Cataract induction in lenses cultured with transforming growth factor-beta

PURPOSE: Anterior subcapsular cataracts are characterized by the appearance of opaque plaques of abnormal cells. Distinctive spindle-shaped cells containing alpha-smooth muscle actin are present and are associated with wrinkling of the overlying lens capsule. Accumulations of extracellular matrix, including type I collagen, also are found. The authors previously reported that transforming growth factor-beta (TGF-beta) induces similar aberrant morphologic changes in lens epithelial explants. More recently, they identified alpha-smooth muscle actin in explants cultured with TGF-beta. The aim of this study was to determine whether TGF-beta induces comparable cataractous changes in whole lenses and to examine the effects of this treatment on the transparency of the lens. METHODS: Whole lenses from 21-day-old rats were cultured in defined serum-free medium with TGF-beta 2 or without added growth factors for 5 days. Lenses were then photographed and prepared for histology and immunolocalization. RESULTS: Lenses cultured with TGF-beta developed distinct anterior opacities just beneath the lens capsule. Histologically, clumps of abnormal cells corresponded with these opacities. Spindle-shaped cells, which contained alpha-smooth muscle actin, were present, and the overlying capsule was often wrinkled. The clumps contained accumulations of type I collagen, laminin, and heparan sulphate proteoglycan. In contrast, lenses cultured without growth factors remained transparent, retained normal lens morphology, and did not accumulate alpha-smooth muscle actin or type I collagen. CONCLUSIONS: These results show that TGF-beta induces whole lenses to form opacities that contain morphologic and biochemical markers for subcapsular cataract.     

The cytotoxic effect of topical mitomycin C on the ciliary body in rabbits]

In order to know the initial lens changes that accompany atopic dermatitis (AD), 99 patients diagnosed dermatologically to have AD without any or with slight external ocular inflammations and with no habit of rubbing the eyelid due to severe itching were examined opthalmologically. Clinically, none of them showed any cataractous changes in their eyes. For the sake of comparison with the above population, 4 AD patients with cataractous eyes, 49 renal transplantation patients who were administered steroids over a long period of time but clinically had no cataractous lenses, and 94 healthy individuals with transparent lenses were also selected as subjects. The crystalline lenses of the subjects were examined using an anterior eye segment analysis system and specular microscopy. From Scheimpflug slit images of the lens, light scattering intensity of different lens layers was measured as an indicator of lens transparency changes. The subcapsular basement membrane and changes in the lens epithelial layers were analyzed from specular images of these areas by image processing. Results and considerations from the investigations were: (1) Initial lens changes in cases with AD which may be occult cataractous findings were often detectable. (2) Cataract associated with AD can be accelerated by steroid administration or the habit of strongly rubbing the eyelid, but this may not be the original cause of cataract formation. (3) Two types of cataract are seen in patients with AD: (a) anterior subcapsular plaque formation and (b) anterior and posterior subcapsular opacity formation. The latter type, however, is also accompanied by epithelial damage from the early stage. (4) Significant numbers of patients with AD who have not yet shown manifest lens changes were found among the subjects.     

