Involvement of interleukin 18 in cataract development in hereditary cataract UPL rats

Our previous studies have demonstrated that lens epithelial damage by excessive nitric oxide causes an elevation in lens opacification in UPL rats, and it has been reported that interferon-gamma production in lens epithelial cells is involved in cataract development. In this study, we investigated the involvement of interleukin (IL)-18, which leads to interferon-gamma, in UPL rat lenses. The opacification of UPL rat lenses starts at 39 days of age. The gene expression levels causing IL-18 activation (IL-18, IL-18 receptor and caspase-1) are increased at 32 days of age, and the expression of mature IL-18 protein in the UPL rat lenses also increases with ageing. On the other hand, the interferon-gamma levels in UPL rat lenses are increased, and the increase in interferon-gamma levels in UPL rat lenses reaches a maximum at 39 days of age. Mature IL-18 expression and interferon-gamma production are achieved prior to the onset of lens opacification. In conclusion, the expression levels of IL-18 in the lenses of UPL rats are increased with aging. In addition, interferon-gamma levels in the lenses of UPL rats are also increased. It is possible that interferon-gamma generated by the activated IL-18 may induce cataract development in UPL rats.     

Mitochondria induce oxidative stress, generation of reactive oxygen species and redox state unbalance of the eye lens leading to human cataract formation: disruption of redox lens organization by phospholipid hydroperoxides as a common basis for cataract disease

The aging eye appears to be at considerable risk from oxidative stress. Lipid peroxidation (LPO) is one of the mechanisms of cataractogenesis, initiated by enhanced promotion of oxygen free radicals in the eye fluids and tissues and impaired enzymatic and non-enzymatic antioxidant defenses of the crystalline lens. The present study proposes that mitochondria are one of the major sources of reactive oxygen species (ROS) in mammalian and human lens epithelial cells and that therapies that protect mitochondria in lens epithelial cells from damage and reduce damaging ROS generation may potentially ameliorate the effects of free radical-induced oxidation that occur in aging ocular tissues and in human cataract diseases. It has been found that rather than complete removal of oxidants by the high levels of protective enzyme activities such as superoxide dismutase (SOD), catalase, lipid peroxidases in transparent lenses, the lens conversely, possess a balance between peroxidants and antioxidants in a way that normal lens tends to generate oxidants diffusing from lenticular tissues, shifting the redox status of the lens to become more oxidizing during both morphogenesis and aging. Release of the oxidants (O(2)(-)·, H(2)O(2) , OH·, and lipid hydroperoxides) by the intact lenses in the absence of respiratory inhibitors indicates that these metabolites are normal physiological products inversely related to the lens life-span potential (maturity of cataract) generated through the metal-ion catalyzed redox-coupled pro-oxidant activation of the lens reductants (ascorbic acid, glutathione). The membrane-bound phospholipid (PL) hydroperoxides escape detoxification by the lens enzymatic reduction. The lens cells containing these species would be vulnerable to peroxidative attack which trigger the PL hydroperoxide-dependent chain propagation of LPO and other damages in membrane (lipid and protein alterations). The increased concentrations of primary LPO products (diene conjugates, lipid hydroperoxides) and end fluorescent LPO products were detected in the lipid moiety of the aqueous humor samples obtained from patients with cataract as compared to normal donors. Since LPO is clinically important in many of the pathological effects and aging, new therapeutic modalities, such as patented N-acetylcarnosine prodrug lubricant eye drops, should treat the incessant infliction of damage to the lens cells and biomolecules by reactive lipid peroxides and oxygen species and "refashion" the affected lens membranes in the lack of important metabolic detoxification of PL peroxides. Combined in ophthalmic formulations with N-acetylcarnosine, mitochondria-targeted antioxidants are promising to become investigated as a potential tool for treating a number of ROS-related ocular diseases, including human cataracts.     

