Inhibitory effect of dexamethasone on TGF-beta1 expression of rabbit ciliary pigment epithelia cultured in vitro.

In order to elucidate the effect of dexamethasone on the expression of transforming growth factor-beta1 (TGF-beta1) in ciliary pigment epithelial (CPE) cells cultured in vitro, rabbit CPE cells were cultured in vitro, treated with DMEM medium containing 0, 1 x 10(-8), 5 x 10(-8), 10 x 10(-8) and 50 x 10(-8) mol/L dexamethasone respectively for 5 days. The TGF-beta1 expression was detected by immunohistochemistry Supervision methods and analyzed semi-quantitatively by HMIAS-2000 image system. As opposed to in vivo, rabbit CPE cells expressed TGF-beta1 under cultured circumstance in vitro. The gray scales of the positive yellow staining in the groups of 1 x 10(-8), 5 x 10(-8), 10 x 10(-8) and 50 x 10(-8) mol/L dexamethasone were 136.57 +/- 4.43, 140.20 +/- 6.10, 142.98 +/- 2.99, 146.80 +/- 1.68 and 150.05 +/- 1.94 respectively. When the concentrations of dexamethasone were equal to or higher than 5 x 10(-8) mol/L and, the expression of TGF-beta1 was inhibited. 10(-7) mol/L dexamethasone showed a significant inhibition. It was suggested that CPE cells possess the potential ability of synthesizing and expressing TGF-beta1. The inhibition of TGF-beta1 expression by dexamethasone may be beneficial to the treatment of proliferative vitroretinopathy, also exert some influence on the secretion of aqueous humor and ciliary inflammation.

Expression of transforming growth factor-beta in cultured normal human lens epithelia cells.

In order to investigate whether cultured normal human lens epithelial cells (LEC) express transforming growth factor beta (TGF-beta), reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical methods were used for detection of TGF-beta mRNA and protein in cultured normal human LEC. The results showed that a single RT-PCR amplified product about 310bp was obtained, and the sequence was homologous to the known sequence. TGF-beta immunostain was positive in the plasma of LEC. It was suggested that normal human LEC could produce TGF-beta, and LEC could be affected by TGF-beta through autocrine action.

Effects of visible light on cultured bovine trabecular cells.

To explore the biological effects of light on trabecular cells, cultured bovine trabecular cells were exposed to visible light of different wavelength with different energy. Cellular morphology, structure, proliferation, and phagocytosis were observed. The cells showed no remarkable changes when the energy was low. When the exposure energy reached 1.12 mW/cm2, the cytoplasm showed a rough appearance, and cell proliferation and phagocytosis decreased. This phototoxicity was strong with white light (compound chromatic light), moderate with violet light or yellow light, and mild with red light.

Expression of tissue transglutaminase in cultured bovine trabecluar meshwork cells.

To study whether cultured bovine trabecluar meshwork cells (BTMC) are capable of expressing tTG in protein and at mRNA level, BTMC were cultured in vitro and passaged three times, then the cells were transferred onto or cultured on sterile cover or submitted to isolation of RNA with Trizol, and the expression of tTG was detected by immunohistochemical technique and reverse transcription polymerase chain reaction (RT-PCR) respectively. Our results showed that tTG immunostaining was positive in the cytoplasm and rarely in the nucleus of cultured BTMC. No immunostaining was seen in the negative control. Moreover, a single RT-PCR amplified product whose sequence and size were in accordance with our known parameters was obtained. The expression of tTG in cultured BTMC was confirmed in protein and at mRNA level. BMTC is available more readily for the investigation of the relationship between tTG and primary open-angle glaucoma.

Apoptosis of human trabecular meshwork cells induced by transforming growth factor-beta2 in vitro.

Whether transforming growth factor-beta2 (TGF-beta2) induces apoptosis of human trabecular meshwork cells was investigated in vitro. Cultured 3-5 passage human trabecular meshwork cells were treated with 0 (control), 0.32, 1, 3.2 ng/ml TGF-beta2 for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmission electron microscopy, TUNEL technique and flow cytometry. The results showed characteristic morphologic changes of apoptotic cells were observed under transmission electron microscopy. DNA fragmentation of human trabecular meshwork cells was found by TUNEL technique. Quantitative analysis of flow cytometry showed that percentages of apoptotic human trabecular meshwork cells were (2.79 +/- 0.44)%, (4.43 +/- 1.17)% and (9.60 +/- 2.05)% respectively with different concentrations [1 ng/ml (P<0.05), 3.2 ng/ml (P<0.01)] of TGF-beta2 with the difference being significant between experimental group and control group [(1.41 +/- 0.34)%]. It was concluded that TGF-beta2 can induce apoptosis of human trabecular meshwork cells in vitro and may be involved in the decrease of trabecular meshwork cells in the patients with primary open angle glaucoma and aging of normal people.

