Lawrence E. Stramm

β-Glucuronidase mediated pathway essential for retinal pigment epithelial degradation of glycosaminoglycans. Disease expression and in vitro disease correction using retroviral mediated cDNA transfer

A beta-glucuronidase mediated pathway for the degradation of glycosaminoglycans is present in the retinal pigment epithelium. The pathway has been defined using ocular tissues and cultured cells from mutant animals having a recessively inherited deficiency of the lysosomal enzyme. In situ, storage products accumulate in secondary lysosomes of the retinal pigment epithelium, the cytoplasm fills with inclusions and the cells hypertrophy; severity of the disease increases with aging. Deficient activity of beta-glucuronidase is present in primary and second passage cultures. Radiolabel studies with 35SO4 show a significant retention of cell layer label by mutant retinal pigment epithelial cells during a 72-hr pulse or 24-hr chase period. The labels is in newly synthesized chondroitin sulfate and heparan sulfate, which are natural substrates for the deficient enzyme. There is no difference from normal in the total radioactivity and electrophoretic profile of the glycosaminoglycans that are synthesized and released into the media. A retroviral vector was used to transfer normal rat beta-glucuronidase cDNA into the mutant cells. The vector treatment results in restoration of enzyme activity and correction of the degradative defect; 35SO4 labeling shows that chondroitin sulfate and heparan sulfate levels return to normal. The vector treatment studies indicate that a single gene defect determines the abnormal beta-glucuronidase mediated pathway in the mutant retinal pigment epithelium.

Tissue culture of cat retinal pigment epithelium.

To culture retinal pigment epithelial (RPE) cells from normal cats, the cells were enzymatically dissociated from the eyecup and grown in either Hams F-10 Nutrient Mixture or Eagles Minimum Essential Media supplemented with 20% fetal calf serum. Cultures reached confluency between 6 and 10 days and contained monolayers of polygonally shaped cells. Light and electron microscopy demonstrated that most of the normal morphological characteristics of cat RPE cells in vivo were maintained in vitro; these included apical microvilli, apicolateral junctional specializations, basal infoldings and intracellular organelles. Pigment granules appeared to be diluted by cell division. No evidence of a basal membrane formation was seen; however, a fine granular or fibrillar extracellular matrix was observed in some cultures and was located between the culture plate surface and the basal surface of the RPE. Primary cultures were viable for up to 145 days. The activities of two lysosomal hydrolases (arylsulfatase A and arylsulfatase B) involved in the metabolism of sulfatide and dermatan sulfate were measured in confluent cultures. Mean arylsulfatase A activity was 1297 nmol nitrocatechol/mg protein/hr and arylsulfatase B activity was 553 nmol nitrocatechol/mg protein/hr. These activities were approximately 5 to 10-fold higher than present in cat peripheral leukocytes and skin fibroblasts in vitro. This in vitro system will facilitate studies on normal function and in conditions where the RPE has been compromised by inherited diseases (i.e. gyrate atrophy, mucopolysaccharidosis I and VI).

Ascorbate transport in cultured cat retinal pigment epithelial cells

Transport of ascorbate by primary cultures of cat retinal pigment epithelial cells (RPE) was studied. Confluent primary cultures were incubated with 10-500 microM L-[carboxyl-14C] ascorbic acid in balanced salt solution (BSS) at 37 degrees C for 1 to 40 min. The uptake of radioactive ascorbate followed saturation kinetics with a Km of 42 microM and Vmax of 117 pmol min-1 microgram-1 DNA. Cells incubated with 10 microM radioactive ascorbate for 40 min showed a ratio of intracellular to extracellular radioactive ascorbate of greater than 40. The transport of ascorbate was sodium- and energy-dependent. Replacement of 150 mM NaCl in BSS with 150 mM LiCl reduced ascorbate uptake significantly. Ouabain, 2,4-dinitrophenol, alpha-D-glucose, 3-O-methyl-D-glucose, and the ascorbate analogues, D-isoascorbate and dehydroascorbate, each inhibited ascorbate uptake into RPE cells. The efflux of radioactivity into the incubation media was slow when cells were preloaded with either 50- or 500 microM radioactive ascorbate, but increased when cells preloaded with 50 microM ascorbate were incubated in the presence of excess non-radioactive ascorbate. These studies demonstrated that a sodium-dependent carrier system is involved in transport of ascorbate in primary cultures of cat RPE.

Extracellular matrix production by cat retinal pigment epithelium in vitro: characterization of type IV collagen synthesis.

Feline retinal pigment epithelial cells (RPE) produced an extracellular matrix (ECM) in vitro which was located between the basal surface of the RPE and the culture plate. This ECM had three morphological components: bundle, granular and fibrillar. After 14 days in culture the basal extracellular space contained small amounts of bundle material; granular and fibrillar material were infrequently observed at this time. The amount of ECM material increased with increasing time in culture. The accumulation of the granular component extracellularly was greatest between 60 and 108 days. Fibrillar material, although occasionally observed in the ECM, appeared to be an infrequent component. By 145 days, the ECM filled the extracellular space between the RPE and the culture plate. The time-dependent increase of the ECM indicated continued synthesis and secretion of ECM into the basal extracellular space by the RPE. Confluent RPE cultures, or choroidal/scleral fibroblasts, were incubated for 24 hr with [14C]-proline. Newly synthesized collagen, either in the culture medium or the cell layer, was co-precipitated with added carrier collagen by (NH4)2 SO4. The samples, with or without reduction and alkylation, were digested with pepsin and fractioned by selective salt precipitation and carboxymethyl(CM)-cellulose chromatography. The resulting fractions were further analyzed, or purified for thin layer chromatography (TLC) amino acid analysis, by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Cultured RPE cells, but not choroidal/scleral fibroblasts, produced labelled peptides which were characterized as alpha 1 (IV), and alpha 2 (IV) collagen chains by CM-cellulose chromatography, SDS-PAGE, proline: hydroxyproline ratios and sensitivity to bacterial collagenase. In contrast, choroidal/scleral fibroblasts produced labelled alpha 1 (I), beta 12 (I) and alpha 2 (I) collagen chains. The synthesis of type IV collagen by RPE cells may reflect the production of ECM observed by electron microscopy in cultured feline RPE cells.