Diverse roles of Eph/ephrin signaling in the mouse lens

Recent genetic studies show that the Eph/ephrin bidirectional signaling pathway is associated with both congenital and age-related cataracts in mice and humans. We have investigated the molecular mechanisms of cataractogenesis and the roles of ephrin-A5 and EphA2 in the lens. Ephrin-A5 knockout (-/-) mice often display anterior polar cataracts while EphA2(-/-) lenses show very mild cortical or nuclear cataracts at weaning age. The anterior polar cataract of ephrin-A5(-/-) lenses is correlated with multilayers of aberrant cells that express alpha smooth muscle actin, a marker for mesenchymal cells. Only select fiber cells are altered in ephrin-A5(-/-) lenses. Moreover, the disruption of membrane-associated β-catenin and E-cadherin junctions is observed in ephrin-A5(-/-) lens central epithelial cells. In contrast, EphA2(-/-) lenses display normal monolayer epithelium while disorganization is apparent in all lens fiber cells. Immunostaining of ephrin-A5 proteins, highly expressed in lens epithelial cells, were not colocalized with EphA2 proteins, mainly expressed in lens fiber cells. Besides the previously reported function of ephrin-A5 in lens fiber cells, this work suggests that ephrin-A5 regulates β-catenin signaling and E-cadherin to prevent lens anterior epithelial cells from undergoing the epithelial-to-mesenchymal transition while EphA2 is essential for controlling the organization of lens fiber cells through an unknown mechanism. Ephrin-A5 and EphA2 likely interacting with other members of Eph/ephrin family to play diverse functions in lens epithelial cells and/or fiber cells.     

Cellular changes in cultured lenses.

Observations were made on the frog lens epithelium after culture of the entire lens or of capsular explants. General deviations from normal lens structure as well as specific changes in two media were studied. DNA synthesis and mitosis were induced in the central epithelial cells. Disruption of the orderly, single, epithelial layer that is characteristic of normal lenses was accompanied by the appearance of multilayered plaques of epithelial cells and invasion of vacuolated regions of the lens fibers by epithelial cells. Cells that are fibroblast-like in appearance were observed in regions of the capsule depleted of cells and at the free edges of epithelial sheets in cell culture. Epithelial cells were surrounded by capsule-like material even situated in the lens interior. Nuclie derived from central epithelial cells of lenses cultured in L-15 medium and medium 199 had served as donors in previous nuclear transfer experiments in this laboratory. In our current observation of L-15-cultured lenses, cells were sparsely distributed on the capsule and nuclei were abnormally shaped; in 199-cultured lenses, cells were more densely distributed and nuclei resembled those of normal lenses. Medium 199 without serum could better maintain normal lens structure than L-15 medium without serum. In addition, the percentage of epithelial explants demonstrating cellular outgrowth was greater in medium 199. The differences in cellular behavior were shown not to be the result of different sugars, pH, or the presence of CO2. The nuclear transfer results may reflect the structural changes in the epithelium after lens culture in the two media.     

Cytoskeletal and contractile structures in bovine lens cell differentiation

Bovine lenses from animals of different ages were separated into two epithelial sections, a cortical region and the lens nucleus. Both the 10000 g supernatant fraction and pellet of these sections were analysed by electrophoresis in SDS-containing polyacrylamide gels. When comparing total protein patterns of the cytoskeletal preparations from the different parts of lenses of different ages a decrease in the amount of vimentin, the protein subunit of lens intermediate-sized filaments (IF), was observed upon lens cell differentiation and aging. Amounts of monomeric (G) and filamentous (F) actin in the different stages of lens cell differentiation were quantitated using the DNase I inhibition technique. A significant increase in the relative amount of F-actin was observed upon fibre cell formation. A slight, but significant increase in the total amount of actin relative to the total amount of cellular protein was observed when passing from the central part of the lens epithelium to the epithelial cells in the elongation zone. In the fibre cells the amount of total actin decreased from cortex to nucleus. A possible function of microfilament-assembly in the process of lens cell differentiation is suggested.     

