were higher than that of BMPRIB in the cytoplasm and cell membrane of HSFs in vitro. Western blot further verified the results of immunocytofluorescence. In human sclera, BMP2, BMPR IB and BMPR II were found to be expressed in the cytomatrix of HSF, and weak signal was detected about BMPRIA. CONCLUSION: BMP-2 and all three subtypes of BMPRs were found in HSFs and may play a role in scleral remodeling.

Expression of adenosine receptors in human retinal pigment epithelium cells in vitro.

BACKGROUND: Adenosine receptors (ADORs) have been reported to play a role in experimental myopia. This study aimed to determine the distribution of ADORs in human retinal pigment epithelium (RPE) cells cultured in vitro. METHODS: Human RPE cells (cell line D407) were cultured in vitro. ADOR mRNA in RPE was detected by reverse transcription polymerase chain reaction. ADOR protein expression in RPE was confirmed by Western blotting analysis of cell lysates. Confocal fluorescence microscopy was used to study the subcellular distribution of ADORs. RESULTS: All four subtypes of ADORs mRNA and protein were expressed in human RPE. This was confirmed by Western blotting analysis. The ADOR subtypes were differently distributed within the cells. ADORA1 was expressed in nucleus, perinucleus and cytoplasm of RPE. ADORA2A was concentrated mainly in one side of the perinucleus and cytoplasm of RPE. ADORA2B was strongly expressed in the nucleus, perinucleus and the cytoplasm, and ADORA3 was expressed weakly in the cytoplasm of RPE. CONCLUSIONS: ADORs are expressed in human RPE. The different distribution at the subcellular level suggests different functions of ADOR subtypes.

[Effect on of pirenzepine on expression of mAChRs in the ocular tissues of guinea pig with form-deprived myopia].

OBJECTIVE: To study effects of vitreous injecting M(1)-selective muscarinic antagonist, pirenzepine, on expression of M(1) and M(4) receptor in retina, choroid, sclera and iris-ciliary body of guinea pig with form-deprived myopia. METHODS: Twenty-four 1 - 2 week-old pigmented guinea pigs were randomly divided into four groups. Group1: normal control (N) (n = 6); group 2: simple form-deprived myopia (FDM) (n = 6); group 3: drug control (S) (n = 6); group 4: pirenzepine (P) (n = 6). Expression changes of M(1) and M(4) muscarinic receptors at mRNA level were detected by semi-quantitative RT-PCR in retina, choroid, sclera and iris-ciliary body. RESULT: 1. After 21 days' treatment, FDM group produced relative myopia of -4.92 D and an axial length of 0.29 mm with significance (P < 0.001), compared with N group. Compared with group S, the relative refractive error in group P was +0.88 D, and axial length decreased 0.30 mm with respectively significance (P < 0.001). Differences in refractive error between group S and group FDM were not significant, but in axial length were significant (0.08 mm vs. 0.29 mm) (P < 0.05). 2. Semi-quantitative RT-PCR: M(1) and M(4) mRNA expression showed no significant differences in the retina, choroid and iris-ciliary body of group P compared with group S (P > 0.05). Of interest, in the posterior sclera, mRNA expression of group P was significantly greater than that of group S for the M(1) (P < 0.05) and M(4) subtypes (P < 0.05). The M(1) and M(4) subtype in group P was increased by +19.16% and +64.29% respectively. CONCLUSION: M(1)-selective muscarinic antagonist, pirenzepine, can effectively inhibit form-deprived myopia in guinea pig. M(1) and M(4) subtype in sclera and their cholinergic signaling may participate in muscarinic antagonist inhibition of myopic development.

[Expression of M4 receptor in normal guinea pigs retina].

