Canine Lens Epithelial Cells Research Articles

Evaluation of advanced glycation end-products in diabetic and inherited canine cataracts

The receptor for advanced glycation end-products (RAGE) increases in the human cataract and should correlate with increased DNA damage and proliferation of lens epithelial cells (LECs). The purpose of this study was to measure and immunolocalize RAGE in normal and cataractous canine LECs, and to determine whether there was a correlation between RAGE and DNA damage (gadd45), cell-cycle regulation (p21), and LEC proliferation (proliferating cell nuclear antigen, PCNA). Thirty-two anterior lens capsules from 22 dogs that underwent cataract surgery and 10 lenses from dogs with normal eyes were evaluated. Eleven of the cataractous lenses were from diabetic patients (n=16), and eleven were from patients with inherited cataracts (n=16). Standard immunohistochemical staining was performed using antibodies against RAGE, gadd45, p21, PCNA, alpha-smooth muscle actin, and TGF-beta. Immunostaining intensity for each antibody was given a score of 0-4+. Standard Western blot analysis on normal and cataractous lens capsules was performed using the same antibodies as in the immunohistochemical staining. Comparisons were also made based on age and sex. Real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed for RAGE. There was an increase in RAGE expression with age in normal LECs, but no significant difference was seen when normal adult LECs were compared to cataractous LECs. The stage of the cataract and the presence of LIU were not associated with a significant increase in RAGE expression. There was no age-dependent difference in the normal lenses for gadd45, p21, or PCNA. Significant up-regulation of p21 (P < 0.05) and PCNA (P < 0.05) was seen in diabetic cataracts compared to inherited cataracts. RAGE and PCNA expression did not increase with cataractogenesis, possibly due to overexpression associated with normal aging and constant exposure to oxidative stress from sunlight-related ultraviolet irradiation, respectively. However, p21 and PCNA increased in diabetic cataractogenesis suggesting cell cycle and proliferation dysregulation. This may be related to the rapid onset in this type of cataract compared with the more chronic and slower-to-develop inherited cataracts.

Differential Degradation of Ferritin H- and L-Chains: Accumulation of L-Chain-Rich Ferritin in Lens Epithelial Cells

The storage of iron by ferritin is determined by tissue-specific composition of its 24 subunits, which are designated as either heavy (H) or light (L). For a better understanding of how lens epithelial cells regulate their ferritin subunit makeup, the degradation pattern of each subunit type was analyzed. In addition, age-related changes in ferritin concentration and subunit makeup were determined. Ferritin turnover in primary cultures of canine lens epithelial cells was determined by metabolic labeling with [(35)S]-methionine. Transient transfection with vectors containing coding sequences for either H- or L-chains were used to modify ferritin subunit makeup. Ferritin concentration was measured by ELISA. Immunodetection and fluorescence immunocytochemistry were used to study age-related changes in ferritin chain concentration. Inhibition of the proteasomal protein degradation pathway by clastolactacystin-beta-lactone had no effect on ferritin degradation, whereas inhibition of lysosomal degradation markedly increased ferritin levels, confirming that this system is involved in ferritin turnover. H-chain ferritin degraded at a faster rate than the L-chain. L-chain-rich ferritin in L-chain-transfected cells formed inclusion bodies that were localized to the cytosol. Similar inclusion bodies were found in older lens cells kept in cell culture for more than 8 days. Steady degradation of H-chain ferritin contributed to the maintenance of a constant level of this chain within the lens epithelial cells. In contrast, slower turnover of the L-chain resulted in accumulation of L-chain-enriched ferritin associated with cytoplasmic inclusion bodies. These L-chain-containing inclusion bodies were found in the cytosol of cells overexpressing L-ferritin chain and in nontransfected cells maintained in culture for 8 to 35 days. Overexpression of the L-chain has been associated with the formation of premature cataracts in humans with hereditary hyperferritinemia cataract syndrome. The formation of inclusion bodies in older lens epithelial cells, as demonstrated in the current investigation, is intriguing and could point to possible involvement of cytoplasmic L-chain-enriched ferritin aggregates in the formation of age-related cataract.