Human age-related cataract and lens epithelial cell death

PURPOSE: To evaluate the importance of lens epithelial cell death in age-related cataract. To determine whether the large percentage of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive lens epithelial cells previously reported in human capsulotomy specimens results from apoptosis or necrosis. METHODS: Capsulotomy specimens from patients who had undergone cataract surgery and epithelia from cataractous lenses of eye bank eyes were compared with epithelia from noncataractous lenses of eye bank eyes. DNA fragmentation was assayed using the TUNEL method. Cell membrane integrity was tested using a fluorescent stain for DNA, BOBO-3, that is excluded from living cells. Cell proliferation was assayed by labeling with 5-bromo-2'-deoxyuridine (BrdU). The number of cells in different regions of the lens epithelium was measured by digital imaging and computerized counting of nuclei after staining with methyl green. RESULTS: TUNEL-positive cells were sometimes detected adjacent to denuded regions of capsulotomy specimens, especially when epithelia were not fixed immediately after surgery. TUNEL-stained cells usually stained with BOBO-3, indicating loss of plasma membrane integrity. No BrdU-labeled cells were detected in capsulotomy specimens. Cell density in cataractous lens epithelia was similar to that in normal lens epithelia. In cataractous lenses from eye bank eyes, cell density in the region of the epithelium overlying the cataract was higher than cell density in the region of the epithelium overlying the transparent part of the lens. No correlation was found between cell density and cataract severity or between cell density and age. CONCLUSIONS: TUNEL staining of lens epithelial cells in capsulotomy specimens most likely results from necrotic cell death caused by damage during or soon after cataract surgery. Loss of cells from the lens epithelium, by apoptosis or other mechanisms of cell death, does not seem to play a major role in age-related cataract formation.     

Malonaldehyde, superoxide dismutase and human cataract

UV-spectrophotometry and fluorometry were used to study Malonaldehyde (MDA) and Superoxide Dismutase (SOD) in normal, cataractous human lenses and red blood cells of the patients with cataract. MDA content of senile and complicated cataractous lenses was significantly higher than that of normal human lenses, while that of complicated cataract was significantly higher than that of senile cataract. SOD activity of senile and complicated cataractous lenses was significantly lower than that of normal human lenses, while there was no marked difference between senile and complicated cataractous lenses. Significant correlation between cataractous lenses and red blood cells was not found in MDA content and SOD activity. There was a negative correlation between SOD and MDA in normal human lenses, but no correlation between SOD and MDA in cataractous lenses. The study shows that lipid peroxidation may be one of the possible mechanisms of cataractogenesis in human, and emphasizes the role of SOD in prevention of photoperoxidative damages to the tissues.     

Expression of transforming growth factor-beta (TGF-beta) isoforms in osteosarcomas: TGF-beta3 is related to disease progression

BACKGROUND: Transforming growth factor-beta (TGF-beta) is a multipotent growth factor affecting development, homeostasis, and tissue repair. In addition, increased expression of TGF-beta has been reported in different malignancies, suggesting a role for this growth factor in tumorigenesis. METHODS: Using immunohistochemistry, the expression, prevalence, and distribution of TGF-beta isoforms were evaluated in 25 high grade human osteosarcomas. The Cox proportional hazards models and Kaplan-Meier curves were calculated correlating disease free survival with TGF-beta expression. RESULTS: Expression of one or more TGF-beta isoforms was found in all the osteosarcomas. Immunoreactivity for TGF-beta1 and TGF-beta3 generally was stronger than for TGF-beta2. The cytoplasm of the tumor cells showed stronger staining than their surrounding extracellular stroma. Most notably, osteoclasts showed strong to intense staining for all three isoforms. In 11 of 25 specimens angiogenic activity was noted with staining of multiple small vessels in the tumor stroma. Expression of TGF-beta3, but not of TGF-beta2 or TGF-beta1, related to disease progression, such that there was a statistically significant decrease in the disease free interval as the immunoreactivity for TGF-beta3 increased. CONCLUSIONS: All osteosarcomas expressed TGF-beta in the cytoplasm of the tumor cells as well as in their extracellular stroma. The presence of TGF-beta in the endothelial and perivascular layers of small vessels in the tumor stroma suggests angiogenic activity of this growth factor. The expression of TGF-beta3 was correlated strongly with disease progression (P = 0.027). These data suggest that increased expression of TGF-beta isoforms, especially TGF-beta3, may play a role in osteosarcoma progression.     

Elevated plasma levels of transforming growth factor (TGF)-beta1 and TGF-beta2 in patients with disseminated malignant melanoma

Overexpression of transforming growth factor-beta isoforms (TGF-beta1, -beta2, -beta3) has been previously reported in human melanoma cell lines and tumours. The aim of the present study was to evaluate the plasma levels of TGF-beta isoforms in melanoma patients. Significantly elevated levels of TGF-beta1 (4.2 x the controls, P = 0.0094) and of TGF-beta2 (1.5 x the controls, P = 0.012) but not of TGF-beta3 were measured in patients with disseminated but not locoregional melanoma. These results indicate systemic circulation of potentially immunosuppressive peptides of the TGF-beta family in end-stage melanoma patients.     

