Protein tyrosine phosphatase 1B regulates migration of ARPE-19 cells through EGFR/ERK signaling pathway.

AIM: To evaluate whether protein tyrosine phosphatase 1B (PTP1B) contributed to initiate human retinal pigment epithelium cells (A)-19 migration and investigate the signaling pathways involved in this process. METHODS: ARPE-19 cells were cultured and treated with the siRNA-PTP1B. Expression of PTP1B was confirmed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). AG1478 [a selective inhibitor of epidermal growth factor receptor (EGFR)] and PD98059 (a specific inhibitor of the activation of mitogen-activated protein kinase) were used to help to determine the PTP1B signaling mechanism. Western blot analysis verified expression of EGFR and extracellular signal-regulated kinase (ERK) in ARPE-19 cells. The effect of siRNA-PTP1B on cell differentiation was confirmed by immunostaining for α-smooth muscle actin (α-SMA) and qRT-PCR. Cell migration ability was analyzed by transwell chamber assay. RESULTS: The mRNA levels of PTP1B were reduced by siRNA-PTP1B as determined by qRT-PCR assay. SiRNA-PTP1B activated EGFR and ERK phosphorylation. α-SMA staining and qRT-PCR assay demonstrated that siRNA-PTP1B induced retinal pigment epithelium (RPE) cells to differentiate toward better contractility and motility. Transwell chamber assay proved that PTP1B inhibition improved migration activity of RPE cells. Treatment with AG1478 and PD98059 abolished siRNA-PTP1B-induced activation of EGFR and ERK, α-SMA expression and cell migration. CONCLUSION: PTP1B inhibition promoted myofibroblast differentiation and migration of ARPE-19 cells, and EGFR/ERK signaling pathway played important role in migration process.

Natamycin in the treatment of fungal keratitis: a systematic review and Meta-analysis.

AIM: To review published clinical studies examining the effect of natamycin in the treatment of fungal keratitis. METHODS: We selected the publications in CENTRAL, MEDLINE, EMBASE, CNKI, and CBM. This study systematically reviewed published randomized controlled trials (RCTs) that compared natamycin to other antifungal agents, and conducted feasible Meta-analysis of efficacy results using Revman 5.2 software. RESULTS: We included seven trials which were mainly carried out in developing countries of Asia, with five trials conducted in India, one each in China and Bangladesh. A total of 804 participants were randomized to following comparisons: 2% econazole versus 5% natamycin showed little difference in the effects of treatment of fungal keratitis [RR=0.99, 95% confidence interval (CI), 0.8 to 1.21]; chlorhexidine gluconate versus 5% natamycin indicated that the results on healing of the ulcer at 21d was less conclusive (RR=0.77, 95% CI, 0.55 to 1.08; I (2)=0%); 1% voriconazole versus 5% natamycin suggested that natamycin treatment appeared to be significantly better outcomes than voriconazole (regression coefficient =-0.18 logMAR; 95% CI, -0.30 to -0.05; P=0.006), especially in Fusarium cases (regression coefficient=-0.41 logMAR; 95% CI, -0.61 to -0.20; P<0.001); natamycin versus fluconazole showed a significant difference in cure rate (χ(2)=5.048, P<0.05) and natamycin group was more effective than fluconazole in average period of therapy (t=7.94, P<0.01). CONCLUSION: Natamycin was a preferable choice in the treatment of fungal keratitis, especially in the early period of Fusarium cases.

TREM-1 expression in rat corneal epithelium with Aspergillus fumigatus infection.

