David A. Newsome

Experimental Transplantation of Human Retinal Pigment Epithelial Cells on Collagen Substrates

We studied the use of human retinal pigment epithelial cells cultured on a collagen support as a potential transplantation therapy to replace diseased or damaged retinal pigment epithelium. Using a transvitreal approach, we transplanted human retinal pigment epithelial cells attached to either a sheet of noncross-linked or cross-linked type I collagen into the subretinal space of New Zealand white rabbits, whose eyes lack pigment. Animals were killed after six weeks, and the eyes were fixed for light microscopy. The results demonstrated that, in eyes receiving the noncross-linked collagen support, a layer of pigmented donor retinal pigment epithelium was visible within the subretinal space, with a normal-appearing retina and no evidence of proliferative vitreoretinopathy or graft rejection. We believe this method may be applicable to replace dysfunctional retinal pigment epithelial cells in humans.

Zinc content of human retinal pigment epithelium decreases with age and macular degeneration, but superoxide dismutase activity increases

We previously reported an age-related decline of catalase activity and metallothionein (MT) in human retinal pigment epithelium (RPE) isolated from donor eyes. MT content and catalase activity also were shown to decrease in RPE cells cultured in zinc-deficient medium. We now sought to determine whether zinc content and superoxide dismutase activity change with age and signs of macular degenerative disease in isolated human RPE. Eyes from 57 donors were graded with respect to disease by the presence or absence of visible drusen and macular pigment changes. RPE was collected from macular and peripheral regions. Tissues from 39 donors were separated into soluble and pigment granule fractions and zinc content determined by flame atomic absorption spectroscopy. RPE from 18 separate donors was used for SOD activity analysis. Total RPE zinc was 9% lower in eyes from donors >70 years of age compared to that from donors age 70 and showed a greater decline in the soluble fraction of macular RPE in eyes with signs of macular disease. Total SOD activity increased significantly (P < .05) with donor age. We conclude that zinc content decreased dramatically in the soluble fraction of macular RPE with age and signs of macular degeneration. Total SOD activity correlated negatively with changes in zinc content. It remains to be determined whether the age-related decline in macular zinc is causal with respect to previously observed decreases in cellular MT and catalase.

Influence of zinc on selected cellular functions of cultured human retinal pigment epithelium

Zinc is a necessary micronutrient, usually abundant in human RPE. Our study was undertaken to determine the effects of short-term, zinc deficiency on human retinal pigment epithelium (RPE) using a culture model of fetal human RPE cells. Human fetal RPE cells were isolated and cultured in Coons modified Hams F-12 medium. For zinc depletion studies, cells were cultured for 1 week in Chelex-treated Dulbeccos modified Eagles medium containing low (0.25 microM) or physiologic (11 microM) total zinc concentrations as determined by flame atomic absorption spectroscopy. Protein synthesis was determined by incorporation of 35S-cysteine/methionine and labeled proteins analysed by polyacrylamide gel electrophoresis. Several cell parameters and enzymes were significantly reduced below control when cultured in low zinc: zinc content (40%), proliferation (63%), protein/well (50%), catalase activity (68%), alkaline phosphatase activity (61%), alpha-mannosidase activity (68%), and metallothionein (82%). No statistically significant decline was seen in acid phosphatase activity, superoxide dismutase activity, glutathione peroxidase activity and dexamethasone induction of metallothionein. Zinc repletion (100 microM, 1 h) increased catalase and alpha-mannosidase activities from 32% and 33% of control to 75% and 73%, respectively. Cycloheximide did not inhibit this short-term zinc-induced repletion of catalase or alpha-mannosidase. Protein synthesis in low zinc medium was depressed, but not significantly, as shown by incorporation of radiolabeled 35S-cysteine/methionine into newly synthesized proteins. The effects of zinc deficiency in cultured human RPE are selective. Adequate intracellular zinc was required for maximal activity of some enzymes. The dependence of catalase activity on zinc was not predicted and may help explain the observed decline in catalase activity seen with age in RPE. Our model of zinc deficiency should prove useful in elucidating the complex effects of zinc deficiency and repletion in human RPE.