The oxidative stress in the cataract formation

The lens of the eye is an avascular tissue surrounded by fluids such as the aqueous humor and vitreous body, with one side facing toward the outside of the body. We investigated peroxidative reactions occurring in cataractous lenses, examining changes within the lens tissues as well as in the surrounding environment. 1. Peroxidative reactions in lenses. 1) Aging and peroxidative reactions. The activity of superoxide dismutase (SOD) began to decrease in the lenses of rats at six months of age. Moreover, the level of lipid peroxide increased significantly in the lenses of rats at 24 months of age. Lipoproteins became increasingly oxidized with age. The levels of Na+, K+, and Ca++, ions that are important to the maintenance of membrane function, also varied significantly with age. In the lenses of six-month-old Senescence Accelerated Mice (SAM), there was a marked decrease in the ability of scavenge active oxygen and a marked increase in the amount of lipid peroxide. In human lenses, the level of autofluorescence increased as the lens fiber structure changed with age. 2) Generation of free radicals inside the lens. We verified that HO. and ascorbic acid radicals were being generated inside cataractous lenses using electron spin resonance (ESR). 3) Changes in oxidation-related substances in cataractous lenses. Senile cataractous lenses and diabetic cataractous lenses were classified as four types, cortical, nuclear, posterior subcapsular, and mature. In cataractous lenses from all types of diabetic patients, the levels of glucose, glycated protein, and lipid peroxide were higher than in senile cataractous lenses. Among the four types of cataracts, the accumulation of peroxides was the greatest in the nuclear type both diabetic and senile cataractous lenses. 4) Transitional metals. Iron ions and copper ions existed in lens tissue. In particular, the subepithelial region of the lens stained strongly for copper ions. The increased level of copper ions in cataractous lenses is likely to be related to the increased peroxidation in this tissue. 5) Changes in membrane. Lowered levels of phospholipids and a higher degree of saturation of fatty acids were observed in senile cataractous lenses as compared with normal lenses. The increased saturation of fatty acids indicated that there was a damage to the membrane structure due to peroxidative reactions. The receptors for low density lipoprotein (LDL) were shown to exist on the epithelium of normal lenses. Acetyl-LDL, a denatured lipoprotein was incorporated into senile cataractous lenses but not into normal lenses, suggesting that the barrier function of the membrane deteriorates in cataractous lenses. Moreover, in diabetic cataractous lenses, the levels of very low density lipoprotein (VLDL) and LDL significantly increased. 2. Change in the environment surrounding the lens and peroxidative reactions. 1) Changes in the levels of oxidation-related substances in blood, aqueous humor, and vitreous body from diabetic patients: all had decreased levels of reduced glutathione and superoxide scavenging activity, and increased levels of lipid peroxide and glycated protein. This may have been due to a reduction in the anti-oxidative potential in the environment surrounding the lens due to the enhanced glycation. Changes in the level of oxidation related substances in the vitreous body in particular, will likely have a significant impact on the lens. 2) Changes in lenses as the surrounding environment deteriorates. Human lenses were cultured for three weeks under conditions similar to those found in vivo utilizing the culture system that we had originally designed and constructed. When protective activity against peroxidation was reduced, the amount of lipid peroxide increased significantly. In the presence of high levels of glucose, the levels of lipid peroxide increased and the amount and activity of SOD decreased. 3. Effects of changes in the external environment on peroxidative reactions.     