Cellular changes in cultured lenses

Summary Observations were made on the frog lens epithelium after culture of the entire lens or of capsular explants. General deviations from normal lens structure as well as specific changes in two media were studied. DNA synthesis and mitosis were induced in the central epithelial cells. Disruption of the orderly, single, epithelial layer that is characteristic of normal lenses was accompanied by the appearance of multilayered plaques of epithelial cells and invasion of vacuolated regions of the lens fibers by epithelial cells. Cells that are fibroblast-like in appearance were observed in regions of the capsule depleted of cells and at the free edges of epithelial sheets in cell culture. Epithelial cells were surrounded by capsule-like material even when situated in the lens interior. Nuclei derived from central epithelial cells of lenses cultured in L-15 medium and medium 199 had served as donors in previous nuclear transfer experiments in this laboratory. In our current observation of L-15-cultured lenses, cells were sparsely distributed on the capsule and nuclei were abnormally shaped; in 199-cultured lenses, cells were more densely distributed and nuclei resembled those of normal lenses. Medium 199 without serum could better maintain normal lens structure than L-15 medium without serum. In addition, the percentage of epithelial explants demonstrating cellular outgrowth was greater in medium 199. The differences in cellular behavior were shown not to be the result of different sugars, pH, or the presence of CO₂. The nuclear transfer results may reflect the structural changes in the epithelium after lens culture in the two media. This work was supported by grants 2RO1 EY 00555-06 and 5SO1 RR 05510-10 from the National Institutes of Health.     

Ultrastructure of traumatic cataractogenesis in the frog: a comparison with mouse and human lens.

Normal and needle-punctured lenses of Rana pipiens were examined with the electron microscope in order to characterize the sequence of ultrastructural changes that follow the injury over a 5-month period. Results were compared with those obtained previously in experimentally injured mouse and accidentally injured human lenses. The normal adult frog lens was found to have a morphology similar to that of mammalian lenses. As in the human, frog lens epithelial cells contained scattered microfilaments and were connected by desmosomes and gap junctions. They differed from mouse cells, which had been shown to lack desmosomes and to have microfilaments organized into dense bundles. These differences are postulated to be related to the degree of accommodative deformation of the lens displayed by these species. After injury, cellular debris and fibrin, accumulated in the wound, were phagocytized by extrinsic cells derived from the blood and ocular tissues. Leucocytes, pigmented cells and fibroblasts remained in the wound for eight weeks, along with epithelial cells which proliferated and migrated from the wound margins.Epithelial cells showed an increase in those organelles associated with protein synthesis and transport, and in microfilaments. In cataractous lenses, epithelial cells showed changes in matrix, and lens fibers became organized into smaller, denser compressed units. At five months, considerable healing had taken place, but localized opacities persisted in many frog lenses.     

Studies on the possible role of vitreous humor on protein synthesis and morphology of organ cultured adult rabbit lens. II. Epithelial cells.

The protein synthetic activities of epithelial cells of lenses organ-cultured without adhered vitreous humor manifest significant increase compared to the epithelial cells of lenses incubated with attached vitreous humor. This effect is not due to trauma of vitreous removal, as the addition of freeze-dried vitreous humor to the culture medium of lenses without attached vitreous humor could inhibit protein synthesis. However, the protein synthesis inhibitor in the vitreous humor has no visible effect on the lens morphology. It was also found that the factor from vitreous humor has no effect on mRNA production or cell-free protein synthesis. Thus, it seems that the effect on protein synthesis must be mediated via some other pathway.     

Aging and cellular maturation cause changes in ubiquitin-eye lens protein conjugates

The eye lens is a useful tissue for studying phenomena related to aging since it can be separated into differentially aged or matured zones. This work establishes correlations between ubiquitin-lens protein conjugating capabilities and age, as well as the stage of maturation of bovine lens tissue. When exogenous 125I-ubiquitin was combined with supernatants of epithelial (least mature), cortex, and core (most mature) tissue, ATP-dependent conjugation of 125I-ubiquitin to lens proteins was most effective with the epithelial tissue preparation. Conjugate formation was greatest when lenses were obtained from young animals. Supernatants from cultured bovine lens epithelial (BLE) cells conjugated more 125I-ubiquitin to lens proteins than any tissue preparation. In all cases the predominant conjugates formed in these cell-free assays were of high molecular mass, although conjugates with masses in the 25-70 kDa range were also observed. Lens tissue and cultured BLE cell preparations were also probed with antibodies to ubiquitin to detect in vivo ubiquitin-lens protein conjugates. There was more free ubiquitin and ubiquitin conjugates in tissue from young as compared with older lenses. The greatest levels of conjugates were observed in cultured BLE cells. Specificity in the ubiquitination system is indicated since some of the conjugates formed in vivo appear identical to those formed in the cell-free assays and in reticulocytes using exogenous 125I-ubiquitin. Upon development and maturation of lens tissue (i.e., core as opposed to epithelium), there is accumulation of lower molecular mass conjugates.     

