Estradiol biosynthesis in canine lens epithelial cells.

PURPOSE: To confirm that lens epithelial cells (LEC) synthesize 17ß-estradiol, active estrogen, and to identify the pathway(s) by which normal and cataractous LEC synthesize 17ß-estradiol. METHODS: ELISA was used to measure estradiol in aqueous humor; immunohistochemical staining was used to localize estradiol, testosterone and sulfatase; tritiated water release assay was used to measure aromatase activity; and qRT-PCR was used to quantify expression of aromatase and sulfatase in normal and cataractous canine and human LEC. RESULTS: Canine eyes with and without cataracts had no differences in aqueous humor estradiol levels; however, cataractous LEC had more intense immunoreactivity for estradiol than normal LEC. There were little to no differences in canine sulfatase protein and mRNA expression when normal and cataractous LEC were compared. qRT-PCR demonstrated that canine cataractous LEC had significantly higher expression of aromatase; this was confirmed with the tritiated water release assay. Similar to dogs, human cataracts had both sulfatase and aromatase mRNA expression. CONCLUSIONS: Normal and cataractous LEC can synthesize estradiol by the sulfatase pathway; however, cataractous LEC appear to use the aromatase pathway as well. Because no differences in aqueous humor estradiol levels were detected, we suspect that estradiol synthesized by the sulfatase pathway is secreted into the aqueous humor; whereas, estradiol synthesized by the aromatase pathway is used for, as yet unknown, intracrine purposes.

The effect of phosphorylated Akt inhibition on posterior capsule opacification in an ex vivo canine model.

PURPOSE: To evaluate whether inhibition of phosphorylated Akt (pAkt) would reduce or prevent posterior capsule opacification (PCO) in an ex vivo canine lens capsule model. METHODS: Normal and cataractous lenses (n=6) were evaluated for pAkt via immunohistochemistry and immunoblotting. Primary cultures of lens epithelial cells (LEC) were exposed to ultraviolet light (UV) to induce pAkt. Cultures were then incubated in 0, 2.5, 5, or 10 µM (n=6) of a novel Akt inhibitor (AR-12) for either 8 or 24 h. Cultures were harvested and pAkt expression and telomerase activity examined by immunoblotting and telomeric repeat amplification protocol (TRAP)-enzyme linked immunosorbent assay (ELISA), respectively. Lens capsules were harvested post-sham cataract surgery and exposed to 0, 2.5, 5, 7.5, or 10 µM (n=8) of AR-12 for a total of 14 days treatment. Additional lens capsules (n=6) were exposed to 10 µM of AR-12 for 1 week followed by media alone for 1 week; or exposed to media alone for 1 week followed by 10 µM of AR-12 for 1 week. Histopathology and immunohistochemical staining were performed to evaluate PCO formation. Analysis of telomerase activity on the lens capsules was performed by TRAP-ELISA. RESULTS: pAkt protein expression was increased in clinical samples of canine cataracts compared to normal lenses. Following exposure to UV, cultures of LEC significantly (p<0.05) increased expression of pAkt and telomerase activity. Treatment with AR-12 for both 8 and 24 h following UV irradiation significantly (p<0.01) decreased pAkt expression. When UV-exposed LEC were allowed to recover in the presence of either 5.0 or 10.0 µM AR-12, there was a significant (p<0.05) decrease in telomerase activity. In the ex vivo model of PCO, within the region of the capsulorhexis, PCO inhibition was maximally achieved with 10 µM of AR-12. A significant decrease in LEC was noted on the posterior capsules containing 5.0, 7.5, and 10 µM AR-12 compared to the control capsules (p<0.01). Telomerase activity decreased in a dose-dependent manner. One week of treatment with 10 µM AR-12, immediately following capsule excision, was sufficient to inhibit PCO formation, while a delay in exposure to AR-12 after 1 week of media incubation alone did not prevent PCO formation. CONCLUSIONS: pAkt is known to have roles in cell survival, proliferation, and migration, and this study suggests its inhibition immediately following cataract surgery may be a useful approach to prevent PCO.