Nursing practice and patient care in a changing home healthcare environment

In this study, lenses of autopsy eyeballs, anterior capsules including lens epithelium taken during operation for cortical cataract, after cataract tissue obtained at the time of operation, and Elschnig's pearles and Soemmerring's ring from autopsy eye-balls were examined for a variety of factors, such as growth factors, cytokines, bioactive substance factors, cytoskeleton proteins and extracellular matrices by immunocytohistochemistry. Preoperative lens epithelium expressed epidermal growth factor (EGF), EGF-receptor (R), fibroblast growth factor (FGF), FGF-R, interleukin (IL)-1-RII, tumor necrosis factor-alpha (TNF-alpha), plasminogen activator inhibitor type-1 (PAI-1), keratin and laminine. In addition to the above factors, opacified fibrous capsule in after cataract expressed transforming growth factor-beta (TGF-beta), insulin-like growth factor-II (IGF-II), platelet derived growth factor-AB (PDGF-AB), IL-6, prostaglandin-E2 (PG-E2), alpha smooth muscle actin, fibronectin, and I and III to VI type collagen. Elschnig's pearls expressed FGF-R, TNF-alpha, and laminin. Soemmerring's ring expressed EGF, FGF, FGF-R, IL-1-RII, keratin, tissue-PA, and PAI-1.     

Distribution and quantification of pyridinium cross-links of collagen within the different maturational zones of the chick growth plate

The levels of alanine, aspartate and glutamine transaminase increase considerably in some diseases. We measured the activity of these enzymes and of the transaminase of 3-hydroxykynurenine, an aminoacid, which acts as a UV lens filter. Alanine and glutamine transaminases (carboxypeptidase) were not detected in normal and cataractous human lenses, and aspartate transaminase was found only in the cortex of normal lenses. 3-hydroxykynurenine transaminase was not found in lenses from persons below thirty years of age, but was found in lenses at about fifty years of age, and in cataractous lenses. Transamination of 3-hydroxykynurenine leads to the formation of xanthurenic acid and its derivatives. These substances appear to be responsible for the increase of lens fluorescence during cataract development.     

Capsular tension ring in a patient with Weill-Marchesani syndrome

Weill-Marchesani patients with cataractous lenses may presnet a surgical challenge in the presence of zonular weakness and microsherophakia. A 52-year-old Weill-Marchesani patient developed zonular dehiscence during capsule contraction after cataract extraction in her right eye. Use of a poly(methyl methacrylate) capsular tension ring in the second eye facilitated lens removal and intraocular lens placement. Postoperative results suggest the capsular tension ring provides long-term zonular stabilization by maintaining an internal force against the capsule.     

Lens epithelium: a primary target of UVB irradiation

Cultured rabbit lenses and cultured rabbit lens epithelial cells were irradiated with UV to correlate morphological changes in the epithelium with physiological changes in the whole lens during the development of UV-induced cataract. Two UV spectral ranges were utilized; one spanned 290 to 340 nm and was designated near-UV, the other was a narrower, pure UVB region: 303 to 313 nm, designated UVB. Irradiation with either spectrum of the anterior surface of whole lenses caused opacification and a dose-dependent loss of ion homeostasis as measured by Na+ and Ca2+ concentrations in whole lenses. It was determined that cation pump activity, assessed by 86Rb uptake, continued to decline steadily during culture after UV irradiation. Whole mount preparations of the epithelial cell layer of UVB-irradiated lenses revealed morphological changes within 2 hr of irradiation and cell death after 20 hr. Following posterior irradiation of whole lenses, the epithelial cells remained viable and lenses remained transparent during 3 days of culture, presumably because UV photons did not reach the epithelium. Absorption of UV photons by posterior fiber cell membranes and proteins did not cause opacification. To learn more about the epithelial damage, cultured rabbit lens epithelial cells were irradiated, UVB treatment retarded growth over a 7-day period in cultured cells. The surviving cells at day 7 were abnormal in appearance and the potassium concentration was approximately 50% less than controls, a finding which may explain the previously reported reduction in protein synthesis by UVB irradiation. Collectively, the data suggest that UV cataract is initiated by damage to the epithelium, including a change in membrane permeability leading to loss of ion homeostasis in the lens.     