OBJECTIVE: To detect the expression and distribution of M4 receptor in normal guinea pigs retina. METHODS: Twenty one normal 4-week-old triad color guinea were selected. Eyes were enucleated. Seven eyes were fixed, paraffin-embedded and cut into sections. Expression and distribution of M4 receptor were detected using immunohisto chemically. The rest eyes were dissected. Tissue of retina were separated. RNA and protein of different tissue were extracted respectively. M4 receptor at mRNA level and protein level were detected by RT-PCR and Western blot methods. RESULTS: receptor at mRNA level was detected in normal guinea retina, with the amplification product at 221 bp. No product was detected in negative control. M4 receptor at protein level was detected in normal guinea retina, with signal band at 38,000. No signal was detect in negative control. As detected by immunohistochemistry, M4 receptor was detected in RPE cells, photoreceptor layer, outer nuclear layer, inner nuclear layer, most ganglion cells and the junctional zone of inner plexiform layer and inner nuclear layer. CONCLUSION: M4 receptor was expressed in normal guinea retina.

Time-course of changes to nitric oxide signaling pathways in form-deprivation myopia in guinea pigs.

The aim of this study was to investigate the time-course change of nitric oxide synthase (NOS) activity and cyclic GMP (cGMP) concentration in the posterior retina, choroid and sclera after differing periods of form-deprivation in guinea pigs. Three groups of guinea pigs were subjected to monocular FD for 7, 14 or 21 days. NOS activity and cGMP concentrations in ocular tissues of FD eyes and control eyes were analyzed by radioimmunoassay. The presence of NOS isoforms was detected by immunohistochemistry. Guinea pigs presented with considerable myopia after 14 days of FD. Retinal NOS activity in the FD group was lower than in the control group after 7 days of FD and was higher than in the control group after 14 and 21 days of FD. The choroidal and scleral NOS activities in the FD groups were higher than in the control groups after 21 days. The cGMP concentrations in the FD groups were higher than in the control groups at 21 days of the retinal, choroidal, and scleral tissues. Furthermore, the retinal cGMP concentration in the FD group was also significantly elevated at 14 days relative to the control group. We detected expression of three NOS isoforms in guinea pig ocular tissues. Our main observations were a change in NOS activity and an up-regulation in cGMP concentrations in posterior ocular tissues during the development of myopia. The function of elevated NOS activity may be mediated by cGMP.

[Expression of 5 muscarinic receptor subtypes in the scleral tissue of immature guinea pigs].

OBJECTIVE: To investigate the expression of 5 muscarinic receptor subtypes in the scleral tissue of immature guinea pigs. METHODS: The scleral tissue was collected from six 2-week-old pigmented guinea pigs to determine mRNA expressions of the muscarinic receptors with RT-PCR. Immunofluorescence was used to observe the protein expressions of the 5 muscarinic receptor subtypes. RESULTS: The mRNAs of the 5 muscarinic receptor subtypes were all detected in the scleral tissue, and the mRNA expression was the highest for M1 subtype. Green fluorescence of M1 to M5 subtypes surrounding the fibroblast nuclei were found in the scleral matrix using laser confocal microscopy. CONCLUSION: Five muscarinic receptor subtypes are present in the scleral tissues of immature guinea pigs, indicating the involvement of the muscarinic receptors in eye growth regulation.

Changes of nitric oxide synthase and cyclic guanosine mono-phosphate in form deprivation myopia in guinea pigs.

BACKGROUND: The form deprivation (FD) reduces spatial contrasts and induces myopia. Nitric oxide and cyclic guanosine monophosphate (cGMP) are involved in visual signal transmission. This study investigated changes in nitric oxide synthase (NOS) activity and cGMP concentration in ocular tissues in acute and chronic form deprivation myopia. METHODS: Guinea pigs had one eye covered by translucent glass for 7, 14 or 21 days. Untreated litter mates were used as controls. NOS activity and cGMP concentrations in the retinal, choroidal and scleral tissues of FD eyes and control eyes were analyzed by radioimmunoassay after various durations of FD. The expression of NOS subtypes was identified by immunohistochemistry. RESULTS: Myopia was successfully induced in FD eyes after 14 days. Compared with control groups, the retinal NOS activity and cGMP concentrations in the FD eyes significantly increased after 14 and 21 days while the retinal NOS activity in the FD eyes was transiently suppressed by 7 days of FD. The NOS activity and cGMP concentrations of choroid and sclera in the FD eyes were higher than in the control groups at 21 days. The three isoenzymes of nitric oxide synthase were detected in the ocular tissues of guinea pigs. CONCLUSIONS: The NOS activity and cGMP concentrations were upregulated after chronic FD and the retinal NOS activity was transiently suppressed at acute FD. The function of elevated NOS activity may be mediated by cGMP.