Role of bacteria in the pathogenesis of recurrent uveitis in horses from the southeastern United States.

OBJECTIVE: To determine the role of intraocular bacteria in the pathogenesis of equine recurrent uveitis (ERU) in horses from the southeastern United States by evaluating affected eyes of horses with ERU for bacterial DNA and intraocular production of antibodies against Leptospira spp. SAMPLE POPULATION: Aqueous humor, vitreous humor, and serum samples of 24 clinically normal horses, 52 horses with ERU, and 17 horses with ocular inflammation not associated with ERU (ie, non-ERU inflammation). PROCEDURES: Ribosomal RNA quantitative PCR (real-time PCR) assay was used to detect bacterial DNA in aqueous humor and vitreous humor from clinically normal horses (n = 12) and horses with chronic (> 3-month) ERU (28). Aqueous humor and serum were also evaluated for anti-Leptospira antibody titers from clinically normal horses (n = 12), horses with non-ERU inflammation (17), and horses with confirmed chronic ERU (24). RESULTS: Bacterial DNA was not detected in aqueous humor or vitreous humor of horses with ERU or clinically normal horses. No significant difference was found in titers of anti-Leptospira antibodies in serum or aqueous humor among these 3 groups. Only 2 horses, 1 horse with ERU and 1 horse with non-ERU inflammation, had definitive intraocular production of antibodies against Leptospira organisms. CONCLUSIONS AND CLINICAL RELEVANCE: In horses from the southeastern United States, Leptospira organisms may have helped initiate ERU in some, but the continued presence of the organisms did not play a direct role in the pathogenesis of this recurrent disease.

Effect of grape polyphenols on oxidative stress in canine lens epithelial cells.

OBJECTIVE: To evaluate whether the effects of oxidative stress could be attenuated in cultures of canine lens epithelial cells (LECs) by incubation with grape seed proanthocyanidin extract (GSE), resveratrol (RES), or a combination of both (GSE+RES). SAMPLE POPULATION: Primary cultures of canine LECs. PROCEDURES: LECs were exposed to 100MM tertiary butyl-hydroperoxide (TBHP) with or without GSE, RES, or GSE+RES. The dichlorofluorescein assay was used to detect production of reactive oxygen species (ROS), and immunoblot analysis was used to evaluate the expression of stress-induced cell-signaling markers (ie, the mitogen-activated protein kinase [MAPK] and phosphoinositide-3 kinase [PI3K] pathways). RESULTS: GSE and GSE+RES significantly reduced ROS production after a 30-minute exposure to TBHP. Only GSE significantly reduced ROS production after a 120-minute exposure to TBHP. Incubation with GSE reduced TBHP-induced activity of the MAPK and PI3K pathways. CONCLUSIONS AND CLINICAL RELEVANCE: GSE inhibited key components associated with cataractogenesis, ROS production, and stress-induced cell signaling. On the basis of the data reported here, there is strong evidence that GSE could potentially protect LECs from the damaging effects of oxidative stress.

Prevention of posterior capsular opacification through cyclooxygenase-2 inhibition.

