DNA methyl transferases are differentially expressed in the human anterior eye segment.

PURPOSE: DNA methylation is an epigenetic mark involved in the control of genes expression. Abnormal epigenetic events have been reported in human pathologies but weakly documented in eye diseases. The purpose of this study was to establish DNMT mRNA and protein expression levels in the anterior eye segment tissues and their related (primary or immortalized) cell cultures as a first step towards future in vivo and in vitro methylomic studies. METHODS: Total mRNA was extracted from human cornea, conjunctiva, anterior lens capsule, trabeculum and related cell cultures (cornea epithelial, trabecular meshwork, keratocytes for primary cells; and HCE, Chang, B-3 for immortalized cells). cDNA was quantified by real-time PCR using specific primers for DNMT1, 2, 3A, 3B and 3L. Immunolocalization assays were carried out on human cornea using specific primary antibodies for DNMT1, 2 and 3A, 3B and 3L. RESULTS: All DNMT transcripts were detected in human cornea, conjunctiva, anterior lens capsule, trabeculum and related cells but showed statistically different expression patterns between tissues and cells. DNMT2 protein presented a specific and singular expression pattern in corneal endothelium. CONCLUSIONS: This study produced the first inventory of the expression patterns of DNMTs in human adult anterior eye segment. Our research highlights that DNA methylation cannot be ruled out as a way to bring new insights into well-known ocular diseases. In addition, future DNA methylation studies using various cells as experimental models need to be conducted with attention to approach the results analysis from a global tissue perspective.

MMP2 activity is critical for TGFß2-induced matrix contraction--implications for fibrosis.

PURPOSE: The fibrotic lens disorder posterior capsule opacification (PCO) develops in millions of patients following cataract surgery. PCO characteristics are extensive extracellular matrix (ECM) production and contraction of the posterior lens capsule, resulting in light-scattering ECM modification (wrinkling). The pro-fibrotic cytokine transforming growth factor beta (TGFß) is central to PCO development. This study aimed to elucidate the role of the ECM modulators matrix metalloproteinases (MMPs) in TGFß-mediated PCO formation. METHODS: The human lens epithelial cell-line FHL-124 and human capsular bag models were employed. Gene expression of MMP family members was determined by oligonucleotide microarray and quantitative real-time RT-PCR. MMP2 and MT1-MMP protein levels were analyzed by ELISA and Western blotting, respectively. Matrix contraction was determined using an FHL-124 patch contraction assay; at end-point, cells were stained with Coomassie brilliant blue and area was determined using image analysis software. Cell coverage and wrinkle formation on the posterior capsule were also assessed using human capsular bag models. RESULTS: Active TGFß2 (10 ng/mL) increased gene and protein levels of MMP2 and MT1-MMP and induced matrix contraction in FHL-124 cells. Specific siRNA inhibition of MT1-MMP did not suppress TGFß2-induced matrix contraction. Active TGFß2-mediated contraction was prevented by broad-spectrum MMP inhibitor GM6001 (25 µM), MMP2 siRNA, and MMP2 neutralizing antibody (4 µg/mL). TGFß2-induced wrinkle formation was attenuated in human capsular bags treated with MMP2 neutralizing antibody (20 µg/mL). CONCLUSIONS: MMP2 plays a critical role in TGFß2-mediated matrix contraction, which appears to be independent of MT1-MMP. MMP2 inhibition provides a novel strategy for the treatment of PCO and potentially other fibrotic disorders.

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.

LOXL1 expression in lens capsule tissue specimens from individuals with pseudoexfoliation syndrome and glaucoma.