Identification of a Mechanism by Which Lens Epithelial Cells Limit Accumulation of Overexpressed Ferritin H-chain

The primary cultures of canine lens epithelial cells were transiently transfected with cDNAs for dog ferritin H- or L-chains in order to study differential expression of these chains. By using chain-specific antibodies, we determined that at 48 h after transfection overexpression of L-chain was much higher (9-fold over control) than that of H-chain (1.7-fold). We discovered that differentially transfected cells secrete overexpressed chains as homopolymeric ferritin into the media. Forty-eight hours after transfection accumulation of H-ferritin in the media was much higher (3-fold) than that of L-ferritin. This resulted in lowering of the concentration of H-chain in the cytosol. Co-transfection of cells with both H- and L-chain cDNAs increased the intracellular levels of H-chain and eliminated secretion of H-ferritin to the media. We concluded that lens epithelial cells differentially regulate concentration of both ferritin chains in the cytosol. The overexpressed L-chain accumulated in the cytosol as predominantly homopolymeric L-ferritin. This is in contrast to H-chain, which is removed to the media unless there is an L-chain available to form heteropolymeric ferritin. These data indicate that the inability of cells to more strictly control cytosolic levels of L-chain may augment its accumulation in lenses of humans with hereditary hyperferritinemia cataract syndrome, which is caused by overexpression of L-chain due to mutation in the regulatory element in the untranslated region of the mRNA of the chain.

The Effect of UVB Irradiation on Ferritin Subunit Synthesis, Ferritin Assembly and Fe Metabolism in Cultured Canine Lens Epithelial Cells ¶

Ferritin is a multimeric protein consisting of heavy and light chains assembled in different tissue-specific ratios, which can protect cells from oxidative stress by storing reactive iron (Fe). Because the lens is constantly exposed to UV irradiation, we studied its effects on ferritin synthesis and Fe metabolism in cultured lens epithelial cells with and without ascorbic acid (Asc). UVB caused a large increase in accumulation of newly synthesized ferritin chains; this increase was additive to that induced by Asc. In contrast to the Asc-induced increase in Fe storage, Fe storage in ferritin was unaltered by UVB. Although UVB increased accumulation of newly synthesized ferritin chains, total ferritin levels were unaltered. In contrast, Asc, which induced a quantitatively similar increase in accumulation of newly synthesized ferritin chains, doubled the total amount of ferritin. Because UVB did not change Fe storage in ferritin or the size of the labile Fe pool, it was hypothesized and then determined that these newly synthesized chains did not assemble into functional holoferritin. Numerous studies detail the effects of various treatments on de novo ferritin synthesis; however, this study provides a cautionary note regarding the conclusions of such studies in the absence of data indicating assembly of functional ferritin molecules.

The Effect of UVB Irradiation on Ferritin Subunit Synthesis, Ferritin Assembly and Fe Metabolism in Cultured Canine Lens Epithelial Cells¶

The Effect of UVB Irradiation on Ferritin Subunit Synthesis, Ferritin Assembly and Fe Metabolism in Cultured Canine Lens Epithelial Cells¶