Heat shock proteins of adult and embryonic human ocular lenses.

We investigated the presence and distribution of heat shock proteins, HSP-70 [Horwitz, J. 1992. Proc Natl Acad Sci 89:10449-10453], HSP-40, HSc-70, HSP-27, and alphabeta-crystallin in different regions of adult and fetal human lenses and in aging human lens epithelial cells. This study was undertaken because heat shock proteins may play an important role in the maintenance of the supramolecular organization of the lens proteins. Human adult and fetal lenses were dissected to separate the epithelium, superficial cortex, intermediate cortex, and nucleus. The water soluble and insoluble protein fractions were separated by SDS-PAGE, and transferred to nitrocellulose paper. Specific antibodies were used to identify the presence of heat shock proteins in distinct regions of the lens. HSP-70 [Horwitz, 1992], HSP-40, and HSc-70 immunoreactivity was mainly detected in the epithelium and superficial cortical fiber cells of the adult human lens. The small heat shock proteins, HSP-27 and alphabeta-crystallin were found in all regions of the lens. Fetal human lenses showed immunoreactivity to all heat shock proteins. An aging study revealed a decrease in heat shock protein levels, except for HSP-27. The presence of HSP-70 [Horwitz, 1992], HSP-40, and HSc-70 in the epithelium and superficial cortical fiber cells imply a regional cell specific function, whereas the decrease of heat shock protein with age could be responsible for the loss of optimal protein organization, and the eventual appearance of age-related cataract.     

Functions of crystallins in and out of lens: Roles in elongated and post-mitotic cells

The vertebrate lens evolved to collect light and focus it onto the retina. In development, the lens grows through massive elongation of epithelial cells possibly recapitulating the evolutionary origins of the lens. The refractive index of the lens is largely dependent on high concentrations of soluble proteins called crystallins. All vertebrate lenses share a common set of crystallins from two superfamilies (although other lineage specific crystallins exist). The α-crystallins are small heat shock proteins while the β- and γ-crystallins belong to a superfamily that contains structural proteins of uncertain function. The crystallins are expressed at very high levels in lens but are also found at lower levels in other cells, particularly in retina and brain. All these proteins have plausible connections to maintenance of cytoplasmic order and chaperoning of the complex molecular machines involved in the architecture and function of cells, particularly elongated and post-mitotic cells. They may represent a suite of proteins that help maintain homeostasis in such cells that are at risk from stress or from the accumulated insults of aging.     

Growth regulation of the mammalian ocular lens by vitreous humor.

Experiments were performed in our laboratory to study the effects of a mammalian 8 kD vitreous humor (VH) factor on the DNA synthesis and mitosis of the epithelial cells of organ cultured rabbit lens. The 8 kD polypeptide factor was purified from mature rabbit vitreous humor by liquid chromatography. Proliferative activities of the epithelial cells of organ cultured lenses were stimulated by 3% rabbit serum. The data from our experiments depicted that the 8 kD VH factor effectively inhibits DNA synthesis and mitosis by the epithelial cells of the organ cultured lens. Our experiments also showed that this 8 kD VH factor can maintain its growth inhibitory activity even when heated for 3 min at 95 degrees C. The growth inhibitory effect of the 8 kD VH factor was dose dependent. Using iodinated vitreal proteins it was demonstrated that the VH proteins are able to enter or bind to lens epithelial cells. The growth inhibitory effect of the 8 kD VH factor was also tested on tissue cultured lens epithelial cells. These experiments showed that the 8 kD VH factor has no growth inhibitory effect on the tissue cultured lens epithelial cells. This experiment has been repeated many times using different concentrations of the factor. These observations suggest that the 8 kD VH factor may have receptors in the lens capsular material (extracellular matrix) and the factor-receptor binding is essential for the growth inhibitory effect.     