Glycosaminoglycan synthesis and secretion by bovine retinal capillary pericytes in culture.

The synthesis and secretion of glycosaminoglycans (GAGs) was characterized in subcultures of bovine retinal capillary pericytes. The GAGs were metabolically labeled with [3H]glucosamine and 35SO4 for 3 days, and then precipitated from the cell layer or media by cetylpyridinium chloride and ethanol, separated by cellulose acetate electrophoresis and further identified by their susceptibility to degradative procedures. The predominant radioactively labeled GAG associated with the pericyte-cell layer was heparan sulfate (HS). Radioactively labeled chondroitin sulfate (CS) and hyaluronic acid (HA) were also present in the pericyte-cell layer. No radioactively labeled dermatan sulfate (DS) was detected. The profile of radioactively labeled GAGs secreted by pericytes into the media differed considerably from that associated with the cell layer. Equal amounts of radioactivity were incorporated into HS and CS. Small quantities of radioactively labeled HA were also present in the media. Although no radioactively labeled DS was detected in the pericyte-cell layer, it was present in the media. The total pericyte-cell layer GAG profile was determined by scanning densitometry of the three bands resolved after cellulose acetate electrophoresis and Alcian Blue staining. The slowest band was identified as HS, and accounted for 17% of the total GAGs. The middle band was identified as DS, and accounted for 34% of the total GAGs. The fastest band was tentatively identified as either DS or chondroitinase AC-resistant CS, and constituted 49% of the total GAGs. The GAGs associated with the fibroblast-cell layer and secreted into the media by fibroblasts also were characterized and compared with those produced by pericytes. The major differences were in the secretion of large amounts of HA into the media by fibroblasts, and the presence of radioactively labeled DS in the cell layer of fibroblasts.

Is rhodopsin the ligand for receptor-mediated phagocytosis of rod outer segments by retinal pigment epithelium?

It has been suggested that rhodopsin may have a direct role in the attachment of shed rod outer segments (ROS) to retinal pigmented epithelial (RPE) cells initiating the events which lead to engulfment. We isolated the soluble tryptic glycopeptide from rhodopsin (GP-T1) and used it as a probe to test this hypothesis. In phagocytic assays using both cultured chick and cat RPE cells, GP-T1 did not inhibit the phagocytosis of ROS. Additionally, mannose glycoconjugates were not effective inhibitors of phagocytosis. However, glycopeptides released by tryptic digestion of intact ROS did inhibit ROS uptake by the RPE cells. The results suggest that phagocytosis of ROS is not mediated through a simple carbohydrate recognition system and that rhodopsin is not the ligand recognized by RPE cells.

Synergistic activation of retinal capillary pericyte proliferation in culture by inositol triphosphate and diacylglycerol

Inositol triphosphate (IP3) and diacylglycerol (DG) are second messengers which control ionic events implicated in cell proliferation in a variety of tissues. In order to determine if these two second messengers control the proliferation of bovine retinal capillary pericytes (BRCP) or feline retinal pigment epithelial cells (FRPE) in culture, both intact BRCP or FRPE or BRCP or FRPE made permeable by saponin were used to study the effects of IP3 and DG on [3H]thymidine incorporation into DNA. [3H]Thymidine incorporation by BRCP made permeable to saponin showed specific IP3 dose-dependence; the apparent Km was 0.3 microM of IP3. Similar effects of A23187, a Ca2+ ionophore, or synthetic DG (1-oleoyl-2 acetyl-glycerol) were also observed. The combination of synthetic DG (0-,2-,4-, 8 micrograms ml-1) and 1 microM A23187 produced greater stimulation of [3H]thymidine incorporation by intact BRCP than was seen with DG or A23187 alone. In contrast to BRCP. [3H]thymidine incorporation by FRPE was not stimulated by IP3, A23187 or synthetic DG. The synergistic activation of IP3 and DG provided direct evidence to support the view that BRCP proliferation in vitro were regulated by the levels of the two second messengers.

Mechanisms of Hereditary Visual Cell Disease

Hereditary and developmental diseases of the photoreceptorpigment epithelium complex account for a significant percentage of blindness in young adults. Over 100,000 people demonstrate some form of visual impairment in the USA as a result of these diseases; up to 2% of the population carry the gene(s) for these disorders even though they themselves enjoy normal vision (Boughman et al., 1980). In the human, the term Retinitis Pigmentosa (RP) has been applied to the family of such hereditary diseases which causes degeneration of the photoreceptor cells and subsequent degeneration of other retinal cell types. Several distinct genetic classes of this condition are now known with differing times of onset, levels of severity and clinical courses. It thus seems clear that RP is a family of allied diseases of different etiologies whose final common pathway leads to retinal malfunction and blindness.