PURPOSE: To study lysyl oxidase-like 1 (LOXL1) expression in freshly collected lens capsules from pseudoexfoliation syndrome (XFS), pseudoexfoliation glaucoma (XFG), and normal cataract control individuals. We also investigated the effects of four glaucoma drug medications on LOXL1 expression in primary human lens epithelial cell cultures to see if they could affect LOXL1 expression. METHODS: Lens capsules were collected at the time of cataract surgery. Controls were matched to age, sex, and ethnicity. Total RNA was isolated from individual lens capsule samples and real-time PCR was performed on each sample using primers flanking the sixth exon of the LOXL1 gene. Cell cultures were grown to confluence in four separate six-well plates at 37 °C in 5% CO2. Each plate was then treated with one of four different glaucoma drugs (brinzolamide 1%, brimonidine tartrate 0.1%, timolol maleate 0.5%, and latanoprost 0.005%) once daily for seven days (at both 1:1,000 and 1:100 concentrations relative to media). Controls were not treated with any drug but media was changed in the same manner. After one week of treatment, cells were harvested and total RNA isolated. Real-time PCR was performed on each group of cells. RESULTS: Seven XFS, seven XFG, and ten cataract control specimens were analyzed. LOXL1 expression was detected in the lens capsule specimens from each of the four groups. Significant expression differences were found between the control and XFG groups and XFS and XFG groups. No significant difference was observed between the control and XFS group. No significant decrease in LOXL1 expression was seen with drug incubation of the four medications (Brinzolamide, Timolol, Latanoprost, and Brimonidine) at the 1:1,000 drug:media concentrations versus controls. At 10-fold higher concentrations (1:100 drug:media), brinzolamide, timolol maleate, and latanoprost showed small increases in LOXL1 expression relative to controls. This effect was not observed with brimonidine tartrate. CONCLUSIONS: These results establish that LOXL1 expression is reduced in lens capsule specimens from XFG individuals but not XFS. The drug treatment incubation studies suggest that the change in LOXL1 expression observed in XFG is not attributable to glaucoma drug therapy. If a causative functional relationship can be validated, modification of LOXL1 expression in affected tissues may represent a novel treatment strategy for this disorder.

Temporal changes in MMP mRNA expression in the lens epithelium during anterior subcapsular cataract formation.

Transforming growth factor beta (TGFbeta) has been known to play a role in anterior subcapsular cataract (ASC) formation and posterior capsule opacification (PCO), both of which are fibrotic pathologies of the lens. Several models have been utilized to study ASC formation, including the TGFbeta1 transgenic mouse model and the ex-vivo rat lens model. A distinct characteristic of ASC development within these models includes the formation of isolated fibrotic plaques or opacities which form beneath the lens capsule. A hallmark feature of ASC formation is the epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) into myofibroblasts. Recently, the matrix metalloproteinases (MMPs) have been implicated in the formation of these cataracts through their involvement in EMT. In the present study, we sought to further investigate the role of MMPs in subcapsular cataract formation in a time course manner, through the examination of gene expression and morphological changes which occur during this process. RT-QPCR and immunohistochemical analysis was carried out on lenses treated with TGFbeta for a period of 2, 4 and 6 days. Laser capture microdissection (LCM) was utilized to specifically isolate cells within the plaque region and cells from the adjacent epithelium in lenses treated for a 6 day period. Multilayering of LECs was observed as early as day 2, which preceded the presence of alpha smooth muscle actin (alpha-SMA) immunoreactivity that was evident following 4 days of treatment with TGFbeta. A slight reduction in E-cadherin mRNA was detected at day 2, although this was not significant until the day 4 time point. Importantly, our results also indicate an early induction of MMP-9 mRNA following 2 days of TGFbeta treatment, whereas MMP-2 was found to be upregulated at the later 4 day time point. Further experiments using FHL 124 cells show an induction in MMP-2 protein levels following treatment with recombinant MMP-9. Together these findings suggest an upstream role for MMP-9 in ASC formation.

Matrix metalloproteinase-9 activity in human lens epithelial cells of cortical, posterior subcapsular, and nuclear cataracts.