The effect of epidermal growth factor on the growth potential of epithelial cells from human lens

The growth promoting effect of epidermal growth factor (EGF) was studied in cultures of epithelial cells from human normal and cataractous lenses. The growth potential of lens epithelial cells was measured by MTT assay. The concentration of EGF in culture medium were classified into 7 groups (0 ng/ml-10(3) ng/ml). When the concentration of EGF was 1 ng/ml, EGF induced the highest increase of growth potential epithelial cells compared with an EGF-free group.     

Role of small GTP-binding proteins in lovastatin-induced cataracts

PURPOSE: To investigate the biochemical mechanisms involved in the cataract induced by lovastatin, a commonly used cholesterol-lowering agent. METHODS: The effects of lovastatin on lens transparency and on lens epithelial cell proliferation and structure have been investigated using organ-cultured rat lenses and cultured epithelial cells from human and rabbit lenses, respectively. Lens histologic and morphologic changes were recorded microscopically. Small GTP-binding protein profiles were determined by [alpha-32P] GTP overlay assays. RESULTS: Rat lenses organ cultured for 7 days with lovastatin, a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, developed frank subcapsular opacity. Lens epithelial cells (both human and rabbit) demonstrated extensive morphologic changes and inhibition of proliferation when treated with lovastatin. Histologic sections of lovastatin-treated lenses showed partial to complete degeneration of the central epithelium, distortion of elongating epithelial cells, and extensive vacuole formation in the equatorial regions of the cortex. Supplementation of the medium with DL-mevalonic acid (a precursor of isoprenoids whose synthesis is inhibited by lovastatin) prevented the lovastatin-induced changes in whole lenses or in lens epithelial cell cultures, whereas supplementation with cholesterol had no such effect. GTP-binding proteins accumulated in the soluble fractions of lovastatin-treated lens epithelial cells. This was consistent with a blockade in isoprenylation preventing normal association with membranes. CONCLUSIONS: The findings suggest that impairment of the function of small GTP-binding proteins, due to a lovastatin-induced blockade in their isoprenylation, affects lens cell structure and proliferation in tissue culture and induces lens opacity in organ culture. These findings are consistent with the proposed roles of small GTP-binding proteins as molecular switches that regulate fundamental cellular processes, including growth, differentiation, and maintenance of cell structure.     

Effects of transforming growth factor-beta (isoforms 1-3) on amyloid-beta deposition, inflammation, and cell targeting in organotypic hippocampal slice cultures

The transforming growth factor-beta (TGF-beta) family consists of three isoforms and is part of a larger family of cytokines regulating differentiation, development, and tissue repair. Previous work from our laboratory has shown that TGF-beta1 can increase amyloid-beta protein (Abeta) immunoreactive (Abetair) plaque-like deposits in rat brain. The aim of the current study was to evaluate all three isoforms of TGF-beta for their ability to affect the deposition and neurotoxicity of Abeta in an organotypic, hippocampal slice culture model of Abeta deposition. Slice cultures were treated with Abeta either with or without one of the TGF-beta isoforms. All three isoforms can increase Abeta accumulation (over Abeta treatment alone) within the slice culture, as determined by ELISA. However, there are striking differences in the pattern of Abetair among the three isoforms of TGF-beta. Isoforms 1 and 3 produced a cellular pattern of Abeta staining that colocalizes with GS lectin staining (microglia). TGF-beta2 produces dramatic Abeta staining of pyramidal neurons in layers CA1-CA2. In addition to cellular Abeta staining, plaque-like deposits are increased by all of the TGF-betas. Although no gross toxicity was observed, morphological neurodegenerative changes were seen in the CA1 region when the slices were treated with Abeta plus TGF-beta2. Our results demonstrate important functional differences among the TGF-beta isoforms in their ability to alter the cellular distribution and degradation of Abeta. These changes may be relevant to the pathology of Alzheimer's disease (AD).     