AIM: To investigate the expression of triggering receptor expressed on myeloid cells-1 (TREM-1) in the aberrant inflammation within the corneal epithelium at early period of fungal infection. METHODS: A total of 65 Wistar rats were randomly divided into control group, sham group and fungal keratitis (FK) group, in which the cornea was infected by Aspergillus fumigatus (A. fumigatus). After executed randomly at 8, 16, 24, 48 and 72h after experimental model being established, the severity of keratomycosis in rats was scored visually with the aid of a dissecting microscope and slit lamp. Then corneas in three groups were collected to assess the expression of TREM-1 through quantitative reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence technique and Western blot analysis. The correlation between FK inflammation and expression of TREM-1 was also analyzed. RESULTS: Corneal inflammation scores increased with time after fungal infection (F=49.74, P=0.000). The inflammation scores in FK group were obviously higher than those in sham group on the whole (F=137.78, P=0.000). Levels of TREM-1 in the infected rat corneal epithelium had elevated at 8h and peaked at 48h (P<0.001, compared with control group). Western blot analysis also showed an obviously elevated TREM-1 level in rat corneal epithelium at 24h and 48h after fungal infection. Immunofluorescence technique showed that TREM-1 mainly existed in corneal epithelium and infected corneal stoma of rat. TREM-1 protein expression was enhanced after fungal infection. Moreover, severity of FK inflammation was significantly related to TREM-1 expression in FK (r=0.942, P=0.000). CONCLUSION: TREM-1 may contribute to amplify the inflammation in the cornea infected with A. fumigatus and play critical roles in the battle against A. fumigatus in the innate immune responses.

Protein tyrosine phosphatase 1B regulates the activity of retinal pigment epithelial cells.

PURPOSE: To determine whether protein tyrosine phosphatase 1B (PTP1B) is expressed in rat retinal pigment epithelium (RPE) cells, to evaluate whether inhibition of PTP1B contributes to initiation of RPE cells into an active state, and to investigate the signaling pathways involved in this process. METHODS: Rat retinas were detached by trans-scleral injection of 1.4% sodium hyaluronate into the subretinal space. Immunocytochemistry evaluated the expression of PTP1B in RPE cells located at normal and detached retinas. From the cultured RPE cells treated with TCS-401, cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetracolium bromide assay, and the protein expression levels of cyclin A and cyclin D1 were determined. The effect of TCS-401 on cell differentiation was confirmed by immunostaining for α-smooth muscle actin and by western blot. Cell migration activity and PTP1B signaling mechanism were determined. Migration Assay was used to evaluate cell migration activity. PTP1B signaling mechanism was determined by use of PD98059 and LY294002. RESULTS: PTP1B was expressed in the RPE layer of the normal retina. After retinal detachment, weak immunolabeling of PTP1B was seen in the RPE cells. TCS-401 promoted the proliferation and expression of cyclin A and cyclin D1 in RPE cells. TCS-401 induced RPE cells to differentiate toward better contractility and motility. A migration assay proved that inhibiting PTP1B improved the migratory activity of RPE cells. TCS-401 activated extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) phosphorylation. Pretreatment with PD98059 and LY294002 abolished TCS-401-induced activation of Erk, Akt, cell proliferation, and cell migration. CONCLUSIONS: PTP1B may be involved in regulating the active state of RPE cells. The inhibition of PTP1B promoted the proliferation, myofibroblast differentiation, and migration of RPE cells, and MEK/Erk and PI3K/Akt signaling pathways played important roles in the proliferation and migration process.

Role of TREM-1 in response to Aspergillus fumigatus infection in corneal epithelial cells.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a cell surface receptor that is highly expressed in inflammatory lesions caused by infectious agents such as bacteria and fungi and amplifies immune responses. The aim of the current study was to investigate TREM-1 expression in corneal epithelial cells infected by Aspergillus fumigatus (A. fumigatus) and evaluate its role. In this study, infection with A. fumigatus upregulated TREM-1 expression in corneal epithelial cells both in vitro and in vivo. Furthermore, treatment with the antagonistic peptide of TREM-1 decreased the levels of inflammatory cytokines that were enhanced by the fungal infection. We speculated that cross-talk occurs between TREM-1 and Toll-like receptor-4 (TLR-4) in cornea fungal infection. Inhibitors of TLR-4 and myeloid differentiation factor 88 (MyD88) could partially inhibit the upregulation of TREM-1 induced by A. fumigatus respectively. In addition, TLR-4 blockade enhanced the inhibitory effect of the antagonistic peptide of TREM-1 on A. fumigatus-induced inflammation. These findings suggest that TREM-1 plays critical roles in fungal infection, and targeting it may represent a novel therapeutic strategy for patients with fungal keratitis.