PURPOSE: To evaluate the level of matrix metalloproteinase-9 (MMP-9) activity in lens epithelial cells (LECs) derived from different types of cataract in patients having phacoemulsification. SETTING: Iladevi Cataract & IOL Research Centre, Memnagar, Ahmedabad, India. METHODS: This observational study of 275 patients having phacoemulsification was performed to evaluate the level of MMP-9 activity in LECs. All anterior lens capsules harboring the LECs and derived from the surgical curvilinear capsulorhexis were obtained during phacoemulsification. The anterior lens capsule samples were processed to analyze MMP-9 activity using a succinylated gelatin assay. The samples were grouped based on age and on pure cataract type. RESULTS: The level of MMP-9 activity in LECs was highest in eyes with cortical cataract. A significant difference in the level of MMP-9 activity was found in different types of cataract (P<.001). The highest level of MMP-9 activity was in patients older than 60 years. The MMP-9 activity increased gradually with age irrespective of cataract type (P<.001). CONCLUSIONS: A significant difference was observed in the level of MMP-9 activity between different types of cataract. The activity of MMP-9 was highest in eyes with cortical cataract. The level of MMP-9 activity increased with age in the LECs of patients with age-related cataract.

Superoxide dismutase activity in the lens capsule of patients with pseudoexfoliation syndrome and cataract.

PURPOSE: To evaluate the activity of the antioxidative enzyme superoxide dismutase (SOD) in the anterior lens capsule of patients with cataract complicating pseudoexfoliation syndrome (PEX) in an attempt to determine the role of the oxidative mechanisms in the etiopathogenesis of this disorder. SETTING: Departments of Ophthalmology and Biochemistry, Ankara University School of Medicine, Ankara, Turkey. METHODS: At the beginning of phacoemulsification surgery, the anterior lens capsules in 21 eyes with PEX and cataract (group A) and those in 24 eyes with cataract alone (group B) were collected with continuous curvilinear capsulorhexis and were frozen at -20 degrees C. At the time of analysis, the specimens were thawed and centrifuged and the supernatants of the homogenate obtained from the samples were analyzed for the activity of the enzyme SOD. RESULTS: The mean age of patients was 74.8 years +/- 5.5 (SD) in group A and 71.7 +/- 5.6 years in group B. The specific activity of SOD was 17.6 +/- 10.8 IU/mg and 4.36 +/- 1.80 IU/mL in group A and 9.9 +/- 12.9 IU/mg and 2.71 +/- 2.61 IU/mL in group B. Superoxide dismutase activity was significantly higher in group A patients than in group B patients (P = .022 and P = .011, respectively). CONCLUSIONS: The increase in SOD activity in the lens capsule of patients with PEX and cataract suggests that oxidative mechanisms play a role in the etiopathogenesis of cataract in PEX. This suggests that SOD activity may be increased as a compensatory mechanism to eliminate this oxidative stress.

Thioredoxin reductase may be essential for the normal growth of hyperbaric oxygen-treated human lens epithelial cells.

We have shown previously with in vivo and in vitro animal models that the lens epithelium, in contrast to the nucleus, is remarkably resistant to hyperoxia. The main purpose of this study was to investigate the mRNA response of cultured human lens epithelial cells (LECs) to challenge by a high level of hyperbaric oxygen. Cells were treated for 3 hr with 50 atm of 99% O2, and then cultured normally for various times up to 11 days. Although the cells appeared normal immediately after the O2-treatment, they failed to grow and suffered 50% cell loss, as well as significant mitochondrial damage, during normal post-culture. Growth of the cells resumed after 3 days and by day 11, the number of O2-treated cells was the same as the controls. Remarkably, the 3 hr O2-treatment produced no immediate effects on either the cellular level of GSH, or on the activities of a number of antioxidant enzymes including glyceraldehyde-3-phosphate dehydrogenase, which is generally regarded as being highly sensitive to oxidation. In contrast, the activity of thioredoxin reductase (TrxR) was severely affected by the O2, decreasing by 51% after the 3 hr exposure. O2-induced death of the cells appeared to be caused by loss of ATP since a 31% decrease in ATP level occurred immediately after the O2-treatment, in spite of a 46% increase in lactate production. Analysis with real-time PCR showed a maximum 3-6-fold increase in mRNA levels 9 hr after the 3 hr O2-exposure for the enzymes heme oxygenase-1 (HO-1), MnSOD and TrxR1 (the cytoplasmic form of TrxR). These results were confirmed with the use of one-step RT-PCR and Northern blotting. Initial upregulation of message for HO-1 occurred a few hours before any upregulation of MnSOD could be detected, suggesting that release of free iron from the degradation of heme by HO-1 may have played a role in the upregulation of the dismutase. No significant changes in mRNA levels were observed for the antioxidant enzymes catalase, CuZnSOD, glutathione reductase and glutathione peroxidase, or for the antioxidant protein thioredoxin. Recovery of TrxR activity over a 4-day period appeared to parallel the return of the cells to a normal rate of growth. The results indicate that damaging effects of hyperoxia on cultured LECs occur primarily in the mitochondria, rather than in the cytoplasm. Cells avoid O2-induced cell death, and return to a normal rate of proliferation by upregulating mRNA levels for HO-1, MnSOD and TrxR1. It appears that full activity of TrxR1, an enzyme required for the production of deoxyribonucletides for DNA synthesis, is essential for the normal growth of O2-challenged LECs.