Insulin-like growth factor-I gene cloning and protein expression in bovine trabecular meshwork tissue and cells.

Whether cultured bovine trabecular meshwork cells and trabecular tissue ex vivo express insulin-like growth factor-I (IGF-I) messenger RNA (mRNA) and protein was investigated. Total RNA of cultured bovine trabecular meshwork cells as well as trabecular meshwork tissue freshly excised from bovine eyes was extracted, and reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect IGF-I mRNA. RT-PCR product was verified by sequencing. Immunohistochemical stain was used to detect IGF-I protein. The results showed that a single PCR amplified product was obtained, and the sequence was homologous to the known sequence.. IGF-I immunostain was positive in the cytoplasm of trabecular meshwork cells. It was concluded that trabecular meshwork cells produce IGF-I and contribute to the presence of IGF-I in trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by IGF-I not only through paracrine, but also autocrine action. Whether abnormal down-regulations in IGF-I production may contribute to the pathogenesis of primary open-angle glaucoma and the possibility of promoting the autocrine action of IGF-I by trabecular meshwork cells to treat the disease is worth further investigation.

Antagonistic effects of tranilast on proliferation and collagen synthesis induced by TGF-beta2 in cultured human trabecular meshwork cells.

Whether tranilast had antagonistic effect on proliferation inhibition and collagen synthesis promotion induced by TGF-beta2 in cultured human trabecular meshwork cells was investigated. Suspension of 1 x 10(4) cultured human trabecular meshwork cells of 3-5 passage was distributed in each well of a 96-well disk and divided into control group and experimental group. After 24 h, 0 microg/ml (control), 12.5 microg/ml, 25 microg/ml, 50 microg/ml tranilast with 3.2 ng/ml TGF-beta2 were added into the incubation medium. Another 24 h later, proliferation and collagen synthesis in cultured human trabecular meshwork cells were examined respectively by using tetrazolium-based semiautomated colormetric (MTT) assay and 3H-proline incorporation with liquid scintillation technique. The results showed absorbance (A) values of the experimental groups were 0.9036 +/- 0.3017, 1.1361 +/-0.1352, 1.2457 +/- 0.1524 according to the different concentrations of tranilast, and 0.8956 +/-0.1903 of the control group. In comparison with the control group, 25 microg/ml (q'= 3.23, P< 0.05), 50 microg/ml (q'=4.70, P<0.01) tranilast significantly antagonized the decrease of the A values induced by TGF-beta2 in the cultured human trabecular meshwork cells. In comparison with the control group [817.37+/-124.21 cpm/10(4) cells], 12.5 microg/ml (620.33+/-80.46 cpm/10(4) cells, q'= 4.26, P<0.05), 25 microg/ml (594.58+/-88.13 cpm/10(4) cells, q'=4.81, P<0.01), 50 microg/ml (418.64+/-67.90 cpm/10(4) cells, q'=8.62, P<0.01) tranilast significantly inhibited the incorporation of 3H-proline into the cultured human trabecular meshwork cells promoted by TGF-beta2 in a dose-dependent manner. It was concluded that tranilast had the antagonistic effect on the proliferation inhibition and collagen synthesis promotion induced by TGF-alpha2 in the cultured human trabecular meshwork cells.

[Expression of insulin-like growth factor-I in bovine trabecular meshwork cells in vitro].

OBJECTIVE: To determine whether cultured bovine trabecular meshwork cells in vitro would express insulin-like growth factor-I (IGF-I). METHODS: Newborn bovine trabecular meshwork cells of the third passage were cultured in vitro. The whole RNA from 10(6) cells was extracted with Trizol reagent, and specific oligonucleotide primer pair and reverse transcription polymerase chain reaction (RT-PCR) were used for detection of IGF-I messenger RNA. Immunohistochemical stain was used to detect IGF-I protein. RESULTS: Cultured cells had the specific characteristics of bovine trabecular meshwork cells. A single RT-PCR amplified product whose sequence was homologous to the known sequence was obtained. IGF-I immunostain was positive. CONCLUSIONS: Trabecular meshwork cells may produce IGF-I. This finding strongly supports the possibility that IGF-I may affect trabecular meshwork cells through paracrine and autocrine mechanisms.

[Analysis of the quality of clinical trials about therapeutic research using the standard of evidence-based medicine].