PURPOSE: To determine if cyclooxygenase-2 (COX-2) is upregulated when lens epithelial cells (LEC) in clinical samples of cataracts and posterior capsule opacification (PCO) undergo epithelial-mesenchymal transition (EMT)-like changes. We also wanted to learn if inhibition of the enzymatic activity of COX-2 could prevent PCO formation. METHODS: To ensure that EMT-like changes were occurring in LEC, real-time RT-PCR was used to examine expression of EMT markers. Clinical samples of canine cataracts and PCO were examined for COX-2 expression using immunohistochemistry, western blot analysis, and real-time RT-PCR. The COX-2 inhibitors, rofecoxib and celecoxib, were used in an ex vivo model of PCO formation, and the effects on cellular migration, proliferation, and apoptosis were analyzed using immunohistochemistry and western blots. Prostaglandin E2 (PGE2) expression was examined with ELISA. RESULTS: Markers of EMT, such as lumican, Snail, Slug, and COX-2 were expressed in LEC. In clinical samples of cataracts and PCO, there was overexpression of COX-2 protein and mRNA. Both rofecoxib and celecoxib were effective at inhibiting PCO formation in our ex vivo model. Prevention of PCO with the COX-2 inhibitors appeared to work through decreased migration and proliferation, and increased apoptosis. Neither of the drugs had a toxic effect on confluent LEC and appeared to inhibit PCO through their pharmacologic action. Synthesis of PGE2 was inhibiting in the capsules treated with the COX-2 inhibiting drugs. CONCLUSIONS: Extracapsular phacoemulsification cataract surgery is the most common surgical procedure performed in human and veterinary ophthalmology. The most frequent postoperative complication is PCO. The LEC that remain adhered to the lens capsule undergo EMT-like changes, proliferate, and migrate across the posterior lens capsule causing opacities. We have shown that COX-2, a protein associated with EMT, is upregulated in canine cataracts and PCO. Inhibiting the enzymatic activity effectively prevented EMT of LEC in our ex vivo model of PCO through pharmacologic action, and not acute toxicity. These findings indicate that using COX-2 inhibitors in vivo may be an effective technique in preventing PCO.

Ultraviolet irradiation up-regulates telomerase transcription and activity in lens epithelial cells.

PURPOSE: Ultraviolet irradiation (UVR) increases telomerase activity in various cell types including skin, a sun-exposed organ. The lens is also continually exposed to UVR and we hypothesized that lenses exposed to UVR would have increased telomerase activity, with up-regulated TERT and TR, the two main components of the telomerase holoenzyme. To evaluate whether the cornea would protect lenses from such changes, whole globes, as well as isolated lenses, were exposed to UVR, and lenses were evaluated for changes in telomerase activity. METHODS: There were three parts to this project. The first part of this experiment evaluated freshly harvested normal adult canine lenses exposed to 0, 300, 600, or 1200 J/m(2) UVR, and then allowed to recover for 1, 2, 3 and 4 h. Since only 600 J/m(2) UVR increased telomerase activity, four more postexposure recovery time-points for this UVR dose were evaluated: 10 min, 30 min, 8 h and 24 h. The second part of this experiment used freshly enucleated whole canine globes exposed to 0, 50, 100, 150, 300, 600 or 1200 J/m(2) and incubated overnight; lens epithelial cells (LEC) were evaluated for telomerase activity. The third part evaluated lenses that were exposed to 0 or 600 J/m(2) UVR, and then allowed to recover for 8 and 24 h, before TERT and TR mRNA levels were measured. RESULTS: Isolated lenses exposed to 600 J/m(2) UVR had significantly higher telomerase activity than unexposed controls and other UVR doses, at all time-points except 24 h postexposure. Lenses from whole globes exposed to UVR showed a dose-dependent increase in telomerase activity except at 50 J/m(2) and 1200 J/m(2). Isolated lenses exposed to 600 J/m(2) UVR and then allowed to recover for 8 and 24 h significantly up-regulated TERT and TR mRNAs compared to unexposed control lenses. CONCLUSIONS: Telomerase activity is regulated at both the transcriptional and post-translational levels in canine LEC. Previous work in our laboratory showed dose, time, and age-dependent changes in telomerase activity in the lens. The present study showed that TERT and TR mRNA transcription was increased for up to 24 h following an acute dose of UVR. Both telomerase activity and TERT and TR mRNA levels were elevated until 24 h post-UVR exposure, TERT in combination with TR functions in proliferation-related telomerase activity, but TERT alone has an anti-apoptotic function and its up-regulation may protect LEC from the acute effects of UVR. We are continuing to evaluate the mechanisms by which telomerase is regulated in normal and cataractous LEC.