Effects of variation in superoxide dismutases (SOD) on oxidative stress and apoptosis in lens epithelium.

Among the critical antioxidant enzymes that protect the cells against oxidative stress are superoxide dismutases: CuZnSOD (Sod1) and MnSOD (Sod2). The latter is also implicated in apoptosis. To determine the importance of these enzymes in protection against reactive oxygen species (ROS) in the lens, we analysed DNA strand breaks in lens epithelium from transgenic and knockout (Sod1) mice following exposure to H2O2 in organ culture. Since Sod2 knockouts do not survive, comparison was made of lenses of partially-deficient (heterozygote) for Sod2 and the wild-type controls which have twice the enzyme level. Antioxidant potential of Sod2 was also studied in human lens epithelial cells (SRA01/04) in which the enzyme was up- and down-regulated by transfection with plasmids containing sense and antisense human cDNA for MnSOD. DNA strand breaks in the epithelium of Sod1 knockouts and Sod2 heterozygotes were much greater than in the corresponding wild-type or in transgenic mice over-expressing the enzymes when the lenses were exposed to H2O2. The functional role of Sod2 in apoptosis was examined in cultured human lens epithelial cells. Cells with higher enzyme levels were more resistant to the cytotoxic effects of H2O2, O2- and UV-B radiation. Furthermore, Sod2-deficient cells showed dramatic mitochondrial damage, cytochrome C leakage, caspase 3 activation and increased apoptotic cell death when they were challenged with O2-. Thus, mitochondrial enzyme (Sod2) deficiency plays an important role in the initiation of apoptosis in the lens epithelium.

The presence of a transsulfuration pathway in the lens: a new oxidative stress defense system.

The finding that a lens under oxidative stress accumulated free and protein-bound cysteine (protein-S-S-cysteine) in the fiber cells prompted us to examine if there is an alternative source for cysteine pools besides the active cysteine transport system in the lens, namely, the transsulfuration pathway of homocysteine-cystathionine-cysteine, which utilises methionine through transmethylation. We examined the presence of the gene for cystathionine-beta-synthase (CBS), the rate limiting enzyme that converts homocysteine to cystathionine in the transsulfuration pathway, in human lens epithelial (HLE) B3 cells using PCR with primers designed based on the sequence of human liver CBS (Forward 5'-CCA CAC TGC CCC GGC AAA AT-3'; Reverse 5'-CTG GCA ATG CCC GTG ATG GT-3'). The purified DNA fragment (586 bp) from PCR analysis was sequenced and confirmed the homology with CBS gene from other human tissues. The CBS protein band (67 kDa) was present in the HLE cells, which reacted positively with the human liver anti-CBS antibody. The enzyme protein was detected in the pig and human lenses with the highest intensity in the epithelial layer, lower but equal quantities of CBS was present in the cortical and nuclear regions. Human nuclear CBS increased while epithelial CBS decreased with aging. Oxidative stress transiently upregulated the gene expression of CBS both in HLE cells (0.1 mMH2O2) and in pig lens cultured in TC 199 medium (0.5 mMH2O2). The catalytic activity for CBS, which was assayed by measuring the production of C14-cystathionine from C14-serine in the presence of homocysteine, S-adenosyl-methionine and pyridoxal phosphate, was detectable in the HLE cells and transiently activated with H2O2. Free cystathionine accumulated when HLE B3 cells were treated with propargylglycine (PGG), an inhibitor of cystathionase, the downstream enzyme that converts cystathionine to cysteine. More cystathionine accumulation occurred when the cells were simultaneously exposed to PGG and 0.1 mMH2O2. We have shown that oxidative stress of H2O2 could increase the flux of this transsulfuration pathway by committing more homocysteine to cysteine and glutathione production as H2O2 (0.1 mM) inhibited the remethylation enzyme of methionine synthase while concurrently activating the CBS enzyme. This is the first evidence that a transsulfuration pathway is present in the lens, and that it can be upregulated under oxidative stress to provide additional redox potential for the cells.