Telomerase Activity in Lens Epithelial Cells of Normal and Cataractous Lenses

Telomerase is a ribonucleoprotein responsible for maintaining telomere length, preventing chromosomal degradation and recombination, and repairing DNA strand breaks. These activities are believed to be important in preventing cell senescence. Telomerase activity is normally found in germinal, neoplastic and stem cells, but not any ocular tissue studied to date. The epithelium of the crystalline lens is comprised of a population of cells with diverse mitotic potential including the germinative epithelium which contains cells with the potential for unlimited replicative capacity, equatorial cells which terminally differentiate into lens fibers, and the central epithelium which are considered to be quiescent and nonreplicative under normal circumstances.We speculated that the germinative region of lens epithelial cells might have telomerase activity, and that dysregulation of its activity might be associated with cataractogenesis. We investigated these hypotheses in lens capsule specimens from normal and cataractous dogs and from cultures of canine lens epithelial cells using standard assays for telomerase activity and telomere length. Telomerase activity was found in normal canine lens epithelial cells in the central, germinative and equatorial regions of the anterior lens capsule at equivalent levels. Similar findings were made in feline and murine lens epithelial cells, indicating that the presence of telomerase activity in the lens was not species specific. Lens fiber cells, corneal epithelium and endothelium and nonpigmented ciliary epithelium were telomerase negative. Telomerase activity and telomere lengths were significantly greater in lens epithelia from cataractous lenses when compared with normal lenses.Since telomerase activity is associated with an immortal phenotype, the presence of telomerase activity in the lens epithelial cells may function to prevent conversion to senescence. It was, therefore, difficult to explain why these cells cannot be passaged more than four times in culture. We found that telomerase activity and telomere lengths gradually decreased with increased passages until telomerase activity was no longer present at passage two. Consistent with these findings, there were no senescent cells present on the lens capsule when the lens was initially dissected for culture, but an increasing number of cells were senescent with each passage, correlating well with the loss of telomerase activity.Telomerase activity is likely important in the germinative epithelium to maintain its proliferative potential and prevent cell senescence. Telomerase may function in the quiescent, central lens to maintain telomeres damaged by oxidative stress and ultraviolet light exposure, thereby preventing accelerated loss of these elements which triggers cell senescence. It remains to be determined if the increase in telomerase activity in lens epithelial cells from cataractous lenses is a primary dysregulation that may have a role in the development of the cataract, or is secondary to cataract formation.

The Effect of Ascorbic Acid and Ferric Ammonium Citrate on Iron Uptake and Storage in Lens Epithelial Cells

Ferritin is the major intracellular iron storage protein which has been shown to protect cells against oxidative damage. Recent reports that an inherited abnormality in human ferritin synthesis is associated with early bilateral cataracts underscore the importance of understanding ferritin synthesis and iron storage in lens epithelial cells. We previously demonstrated that ascorbic acid greatly increases de novo synthesis of ferritin in lens epithelial cells. The objectives of the present study were to determine: (1) the effects of ascorbic acid and ferric ammonium citrate on iron uptake by canine lens epithelial cells from iron bound to transferrin and from ferric chloride and (2) the incorporation of this element into ferritin. Iron uptake by lens epithelial cells from59ferric chloride was 20 times higher than from59iron-transferrin and iron deposition into ferritin was 8-fold higher when59ferric chloride was the source. Ascorbic acid had a stimulatory effect on iron uptake from transferrin and on incorporation of this element into ferritin. The ascorbic acid-induced increase of iron uptake required de novo protein synthesis but not specifically de novo ferritin biosynthesis. Although ferritin is not directly involved in iron uptake, the level of ferritin protein could control the pool of intracellular iron. The present results indicate that iron homeostasis in lens epithelial cells is affected mainly by changes in apoferritin synthesis, which is greatly stimulated by ascorbic acid, rather than by altering the rate of protein degradation, which is very slow in these cells under all circumstances.Ferric ammonium citrate activates iron uptake from transferrin in a wide range of cell lines by generation of free radicals. Ferric ammonium citrate also increased iron uptake from Tf in lens epithelial cells. Ferric ammonium citrate treated cells incorporated 5 times more iron and deposited 2 times more iron into ferritin than control cells. Increased incorporation of iron into ferritin was due to ferric ammonium citrate-induced stimulation of de novo ferritin synthesis rather than an increased rate of iron deposition into pre-existing ferritin. Ferric ammonium citrate had a different effect on iron uptake from ferric chloride; total iron uptake was not significantly increased while deposition into ferritin was significantly decreased. These results demonstrate that iron homeostasis in lens epithelial cells is regulated by ascorbic acid and by changes in the rate of de novo ferritin synthesis. In addition, the differences in iron uptake from transferrin and ferric chloride and its subsequent incorporation into ferritin suggests that the mechanisms by which iron is incorporated into ferritin are source dependent.