Zinc uptake by primate retinal pigment epithelium and choroid

We studied zinc uptake by nonhuman primate retinal pigment epithelium (RPE) and choroid, using 65Zn as a probe. With intravenously administered 65ZnCl2, virtually all detectable tracer was lost from the plasma after 20 hours but the pigment epithelium-choroid showed prominent uptake and retention. Plasma concentrations of oral 65ZnO remained high 20 hours after feeding. Uptake and retention of orally administered 65Zn as 65ZnO from the bloodstream by the RPE/choroid was avid in both young and old animals. Excretion in urine and feces was minimal. All pigmented ocular tissues took up and retained 65Zn. A survey of total zinc content of human and nonhuman primate ocular tissues showed that the pigmented tissues had consistently higher concentrations of zinc. Our results demonstrate for the first time direct uptake and retention of zinc from the blood by primate RPE and other ocular tissues.

Effects of extracellular matrix and Bruch's membrane on retinal outer segment phagocytosis by cultured human retinal pigment epithelium

The goal of this study was to determine the effect of extracellular matrix components on the phagocytic function of the retinal pigment epithelial (RPE) cell. Cultured human fetal RPE cells were established in culture and plated on three commercially-prepared substrates: collagen IV, fibronectin and laminin and on three native matrices: bovine corneal endothelial cell matrix (BCEM) denuded bovine Bruchs membrane and denuded human Bruchs membrane. Cultured cells were allowed to become confluent and maintained for an additional two weeks before uptake of fluorescent bovine retinal outer segments (ROS) was measured by flow cytometry. Morphology by phase contrast microscopy and melanization was also determined as measures of differentiation. The results showed that morphology, melanization and ROS uptake by cells on collagen IV, laminin and fibronectin were not different from control cells plated on tissue culture plastic. However, ROS uptake by cells plated on BCEM was significantly less than that of cells cultured on plastic and melanization was greater. ROS uptake by cells plated on both types of Bruchs membrane was also significantly less than control cells. Treatment of cells plated on tissue culture plastic with 44 mM NaHCO3, which increased melanization, also reduced ROS uptake. We conclude that native matrices seem to contain components that significantly depress ROS uptake in culture. The inhibition is not mimicked by collagen, laminin or fibronectin coated wells. The ECM may play a significant role in controlling phagocytosis of ROS either by determining morphology, increasing differentiation or by directly influencing intracellular metabolism, and thus serve as another level of control for this RPE function which may not occur in cells plated on tissue culture plastic. These results may also have implications for the effects of aging or disease in which there are changes in the ECM.

Metallothionein in human retinal pigment epithelial cells: Expression, induction and zinc uptake

The retinal pigment epithelium (RPE) plays several important roles in the continual support and renewal of photoreceptor outer segments. In the present study, we have demonstrated that RPE cells contain a low molecular weight protein with a high capacity for zinc binding that is dependent on available sulfhydryl groups. This protein is inducible by a 24 hour incubation of cultured RPE in medium supplemented with zinc, cadmium, or dexamethasone. The induction of this protein is correlated with an increased capacity for zinc-65 uptake into cultured RPE. Analysis with a cDNA probe specific for the human metallothionein II gene corroborated the existence and induction of metallothionein gene products in RPE cells. Based on these properties, we have identified this protein as metallothionein. The induction of metallothionein likely has a critical influence on the zinc economy of the RPE.

Effect of oxidative stress and bioavailable zinc on metallothionein in cultured human retinal pigment epithelial cells (RPE)

Numerous reports have shown metallothionein protects tissue from reactive oxygen intermediates, considered to be a factor in aging. Because zinc stimulates MT induction and because loss of metal increases proteolysis of the apoprotein, zinc may have a protective role and may indirectly protect against aging. Our laboratory has shown age-related decreases in catalase, zinc and metallothionein in human retinal pigment epithelium. This study was undertaken to investigate the role of bioavailable zinc on MT content in RPE cells subjected to an oxidative stress.