Lens aldose reductase inhibiting potential of some indigenous plants.

Cataract is the leading cause of blindness worldover. Diabetes is one of the major risk factors for cataractogenesis and aldose reductase (AR) has been reported to play an important role in sugar-induced cataract. In the present study, the AR inhibitory activity of Ocimum sanctum (OS), Withania somnifera (WS), Curcuma longa (CL), Azadirachta indica (AI) were studied together with their effect on sugar-induced cataractogenic changes in rat lenses in vitro. Aqueous extracts of the plants, procured from Dabur, India, were reconstituted with double distilled water to make various dilutions. AR inhibitory activity of these extracts and their anticataract potentials were evaluated in vitro in rat lenses. AR inhibitory activity of the aqueous extract of different plants was calculated considering the AR activity of normal rat lenses as 100%. The concentration of the plant extract that showed maximum AR inhibitory activity was selected to further study its effect on galactose-induced lens swelling and polyol accumulation in vitro. All the four plants were found to inhibit lens AR activity but to different extent. From dose-response curve, OS was found to be the most effective AR inhibitor followed by CL, AI and WS. The IC(50) values of OS, CL, AI and WS were calculated to be 20, 55, 57 and 89 microg/ml, respectively. OS showed a significant inhibition (38.05%) in polyol accumulation followed by CL and AI (28.4 and 25.04%, respectively). WS did not show any effect on polyol level in rat lenses. None of the plant extracts showed any significant effect on lens water content.OS possesses a significant anticataract activity in vitro and its anticataract potential could be related with its AR inhibitory effect.

Decreased caspase-3 activity in human lens epithelium from posterior subcapsular cataracts.

Apoptosis has been implied in normal lens development in the embryo as well as in lens fibre differentiation. It has also been suggested to play a role in non-congenital cataract and in the formation of posterior subcapsular opacification, but data on the presence of apoptosis in human lens epithelium from cataractous lenses are scarce and conflicting. The present study aimed to investigate apoptosis in lens epithelium from patients undergoing cataract surgery. The amount of apoptosis detected was correlated to age, gender, type of cataract, medications and disease. Moreover, the ability of human lens epithelial cells in culture to respond to the apoptosis-inducing agent staurosporin by activation of caspase-3 was investigated. Human lens capsulotomy specimens were collected immediately after surgery, frozen and later analysed with respect to caspase-3 activity, using the fluorogenic substrate Ac-DEVD-AMC. Generally, the activity of caspase-3 detected in this manner was very low and in 23% of the specimens it was non-detectable. However, there were differences in caspase activity between lens epithelial cells from different types of cataract, where samples from lenses with posterior subcapsular cataract exhibited significantly lower caspase-3 activity than lenses with a clear subcapsular zone. Age, gender or medications did not show any correlation with caspase activity but human capsulotomy specimens from diabetic patients exhibited significantly lower caspase-3 activity. Staurosporin caused a concentration-dependent increase in caspase activity in cultured human lens epithelial cells and the amount of apoptotic nuclei was also increased as viewed by staining with Hoechst 33342, showing chromatin condensation and nuclear fragmentation. Similar results were obtained when fresh human lens capsulotomy specimens were exposed to 1000 nM staurosporin for 24 hr. To conclude, the present data indicate that human lens epithelial cells have the ability to respond to apoptosis-inducing agents with caspase-3 dependent apoptosis, and that even though the general level of apoptosis in human lens epithelium in vivo is low, there are differences in caspase-3 activity levels in lenses with or without posterior subcapsular cataract. The latter finding supports previous studies indicating that this type of cataract may result from defective differentiation, in which apoptosis may play an important role.