Iron uptake by cultured lens epithelial cells

Transferrin and Fe concentrations increase in the intraocular fluids in pathological conditions and the lens accumulates Fe during ocular inflammation. Tissues take up Fe from transferrin by two mechanisms, receptor-medicated endocytosis of diferric transferrin and a process occurring at the cell membrane which may be mediated by an oxido-reductase. However, Fe metabolism, transport and storage have not been previously investigated in the lens. This study was designed to characterize the uptake of Fe from transferrin by lens epithelial cells in culture. Primary, secondary and tertiary cultures of canine lens epithelial cells and cultures obtained from cataractous lenses were studied. Uptake of 59Fe from transferrin by these cultured cells was measured. Transferrin receptor populations were determined in receptor-binding assays. There was a distinct relationship between the amount of Fe-transferrin added and the amount of Fe taken up, which was linear for the primary cultures but significantly reduced for the secondary, tertiary and cataract cultures (252 +/- 21, 169 +/- 14, 153 +/- 14 and 96 +/- 2 ng Fe/mg protein, respectively). Transferring receptor expression in lens cell cultures was reduced 10-fold within 2 days of addition of serum to cells grown in low-Fe, serum-free medium for 1 week. The reduction of Fe uptake by the subcultured and cataract cell lines probably reflects a decrease in transferrin receptor expression and in the activity of an alternative pathway for Fe transferrin uptake occurring over time. This reduced Fe uptake may result from long-term exposure to relatively high Fe concentration in the media. A reduction in the expression of the transferrin receptor after incubation with high concentrations of Fe supports this conclusion.

Regulation of Ferritin Levels in Cultured Lens Epithelial Cells

In most eukaryotic cells, synthesis of the iron storage protein, ferritin is regulated by iron levels and redox conditions. Proper iron storage is important to protect against damaging iron-catalysed free radical reactions. Although iron-catalysed reactions are believed to contribute to oxidative damage and cataractogenesis, little is known about iron storage in the lens. In this study, ferritin concentration was measured in cultured canine lens epithelial cells. Baseline ferritin concentration ranged from 76-163 ng (mg protein)-1; cells cultured in low-iron media had significantly lower ferritin levels than cells cultured in iron-supplemented media. Addition of a large excess of iron as hemin resulted in an eight-fold increase in ferritin concentration. The iron chelator, Desferal, significantly decreased ferritin concentration. The reducing agent dithiothreitol decreased the hemin-induced increase in ferritin levels, but not baseline levels. In contrast, ascorbic acid induced a large increase in ferritin content. Other studies have shown that induction of ferritin synthesis can protect against oxidative damage. Regulation of ferritin levels may represent a mechanism by which the lens epithelium is protected from oxidative damage. In vivo, epithelial cells are normally exposed to much lower iron concentrations than the cultured lens epithelial cells in this study. However, in pathological circumstances, the iron content and redox state of the aqueous humor is dramatically altered and may affect the steady state levels of ferritin within the lens. This remains to be determined.

Polyol and vacuole formation in cultured canine lens epithelial cells

Polyol accumulation and myo-inositol depletion were accompanied by extensive vacuole formation in cultured canine lens epithelial cells that were incubated for up to 96 hr in growth medium supplemented with 30 m m d-galactose or 30 m m d-glucose. These changes did not occur in cells incubated in a hypergalactosemic or hyperglycemic medium which also contained an aldose reductase inhibitor (20 μ m sorbinil). In addition, these changes were not observed in lens cells incubated in growth medium supplemented with either 30 m m mannitol, which is known to enter cells only slowly, or in 30 m m l-galactose, which is not a substrate for aldose reductase. The vacuoles were visible at the ultrastructural level after 6 hr of incubation in 30 m m >d-galactose and increased in both number and size with time. These vacuoles had a unique fine structure. They did not result from swelling of mitochondria or other cell organelles. As demonstrated cytochemically, they did not represent either lysosomes or Golgi saccules. The proliferation pattern of cells incubated with 30 m m d-galactose was clearly different from that of control cells, but approached normal when an aldose reductase inhibitor was added to the incubation medium. Together these findings suggest that vacuole formation and altered cell proliferation were caused by polyol accumulation and/or myo-inositol loss, both of which result from aldose reductase activity.

Ferritin: structure, biosynthesis, and role in iron metabolism.

Ferritin: structure, biosynthesis, and role in iron metabolism.