OBJECTIVE: To investigate the quality of clinical trials of ocular therapeutic efficacy in China in order to provide the facts to support the Evidence-Based Medicine (EBM) and improve the clinical research trials. METHODS: All the articles published in Chinese Journal of Ophthalmology from 1983 to 2002 emphasizing random control trial and clinical control trial were evaluated as per EBM standard. RESULTS: The total number of articles for clinical therapeutic efficacy published in the past 20 years were 152, of which 35 articles (23.0%) were random control trials, and 8 (5.3%) articles were clinical control trials. The disparity of quality of research was found as follows. The method of random/half random was described in 10 articles (23.3%). Placebo-control and open-control were reported in 3 (3/43) and 9 articles (20.9%), respectively. Blinded, double-blinded, and single-blinded research was adopted in 7 (16.3%), 3 articles (7.0%), and 4 articles (9.3%), respectively. Ten articles (23.3%) reported strict diagnostic standard including inclusion and exclusion standards while 30 articles (69.8%) didn't use exclusion criteria. Follow-up visits from a few months to years was found in 30 out of 43 articles, of 25 articles (58.1%) provided detailed follow-up visit data; 19 articles (44.2%) didn't supply any information of the cases lost follow-up, only 7 articles (16.3%) reported the cause and process of the cases lost follow-up. Adverse events were not consistently reported in the articles. Not a single article estimated sample size when the study was designed. Most articles included small or medium size samples, while lacking of multi-center, large sample, randomized, double-blinded and placebo-controlled clinical trials. Statistical significance for either part or all data except P value was not indicated in 15 articles (34.9%). The indexes assessing the clinical significance of clinical therapeutic efficacy include relative risk reduction, absolute risk reduction, number needed to treat, however, these indexes were not used in all the articles. CONCLUSIONS: In summary, the clinical trials related to ocular therapeutic efficacy published between 1983 and 2002 were not very well designed and the accuracy and the reliability of the results were greatly affected. Therefore, it is imperative to improve the quality of the research by enhancing the design and analysis of the research in future.

[The inhibitory effect of tranilast on transforming growth factor-beta(2) expression in cultured human trabecular meshwork cells].

OBJECTIVE: To investigate the effect of tranilast on transforming growth factor-beta(2) (TGF-beta(2)) expression in cultured human trabecular meshwork cells. METHODS: TGF-beta(2) expression in cultured 3-5 passage human trabecular meshwork cells was measured by semi-quantitative RT-PCR after treated with 0.0 micro g/ml (control), 12.5, 25.0 and 50.0 mg/L tranilast for 48 h. RESULTS: The value of TGF-beta(2)/G3PDH of cells treated with 12.5, 25.0 and 50.0 mg/L tranilast was 1.85 +/- 0.35, 1.66 +/- 0.42, 1.16 +/- 0.24, respectively. The difference between these treated groups and that of the control group (3.82 +/- 0.56) was statistically significant (q' = 10.77, 11.80, 14.54, P < 0.01), respectively. The value of TGF-beta(2)/G3PDH in the tranilast treated trabecular meshwork cells decreased in a dose-dependent manner. CONCLUSION: Tranilast could inhibit TGF-beta(2) expression in cultured human trabecular meshwork cells. It is worth to study the using of tranilast in the treatment of primary open-angle glaucoma.

Effect of transforming growth factor-beta(2) on extracellular matrix synthesis in bovine trabecular meshwork cells.

OBJECTIVE: To investigate the effect of transforming growth factor-beta(2) (TGF-beta(2)) in human aqueous humor on extracellular matrix synthesis in cultured bovine trabecular meshwork cells. METHODS: Cultured 3-5 passage bovine trabecular meshwork cells were divided into control group and experimental group. After treated with 0 ng/L (control), 0.32 x 10(3) ng/L, 1.00 x 10(3) ng/L, 3.20 x 10(3) ng/L TGF-beta(2) for 48 h, collagen, fibronectin and hyaluronic acid synthesis in bovine trabecular meshwork cells were examined respectively by (3)H-proline incorportion and liquid scintillation technique, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). RESULTS: In comparison with the control group, TGF-beta(2) significantly promoted the synthesis of collagen at 0.32 x 10(3) ng/L (P < 0.05), 1.00 x 10(3) ng/L (P < 0.01), 3.20 x 10(3) ng/L (P < 0.01), (3)H-proline incorporation increased in a concentration-dependent manner; and 1.00 x 10(3) ng/L (P < 0.05), 3.20 x 10(3) ng/L (P < 0.01) TGF-beta(2) significantly promoted the synthesis of fibronectin in cultured bovine trabecular meshwork cells. 1.00 x 10(3) ng/L (P < 0.01) and 3.20 x 10(3) ng/L (P < 0.01) TGF-beta(2) significantly inhibited the synthesis of hyaluronic acid in the cultured cells. CONCLUSION: The data suggest that TGF-beta(2) play important roles in extracellular matrix age-related changes of normal trabecular meshwork and abnormal deposition of the extracellular matrix in the trabecular meshwork, especially the juxtacanalicular tissue of primary open-angle glaucoma patients.