Epidemiological characteristics and risk factors of diabetic retinopathy in type 2 diabetes mellitus in Shandong Peninsula of China.

AIM: To determine the epidemiological characteristics and estimate the risk factors of diabetic retinopathy (DR) in patients with type 2 diabetes mellitus (T2DM) in Shandong Peninsula of China. METHODS: The cases of T2DM admitted to Affiliated Hospital of Medical College of Qingdao University, Shandong Province, China, from January 2006 to December 2010 were retrospectively reviewed. The epidemiological characteristics of DR were estimated. The cases were divided into two groups according to degrees of retinopathy: non-DR group and DR group. Logistic regression analysis was used to study the related risk factors of DR. RESULTS: The prevalence of DR in patients with T2DM was 25.08% (834/3326). There was significant difference between the average age for men (59.08±15.43 years) and for women (62.92±18.19 years, P=0.0021). The majority of DR occurred in women (female: male ratio=1.76:1, P<0.0001). The incidence rate of DR in urban (489/834) was higher than that in rural area (345/834, P<0.0001). In 834 DR patients, the mean duration of T2DM was 8.90±4.15 years (range: 0-16 years); 440 people (52.76%) had received varying degrees of health education about prevention and primary care of DM; and 473 people (56.71%) suffered from other DM complications confirmed at the same time. In addition, the incidence rate of monocular (551/3326) and binocular retinopathy (283/3326) were statistically different (P<0.0001). Factors associated (P<0.05) with the presence of DR included old age, lower health educational level, intraocular surgery history, longer duration of T2DM, accompanying with other DM complications, no standard treatment procedure, lower body mass index (BMI) and higher fasting plasma glucose (FPG), glycated hemoglobin A(1)C (HbA(1)C), urine albumin (UA), total cholesterol (TC), low-density-lipoprotein cholesterol (LDL-C). The risk factors (P<0.05) independently associated with the presence of DR were: longer duration of T2DM, lower health educational level, higher FPG, higher UA, lower BMI and higher TC. CONCLUSION: DR is highly prevalent in the patients with T2DM in Shandong Peninsula of China. Besides blood glucose, many factors are associated with the present and development of DR.

The effects of sodium orthovanadate-induced phosphatase inhibition on rat retinal pigment epithelium cell activity.

PURPOSE: The purpose of this study was to assess the contribution of sodium orthovanadate (SOV)-induced phosphatase inhibition to the activation of rat retinal pigment epithelium (RPE) cells. METHODS: Confluent cultures of rat RPE cells were treated with the general phosphatase inhibitor SOV. The effects of SOV on the cell cycle were determined by flow cytometry and protein detection of cyclin A and cyclin D1, two different cell cycle regulatory factors. The effects of SOV on cell differentiation were confirmed by immunostaining for α-smooth muscle actin (α-SMA). A migration assay was used to evaluate the effects of SOV on cell migration. RESULTS: SOV could accelerate the cell cycle of RPE cells. Western blotting showed that SOV significantly increased the expression of cyclin A and cyclin D1 in a dose-dependent fashion. The results of α-SMA staining and western blotting demonstrated that SOV induced RPE cells to differentiate toward better contractility and motility. The migration assay indicated that SOV improved the migration activity of RPE cells. CONCLUSIONS: Sodium orthovanadate can improve proliferation, differentiation, and migration of rat RPE cells and can also induce the reentry of contact-inhibited rat RPE cells into the cell cycle.

[The effect of rapamycin liposome gutta inhibiting rat corneal neovascularization].