[Effect of pirenzepine on form deprivation myopia in chicks and its possible mechanism].

OBJECTIVE: To observe the effect of M1-selective muscarinic antagonist, pirenzepine, on form deprivation myopia and investigate the expression of MMP-2 and its inhibitor TIMP-2 in the fibrous sclera in order to better understand the mechanism by which pirenzepine inhibits myopia. METHODS: 40 chicks after birth one day were divided into 4 groups randomly: I. Control group; II. Form deprivation group; III. Vehicle application group; IV. Pirenzepine injected group. Form deprivation myopia was established in right eyes of group II, III, IV by placement of a translucent occluder. The deprived eyes of group III and IV received daily subconjunctival administration of vehicle PBS and pirenzepine respectively. Optical measures such as refraction, axial length, equatorial diameter were made at the end of the experiment. Total RNA and protein were extracted from the posterior fibrous sclera chicks. The expression of MMP-2 and TIMP-2 mRNA and protein were investigated with RT-PCR and Western blot analysis respectively. RESULTS: Refraction status, axial length, equatorial diameter of the eyes in pirenzepine injected group were significantly lower when compared with form deprivation group (P < 0.01), but the parameters were higher when compared with normal control group therefore relatively myopic changes were detected. There were no significant difference between drug control and pirenzepine injected group when optical measures and the expression of MMP-2, TIMP-2 were concerned (P > 0.05). The expressions (mRNA and protein) of both MMP-2 and TIMP-2 were significantly different in form deprivation group when compared with normal control group (MMP-2 mRNA increased by 143.51%, P < 0.01; protein increased by 114.60%, P < 0.01; TIMP-2 mRNA decreased by 55.05%, P < 0.01; protein decreased by 53.73%, P < 0.01). In pirenzepine injected group the relative expression of MMP-2 mRNA and protein were decreased obviously by 41.95% (P < 0.01) and by 36.16% (P < 0.01), while TIMP-2 mRNA and protein expression was increased significantly by 72.46% (P < 0.01) and by 53.05% (P < 0.01) respectively compared with the form deprived group. CONCLUSION: Subconjunctivally administration of the M1 selective muscarinic antagonist, pirenzepine, partly prevents or restrains form deprivation induced myopia. It may exert its inhibitory effect by modulating the expression of MMP-2 and TIMP-2 in fibrous sclera.

[Effects of exogenous p21 gene on cells cycle of human lens epithelial cells].

OBJECTIVE: To investigate the effects of transfected exogenous p21 gene on the cells cycle of HLE-B3 cells line. The feasibility of prevention of secondary cataract by gene therapy was evaluated. METHODS: Total length of human p21 gene cDNA was cloned on the parent's plasmid pcDNA3 to construct the recombinant plasmids of pcDNA3/p21, a large amount of pcDNA3/p21 plasmid DNA was prepared by QIAGEN endofree maxi kit. After harvest of the plasmid DNA, the HLE-B3 cells line was transfected. The cell growth was observed and the cells cycle was analyzed by flow cytometry. The expression of p21 mRNA was detected by RT-PCR and the expression of p21 protein was detected by immunohistochemistry and western blot analysis. RESULTS: Forty eight hours after transfection, the growth of transfected cells became slower, some cells floated and died; the control cells and blank plasmid (blank pcDNA3) transfected cells grew normally. Flow cytometry analysis revealed that the number of cells in G(1) phase increased markedly in transfected cells. The RT-PCR showed that the product of p21 in the transfected cells (dead or alive cells) was obviously higher than that of the controls. Immunohistochemical studies showed few positive cells in the controls, and very high positive signal was detected after transfection. Western blot showed a positive band at the level of 21 000 in transfected cells, no positive band could be found in the controls. CONCLUSION: Exogenous p21 gene can transfect the HLE-B3 cells and can be expressed. The transfection can affect the cells cycle by G(1) arrest and induces cells death through the apoptosis process. This result suggests that it is possible to prevent the occurrence of secondary cataract by gene therapy.