Expression of transforming growth factor-beta 1 messenger ribonucleic acid and the modulation of deoxyribonucleic acid synthesis by transforming growth factor-beta 1 in human endometrial cells

OBJECTIVE: The purpose of this study was (1) to evaluate the potential sites of transforming growth factor-beta 1 synthesis in human endometrium by analyzing separated endometrial glands and stromal cells for transforming growth factor-beta 1 messenger ribonucleic acid by Northern analysis of total ribonucleic acid and (2) to investigate the effects of transforming growth factor-beta 1 on deoxyribonucleic acid synthesis in endometrial epithelial and stromal cells in culture. STUDY DESIGN: Endometrial glands and stroma from proliferative and secretory endometrium were isolated after collagenase treatment of endometrial tissue minces and were analyzed for transforming growth factor-beta 1 messenger ribonucleic acid by Northern analysis. We studied the effects of estradiol-17 beta and transforming growth factor-beta 1 on deoxyribonucleic acid synthesis in endometrial epithelium and transforming growth factor-beta 1 on stromal cells in culture by evaluating tritiated thymidine incorporation into trichloroacetic acid-precipitable material. RESULTS: Transforming growth factor-beta 1 messenger ribonucleic acid was detected for Northern analysis in separated endometrial stromal cells in levels that were greatest during the secretory phase and in greater levels than in epithelial cells from that same tissue. Transforming growth factor-beta 1 messenger ribonucleic acid in glandular epithelium in culture was not increased to detectable levels by treatment with transforming growth factor-beta 1. Deoxyribonucleic acid synthesis in endometrial glandular epithelium was inhibited by transforming growth factor-beta 1, but transforming growth factor-beta 1 stimulated deoxyribonucleic acid synthesis in endometrial stromal cells in culture. After treatment for 5 days with estradiol-17 beta (10(-8) mol/L), deoxyribonucleic acid synthesis in endometrial glands in culture was decreased by 40%. Transforming growth factor-beta 1 (1 ng/ml) did not alter this effect of estradiol-17 beta on deoxyribonucleic acid synthesis. CONCLUSIONS: Transforming growth factor-beta 1 acts to decrease deoxyribonucleic acid synthesis in epithelial cells and to increase it in stromal cells isolated from human endometrium and maintained in monolayer culture. Transforming growth factor-beta 1, potentially of stromal cell origin, could participate in the regulation of endometrial cell proliferation and differentiation in vivo.     

A re-evaluation of the concept of homophilic interactions?

In this histochemical study on the ocular lens, the authors test for the presence of various sorts of the (unsaturated) polycyclic aromatic hydrocarbon (PAH) in 66, both clear and cataractous human lenses. A statistically significant greater amount of PAH is found in the cataractous lenses studied (p < 0.01), with two kind of PAH, phenanthrene and 1,2-benzoanthracene, appearing exclusively in lenses with cataracts. The authors put forward a hypothesis on the cataractogenic effect of PAH on the basis of its interference with lens metabolism and the subsequent production and release of free-radicals.     

Confocal microscopy of human lens membranes in aged normal and nuclear cataracts

PURPOSE: To visualize the structure and determine the continuity of lipid membranes in lens fiber cells (LFCs) from human aged normal and cataractous lenses. METHODS: Thick sections from human nuclear cataracts and aged normal lenses were stained with the lipophilic probe DiI, and then analyzed by confocal microscopy. Staining patterns of membranes were observed in individual optical sections or three-dimensional projections of z-series taken in longitudinal section and cross-section of LFCs from different regions within the lens nucleus. RESULTS: DiI bound to and delineated the plasma membrane of LFCs from all regions of the lens nucleus. Three-dimensional projections of z-series from aged normal and cataractous lenses suggested that some of the stained lipid membranes were not continuous with LFC plasma membrane of cataractous lenses. CONCLUSIONS: The results obtained using these methods demonstrated that lipid membranes, discontinuous with the plasma membrane of LFCs, were indicative of a novel process occurring predominately in cataractous human lenses.