Plasma membrane calcium ATPase gene expression in bovine lens epithelium.

Plasma membrane calcium adenosine triphosphatase (Ca(2+)-ATPase) is an energy-dependent protein responsible for transporting cytosolic calcium across the plasma membrane. Multiple plasma membrane Ca(2+)-ATPase isoforms are expressed from four genes (PMCA1-4) and alternative mRNA splicing. We have studied PMCA gene expression in bovine lens epithelium tissues by reverse transcription-polymerase chain reaction, Southern blot, and Northern blot hybridization. All four PMCA genes are expressed in the lens epithelium, the PMCA3 transcript being the most abundant. The transcripts for PMCA1, PMCA2, and PMCA4 exist in decreasing order of abundance. There is no evidence for the expression of any novel PMCA genes in bovine lens epithelium.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases of fibrous humans lens capsules with intraocular lenses.

PURPOSE: We located immunohistochemically the matrix metalloproteinases (MMP) -1, -2, -3 and -9 and the tissue inhibitors of matrix metalloproteinases (TIMP) -1 and -2 in the fibrous capsule of patients with intraocular lenses (IOLs). METHODS: During vitreoretinal surgery in 10 patients we obtained post-cataract surgery lens capsules with or without an IOL. The mean interval between the previous cataract operation and the extraction of the specimens was 35.2 months (range: 2-120 months). Circular sections of the anterior capsule with lens epithelial cells (LECs) were also obtained during cataract surgery. Specimens were processed for immunohistochemical identification of MMPs and TIMPs by light microscopy. RESULTS: While all the members of MMPs and TIMPs were not detected in the normal anterior capsules, they were detected in the ECM and/or LECs on the lens capsules extracted within 18 months after IOL implantations in all of the 4 patients, but were not observed in specimens obtained 18 months or longer postoperatively. In LECs of 1 capsule specimen 10 years postoperatively, MMP-1, but not other MMPs and TIMPs, was detected. CONCLUSIONS: MMPs and TIMPs were detected in the ECM and/or LECs on post-cataract surgery capsules. These proteins may be remodeling the newly deposited ECM and regulating LEC behavior on residual lens capsules in the early phase of healing after cataract surgery.

Na,K-ATPase in lens epithelia from patients with senile cataracts.

Sodium-potassium-adenosine triphosphatase (Na,K-ATPase) has long been recognized for its role in regulating electrolyte concentrations in the lens, within which the electrolyte balance is vital to lens transparency. In this study, we compared the abundance of the alpha-subunit of Na,K-ATPase in lens epithelia of patients with senile cataracts, in order to examine the role of this enzyme in various types of lens opacity. Human lens epithelia were collected from 27 patients with senile cataracts who had undergone phacoemulsification. The type and the severity of lens opacity were graded and scored according to the Lens Opacities Classification System II. The mean age of the patients was 67.5 years (range, 46-80 yr). Abundance of the Na,K-ATPase alpha-subunit peptide in the lens epithelium was quantified by means of Western immunoblotting. Immunoblotting revealed that the amount of Na,K-ATPase alpha-subunit tended to decrease with increased cataract severity. In hypermature cataracts, the Na,K-ATPase alpha-subunit was barely detectable. The amount of alpha-subunit of Na,K-ATPase was inversely correlated with the overall severity of cataract (r = -0.64, p = 0.002). However, the inverse correlation was significant only in the cortical region (p = 0.027). As the cortex is located adjacent to the lens epithelium, it is directly affected by the loss of function of Na,K-ATPase in the epithelium. Such loss could result in water accumulation, vesicles, water clefts, Morgagnian globule formation, and Morgagnian cataract.

Immunolocalization of prolyl 4-hydroxylase in rabbit lens epithelial cells.