PolyADP-ribosyl polymerase (adenosine diphosphate ribosyl transferase) in bovine crystalline lens; modulation of its activity during aging

PolyADP-ribose polymerase activities were measured in bovine lens. Activities similar to those in brain were found in the epithelial cells; none activity was detected in the fiber cells. During aging ADP-ribosyl transferase activity of epithelial cells raised, the number of polyADP-ribose chains increased while the average chain length decreased. A possible correlation between ADP-ribosylation and cell proliferation or repair is discussed.     

GROWTH OF LENS AND OCULAR ENVIRONMENT: ROLE OF NEURAL RETINA IN THE GROWTH OF MOUSE LENS AS REVEALED BY AN IMPLANTATION EXPERIMENT

The lens of 6-day-old normal mouse was implanted into the lentectomized eye of adult mouse to examine the effect of retina upon the growth of the implanted lens in vivo. The implanted lens grew normally and its transparency was kept for more than 5 months after implantation. The connection between the implanted lens and the ciliary part of the recipient iris was well established with the regeneration of zonular fibers from the recipient. In young lenses implanted reversely into adult eyes, the epithelial cells facing the retina elongated and a new epithelium was formed on the corneal side of the lens within 5 days. Young lenses implanted either in normal or reverse orientation into eyes from which the retina was previously removed did not grow. The cells of the original lens epithelium of these lenses were randomly accumulated beneath the posterior lens capsule, while the anterior portion of the implanted lenses became an epithelial structure without cell elongation. These results suggest that the growth of the implanted lens may be dependent on the retina of the adult eye.     

Unfolded Protein Response (UPR) is activated during normal lens development

The lens of the eye is a transparent structure responsible for focusing light onto the retina. It is composed of two morphologically different cell types, epithelial cells found on the anterior surface and the fiber cells that are continuously formed by the differentiation of epithelial cells at the lens equator. The differentiation of an epithelial precursor cell into a fiber cell is associated with a dramatic increase in membrane protein synthesis. How the terminally differentiating fiber cells cope with the increased demand on the endoplasmic reticulum for this membrane protein synthesis is not known. In the present study, we have found evidence of Unfolded Protein Response (UPR) activation during normal lens development and differentiation in the mouse. The ER-resident chaperones, immunoglobulin heavy chain binding protein (BiP) and protein disulfide isomerase (PDI), were expressed at high levels in the newly forming fiber cells of embryonic lenses. These fiber cells also expressed the UPR-associated molecules; XBP1, ATF6, phospho-PERK and ATF4 during embryogenesis. Moreover, spliced XBP1, cleaved ATF6, and phospho-eIF2α were detected in embryonic mouse lenses suggesting that UPR pathways are active in this tissue. These results propose a role for UPR activation in lens fiber cell differentiation during embryogenesis.     

DIFFERENTIATION AND DEDIFFERENTIATION OF RAT LENS EPITHELIAL CELLS IN SHORT- AND LONG-TERM CULTURES

The crystallin synthesis of rat lens cells in cell culture systems was studied in relevance to their terminal differentiation into lens fibers. SDS-gel electrophoresis combined with several immunological techniques showed that γ-crystallin is a fiber-specific lens protein and is not localized in the epithelium of either newborn or adult lenses. When lens epithelial cells of newborn rats were cultured in vitro , α-crystaIlin was detected in many, but not all, of cells cultured for 10 days. Cells with α-crystallin gradually changed their shape into a flattened filmy form and finally differentiated into lentoid bodies. The differentiation of lentoid bodies was also found in cultures of epithelial cells obtained from adult lenses. The molecular constitution of lentoid bodies was the same as that of lens fibers in situ . The differentiation of lentoid bodies occurred successively for 5 months in cultures of lens epithelial cells. Most of the proliferating cells, however, lost α-crystallin during the culture period. Thereafter, they did not show any sign of further differentiation into lens fibers. Four clonal lines were established from these cells. One protein which is specific to the lens epithelium and the neural retina in situ (tentatively named as β_u-crystallin) was maintained in all lines, suggesting that some specific properties of ocular cells remain in the lined cells.     

