Concentração de proteínas no humor aquoso de pacientes com glaucoma primário de ângulo aberto em tratamento clínico / Aqueous humor protein concentration in patients with primary open-angle glaucoma under clinical treatment

OBJETIVOS: Comparar a concentração total de proteínas no humor aquoso entre pacientes com glaucoma primário de ângulo aberto e sem glaucoma. MÉTODOS: Foram coletadas amostras de humor aquoso de 22 pacientes com glaucoma primário de ângulo aberto (grupo GPAA) no momento da trabeculectomia. Na coleta, 0,1 mL de humor aquoso foi aspirado da câmara anterior através de uma agulha de calibre 26, no início do procedimento cirúrgico. Coleta semelhante foi realizada em 22 pacientes sem glaucoma no início da cirurgia de catarata (grupo controle). A amostra de humor aquoso foi armazenada a -20°C após a coleta. A concentração total de proteínas no humor aquoso foi determinada por meio de um teste colorimétrico. RESULTADOS: A média geométrica da concentração total de proteínas no humor aquoso foi de 32 mg/dL (amplitude: 8-137 mg/dL) no grupo glaucoma primário de ângulo aberto e de 16 mg/dL (amplitude: 2-85 mg/dL) no grupo controle. A razão da concentração total de proteínas no humor aquoso entre estes dois grupos foi de 2,0 (intervalo de confiança de 95 por cento: 1,3 a 3,2; p=0,003). CONCLUSÕES: A concentração total de proteínas no humor aquoso de pacientes com glaucoma primário de ângulo aberto foi aproximadamente duas vezes maior quando comparada aos pacientes sem glaucoma.

PURPOSE: To compare total protein concentration in the aqueous humor of primary open-angle glaucoma and non-glaucomatous patients. METHODS: Aqueous humor samples were obtained from 22 patients just before trabeculectomy for clinically uncontrolled primary open angle glaucoma (POAG group). Aqueous humor (0.1 mL) was aspirated by inserting a 26-gauge needle into the anterior chamber. The same procedure was performed in 22 non-glaucomatous patients just before cataract surgery (control group). Immediately after collection, the aqueous humor was stored at -20°C. Aqueous humor total protein concentration was determined using a colorimetric assay. RESULTS: The geometric mean of total protein concentration of the aqueous humor samples was 32 mg/dL (range: 8-137 mg/dL) in the primary open angle glaucoma group and 16 mg/dL (range: 2-85 mg/dL) in the control group. The ratio of the protein concentration between the two groups was 2.0 (95 percent confidence interval: 1.3 to 3.2; p=0.003). CONCLUSIONS: The total protein concentration in primary open-angle glaucoma aqueous humor was approximately two times higher than that in non-glaucomatous subjects.

Transforming Growth Factor-beta Levels in Human Aqueous Humor of Glaucomatous, Diabetic and Uveitic Eyes

PURPOSE: Transforming growth factor-beta2 is known to be present at elevated levels in the aqueous humor of patients with primary open angle glaucoma (POAG) and diabetes but not in uveitis-related secondary glaucoma. We investigated total TGF-beta2 levels and levels of the active form of TGF-beta2 in the aqueous humor of eyes with different types of glaucoma. METHODS: The concentration of the total and active form of TGF-beta2 was measured in 63 patients with primary open angle glaucoma, neovascular glaucoma complicated with diabetes (NVG), and secondary open angle glaucoma complicated with uveitis (SOAG) using a double antibody 'sandwich-indirect' ELISA method. RESULTS: The levels of total TGF-beta2 in the aqueous samples of POAG, NVG, and SOAG were elevated. The levels of active TGF-beta2 in the aqueous samples of POAG, and NVG were also elevated, whereas the level of active TGF-beta2 was within the normal range in the aqueous samples of SOAG. CONCLUSIONS: These results suggest that the level of TGF-beta2 may play a role in the pathology of various types of glaucoma.

Inhibitory effect of tissue transglutaminase (tTG) antisense oligodeoxynucleotides on tTG expression in cultured bovine trabecular meshwork cells / 华中科技大学学报（医学）（英德文版）

To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides (tTG-ASDON) on tTG expression in cultured bovine trabecular meshwork cells (BTMCs) in vitro and explore a new treatment alternative for primary open angle glaucoma (POAG), the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON and tTG-ASDON2 were significantly decreased as compared with that of the controls (P < 0.05). On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.

Expression of tissue transglutaminase in cultured bovine trabecluar meshwork cells / 华中科技大学学报（医学）（英德文版）

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.

Insulin-like growth factor-I gene cloning and protein expression in bovine trabecular meshwork tissue and cells / 华中科技大学学报（医学）（英德文版）

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.

Detection of specific proteins in the aqueous humor in primary open-angle glaucoma

To elucidate the mechanism of increased intraocular pressure in primary open-angle glaucoma (POAG), the protein profiles of aqueous humor obtained from POAG patients were compared with those of cataract patients as a control group. Aqueous humor proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by the ultrasensitive silver staining technique. In 79% of the samples taken from POAG patients, protein bands of 140,000 or 160,000 daltons were stained, but none were stained from cataract patients. The presence of these protein bands revealed statistically significant differences between the two groups. Protein bands of 140,000 or 160,000 daltons were evenly visible at all ages in POAG patients, and the positivity of bands had no correlation with sex or initial intraocular pressure level. It is possible that the ultrastructural changes of the aqueous outflow pathway in POAG may be related to the changes in the aqueous protein, presence of 140,000 or 160,000 daltons protein bands.