PURPOSE: To localize the enzyme prolyl 4-hydroxylase in the crystalline lens and determine the ability of lens epithelial cells (LECs) to synthesize procollagen. SETTING: Research laboratory, Department of Ophthalmology, Wakayama Medical College, Wakayama, Japan. METHODS: Phacoemulsification and aspiration of the crystalline lens followed by implantation of a poly(methyl methacrylate) intraocular lens (IOL) were performed in 1 eye each of 6 albino rabbits; the eye was enucleated 1 or 2 months later. Crystalline lenses were also extracted from the eyes of 2 rabbits. These samples were processed for immunohistochemical detection of the alpha- and beta-subunits of prolyl 4-hydroxylase. RESULTS: A monolayer of LECs was detected on the inner surface of the intact anterior capsule. Antibodies directed against both subunits of prolyl 4-hydroxylase reacted strongly to LECs proliferating on capsules with IOLs, whereas little or no reaction was observed in quiescent LECs or in the regenerated lenticular structure. CONCLUSION: The presence of prolyl 4-hydroxylase in LECs proliferating on the inner surface of the lens capsule suggests that these cells are involved in the production of procollagen and fibrosis during capsular injury and repair. Suppression of prolyl 4-hydroxylase activity may prevent the capsule opacification that results from cataract removal and IOL implantation.

Expression of collagenase/gelatinase activity from basement-membrane fibronectin--isolation after limited proteolysis of a bovine lens capsule and molecular definition of this thiol-dependent zinc metalloproteinase.

We have isolated the collagenase/gelatinase activity of fibronectin from a bovine lens capsule hydrolysate, using heparin-agarose, gelatin-agarose, immunopurification with polyclonal antibodies directed against bovine plasma fibronectin, and immunopurification with a monoclonal antibody directed against the extra-domain A of cellular fibronectin. The expression of collagenase/gelatinase activity by the purified fibronectin fragment was dependent on the incubation time at 37 degrees C and the addition of gelatin to the purified sample. Under these conditions, the purified fibronectin fragment exhibited collagenase/gelatinase activity, as measured by means of gelatin zymography and the intramolecularly quenched fluorogenic substrate of collagenases (7-methoxycoumarin-4-yl)-acetylprolylleucylglycylleucyl-[3-(2,4-di nitrophenyl)-L-2,3-diaminopropionyl]-alanylarginylamide. This activity was due to proteins of 47 kDa and 37 kDa, as indicated by the gelatin-zymography pattern. When the processing was analyzed, by means of SDS/PAGE under reducing conditions, purified starting material of 66 kDa and 55 kDa was observed, and molecular masses of 45, 30 and 27 kDa were found for the processed samples. Under these conditions, the processing was more significant when a substrate, i.e the fluorogenic peptide or gelatin, was added to the processing mixture. An inhibition-profile study showed a zinc-dependent collagenase activity. Using the 45-kDa chymotryptic fragment from human plasma fibronectin, which contains the collagen-binding site, the same results were obtained. These results allow us to define a thiol-dependent zinc metalloproteinase expressed after limited proteolysis of both basement membrane and plasma fibronectins. This proteinase contains a collagen-binding domain, a zinc-binding sequence, and a cysteine involved in catalysis. This enzyme is a member of the thimet family of zinc metalloproteinases.

Immunolocalization of prolyl 4-hydroxylase subunits, alpha-smooth muscle actin, and extracellular matrix components in human lens capsules with lens implants.