Corneal Cell Adhesion to Contact Lens Hydrogel Materials Enhanced via Tear Film Protein Deposition

Tear film protein deposition on contact lens hydrogels has been well characterized from the perspective of bacterial adhesion and viability. However, the effect of protein deposition on lens interactions with the corneal epithelium remains largely unexplored. The current study employs a live cell rheometer to quantify human corneal epithelial cell adhesion to soft contact lenses fouled with the tear film protein lysozyme. PureVision balafilcon A and AirOptix lotrafilcon B lenses were soaked for five days in either phosphate buffered saline (PBS), borate buffered saline (BBS), or Sensitive Eyes Plus Saline Solution (Sensitive Eyes), either pure or in the presence of lysozyme. Treated contact lenses were then contacted to a live monolayer of corneal epithelial cells for two hours, after which the contact lens was sheared laterally. The apparent cell monolayer relaxation modulus was then used to quantify the extent of cell adhesion to the contact lens surface. For both lens types, lysozyme increased corneal cell adhesion to the contact lens, with the apparent cell monolayer relaxation modulus increasing up to an order of magnitude in the presence of protein. The magnitude of this increase depended on the identity of the soaking solution: lenses soaked in borate-buffered solutions (BBS, Sensitive Eyes) exhibited a much greater increase in cell attachment upon protein addition than those soaked in PBS. Significantly, all measurements were conducted while subjecting the cells to moderate surface pressures and shear rates, similar to those experienced by corneal cells in vivo.     

Localization of neutral and acidic glycosphingolipids in rat lens

Rat lens was found to contain several neutral and acidic glycosphingolipidsin lens epithelia, cortex and nucleus, and showed developmentalchanges in their content and localization. TLC-immunostainingof gangliosides revealed the enrichment of some ganglio-seriesgangliosides (GM3, GM1, GD3 and GD1b) in lens epithelia andthe presence of GM3 and GD3 in the lens nucleus. Immunohistochemicalstudies confirmed the distribution of GM3 and GM1 in anteriorlens epithelial cells and the cortex, with expression decreasingtoward the lens nucleus. Immunoreaction to GD3 was more intensein the lens nucleus than in epithelial cells. In contrast, theexpression of neolacto-series glycosphingolipids was restrictedto the lens nucleus. In order to investigate the pathologicalchanges of glycosphingolipids in cataract, galactose-inducedcataractous lenses were examined. However, no significant changeswere observed in the content and composition of glycosphingolipids.In addition, Lewis^x epitopes found in human cataractous lenseswere not detected in the cataractous lenses of galactosaemicrats and hereditary cataractous Emory mice. cataract gangliosides glcosphingolipids Lewis^x rat lens     

AGEs in human lens capsule promote the TGFβ2‐mediated EMT of lens epithelial cells: implications for age‐associated fibrosis

Proteins in basement membrane (BM) are long‐lived and accumulate chemical modifications during aging; advanced glycation endproduct (AGE) formation is one such modification. The human lens capsule is a BM secreted by lens epithelial cells. In this study, we have investigated the effect of aging and cataracts on the AGE levels in the human lens capsule and determined their role in the epithelial‐to‐mesenchymal transition (EMT) of lens epithelial cells. EMT occurs during posterior capsule opacification (PCO), also known as secondary cataract formation. We found age‐dependent increases in several AGEs and significantly higher levels in cataractous lens capsules than in normal lens capsules measured by LC‐MS/MS. The TGFβ2‐mediated upregulation of the mRNA levels (by qPCR) of EMT‐associated proteins was significantly enhanced in cells cultured on AGE‐modified BM and human lens capsule compared with those on unmodified proteins. Such responses were also observed for TGFβ1. In the human capsular bag model of PCO, the AGE content of the capsule proteins was correlated with the synthesis of TGFβ2‐mediated α‐smooth muscle actin (αSMA). Taken together, our data imply that AGEs in the lens capsule promote the TGFβ2‐mediated fibrosis of lens epithelial cells during PCO and suggest that AGEs in BMs could have a broader role in aging and diabetes‐associated fibrosis.