OBJECTIVE: To explore the inhibiting effect of rapamycin (RAPA) on corneal neovascularization (CNV) of rats and the functional mechanism. METHODS: A design group was adopted. 102 Wistar rats were divided into four groups at random, including rapamycin liposome treated group (24 rats), the rapamycin solved in bean oil treated group (24 rats), blank liposome treated group (24 rats), blank treated group (24 rats) and normal control group (6 rats). All right eyes of 96 rats were induced by alkali cauterization. Rapamycin liposome were prepared by thin film hydration and the major factors were studied by the method of orthogonal design. After alkali burn, cauterized rats were observed by slitlamp biomicroscope every day. On the 1st, 4th, 7th, 14th days after operation, the expression of HIF-1alpha and VEGF were examined by immunohistochemical method and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The analysis of variance and q test groups for analysis were adopted to analyze the results. RESULTS: (1) The bodies of RAPA liposome were intact kinds of spheres, the average diameter was 145.2 nm, and the envelopment rate was 90.02%. (2) After the burn of 14 d, CNV area of B, C, D and E group were (28.289 +/- 0.703), (28.005 +/- 0.801), (20.002 +/- 1.005) and (22.300 +/- 0.853) mm(2) (F = 159.62, P < 0.05). The CNV of both the rapamycin liposome treated group and the rapamycin solved in bean oil group grew slowly and smaller than that of blank liposome treated group and blank treated group (q = 47.80, 46.20, 34.60, 32.90;P = 0.00). While the rapamycin liposome treated group changed more obviously than the rapamycin solved in bean oil group (q = 13.20, P = 0.00). After alkali burn, the expression of HIF-1alpha and VEGF increased dramatically, meanwhile the expression of HIF-1alpha and VEGF were significantly decreased by RAPA. CONCLUSIONS: Liposome body is an excellent medicine carrier for the RAPA. RAPA can obviously suppress the growth of CNV. The possibly mechanism is weakening VEGF expression by inhibiting the transcription factor HIF-1alpha.

[RNA interference inhibits expression of cyclooxygenase-2 and matrix metalloproteinase-2 in rabbit corneal stromal cells].

OBJECTIVE: To research the effect of short hairpin RNA (shRNA) targeting cyclooxygenase-2 (COX-2) on the expression of COX-2 and MMP-2 in rabbit corneal stromal cells in vitro. METHODS: It was a experimental study. Rabbit corneal stromal cells were cultured in vitro. The transfection efficiency of siRNA mediated by HiperFect Transfection Reagent was determined in rabbit corneal stromal cells in order to optimize the transfection condition. shRNAs (shRNA-1,2,3) specific for COX-2 and one negative control nonspecific shRNA were designed, then were transfected by HiperFect Transfection Reagent into both normal rabbit corneal stromal cells and those which were stimulated by IL-1alpha. 6, 12, 24, 48, 72 hours after IL-1alpha added, cells were collected for Real-Time PCR to detect the gene expression of COX-2 and MMP-2, then the results were compared with those of control group. The data of all groups at the same time was analyzed by one-factor analysis of variance, and the comparison of any two groups was carried out by q-test. RESULTS: The siRNA mediated by HiperFect Transfection Reagent can be transfected efficiently into rabbit corneal stromal cells in vitro, and the maximum efficiency was 70%-80%. The expression of COX-2 and MMP-2 mRNA after IL-1alpha stimulated was much higher than that of blank group. shRNA-2 can significantly inhibit the expression of COX-2 and MMP-2 mRNA in corneal stromal cells stimulated with IL-1alpha. The level of COX-2 and MMP-2 was reduced 83.04% and 73.69% respectively, compared with the expression of single IL-1alpha stimulated group, the difference had statistical significance (q = 24.03, P = 0.00; q = 14.76, P =0.00). The difference of COX-2 mRNA among transfection groups of shRNA-1, shRNA-3, negative control shRNA and single IL-1alpha stimulated group had no statistical significance (F = 0.02, P =0.99). The difference of MMP-2 mRNA among those groups also had no statistical significance (F = 0.02, P = 0.98). CONCLUSION: The RNA interference targeting COX-2 can effectively inhibit the expression of COX-2 and MMP-2 in IL-1alpha stimulated rabbit corneal stromal cells in vitro.