Lens capsules become fibrotic after the extraction of a cataract. To understand this phenomenon, we evaluated the immunolocalization of prolyl 4-hydroxylase (an enzyme involved in procollagen hydroxylation), and extracellular matrix components and cytoskeletal components in a normal human lens capsule and in others with intraocular lenses. Lens capsules containing intraocular lenses were removed from a patient with proliferative vitreoretinopathy and three with proliferative diabetic retinopathy during vitreous surgery. Two circular sections of the anterior capsules with lens epithelial cells were obtained by anterior capsulotomy during cataract surgery. In addition, a lens capsular bag was obtained immediately after phacoemulsification. The lens capsules were processed for light microscopic immunohistochemical detection of the alpha and beta subunits of prolyl 4-hydroxylase, extracellular matrix components (including collagen types, laminin and cellular fibronectin) or cytoskeletal components (such as cytokeratin, vimentin and alpha-smooth muscle actin). Monolayer lens epithelial cells were seen on the inner surface of the normal anterior capsules. Each intraocular lens was found to be fixed in the capsular bag. Light microscopic immunohistochemistry showed that these proliferating cells expressed vimentin and alpha-smooth muscle actin; in contrast, quiescent lens epithelial cells did not stain for alpha-smooth muscle actin. Marked immunostaining for subunits of prolyl 4-hydroxylase was detected in lens epithelial cells proliferating on the capsules, while no or only faint prolyl 4-hydroxylase immunoreactivity was detected in quiescent lens epithelial cells immediately after phacoemulsification. Collagen types I, III and VI and cellular fibronectin were observed diffusely in accumulated connective tissue on a capsule with an intraocular lens. Type IV collagen immunoreactivity was seen both in the capsules and in the connective tissue accumulation on the capsules. Collagen V and laminin were detected in association with cellular proliferation. Collagen VII and VIII and laminin 5 were not seen. We concluded that during wound healing of the lens capsule after cataract extraction, the lens epithelial cells that proliferate on the inner surface of the capsule transform it into a myofibroblastic phenotype, expressing prolyl 4-hydroxylase and alpha-smooth muscle actin. These proliferating cells are involved in the production of collagen on the lens capsule. This results in a postoperative fibrotic process and contraction of the lens capsule.

Topographical distribution of lactate dehydrogenase activity in human clear eye lenses and in lenses with different types of senile cataract: a histochemical investigation.

BACKGROUND: Homogenates of human clear lenses show an age-dependent reduction of enzyme activities. Topographical patterns of enzymes in clear and cataractous lenses can be visualized by histochemistry. MATERIAL AND METHODS: Human lenses were characterized by slit-lamp investigations as bearing different types of senile cataracts. Subsequently, lenses were removed by intracapsular extraction. Clear human lenses served as controls. Bovine lenses served to standardize freeze-cutting and incubation for lactate dehydrogenase histochemistry. RESULTS: Bovine lenses show a sharp demarcation between the enzyme reaction of cortical fibers bearing cell nuclei and the non-reacting deeper fibers not exhibiting cell nuclei. Clear human lenses, lenses with deep supranuclear cortical cataracts, and lenses with nuclear cataracts exhibit the same borderline. However, in lenses with a subcapsular cortical cataract only the epithelium and a very thin layer of the most superficially located fibers show positive enzyme reactions. CONCLUSION: In growing clear human and bovine lenses, independent of age, the more peripherally located cortical fibers bearing cell nuclei exhibit strong enzyme-histochemical reactions. More centrally located lens areas lacking cell nuclei increase in volume in an age-dependent manner. These lens regions do not exhibit enzyme activities detectable by our histochemical technique. Therefore the lens areas free of histochemical reaction product become larger with increasing age, whereas the peripherally located lens fibers apparently do not change their enzyme activities with age. Thus, homogenates of total lenses show age-dependent reductions of enzyme activities, although enzyme activities remain at a physiological level in cortical lens fibers with recognizable cell nuclei. In lenses with immature supranuclear cortical and (particularly) in lenses with black nuclear cataracts, cortical fibers still can exhibit high enzyme activities. Unexpectedly, also ruptured and broken fibers in immature deep supranuclear cortical cataracts show strong enzyme activities. In contrast, in lenses with (incipient) subcapsular cortical cataracts only the most superficially located lens fibers exhibit some enzyme activity.

Calpains in lens epithelium from patients with cataract.

Lens epithelium from patients with cataract was obtained during surgery and frozen. The samples were subjected to SDS-electrophoresis and Western blotting. Calpains were quantified using polyclonal antibodies against m- and mu-Calpain could be detected but not the isoenzyme mu-calpain, indicating that m-calpain is the significant most important calpain in human lens epithelium. Quantification of m-calpain showed no relationship to age or gender, but there were significant differences between different types of cataract.