Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

purchased from Amersham Corporation, Arlington Heights, IL. Ham’s nutrient medium (F12), Dulbecco’s minimum essential media (DMEM), Hank’s balanced salt solution, fetal bovine serum (FBS), penicillin and streptomycin and trypsin were purchased from GIBCO BRL, Gaithersburg, MD.

2.2. Establishment and Maintenance of hRPE Cells Cultures

Primary cultures of hRPE were established from three human eyes obtained from Michigan Eye Bank as described previously (Del Monte et al., 1991; Kusaka et al., 1998). Briefly, the anterior segment, vitreous and the retina of eye were surgically removed. The posterior segment was then washed, treated with trypsin and the loosely adherent hRPE cells were detached by gentle brushing and hydrostatic pressure with a sterile Pasteur pipet. The cells were plated in 16-mm Primaria plates and incubated at 37 degrees C in a 95% air/5% CO2 incubator. The medium-1, which contained Ham f12 + 14% FBS, was changed every three days until the cells were confluent.

2.3. Cellular Proliferation

Cellular proliferation of cultured hRPE cells was measured by tritiated thymidine incorporation and trypan blue exclusion methods as described previously (Kusaka et al., 1998). Briefly, hRPE cells at passage 4-8 were trypsinized and plated in 16-mm wells of 24-well plates at 1 ¥ 10,000 cells per well in medium-1. Experimental reagents were added for 2448 hours (after the cells were confluent.). The cells were then analysed using the trypan blue exclusion and 3H-Thymidine incorporation methods separately.

2.4. Immunoprecipitation of 14-C-Methionine-VEGF

14C-methionine incorporation was measured as described previously (Bitar et al., 1998; Kothary et al., 2004). To measure intracellular VEGF synthesis, hRPE cells were labeled by 14-C-methionine and treated with experimental reagents. hRPE cells were then lysed with zwittergent 3-12 and precipitated with anti-VEGF specific antibody and protein-A.

2.5. Immunohistochemical Detection

Immunohistochemical analysis was carried out by the method previously described (Oncogene Science, Inc Brochure, Uniondale, NY). Briefly, to identify intracellular VEGF, hRPE cells which were grown on coverslips were treated with experimental reagents. The hRPE cells were then incubated with anti-VEGF and then with anti-rabbit-rhodamine. Coverslips then were dried and mounted on a slide using gel mount.

2.6. Statistical Analysis

All values, representing the mean ± SEM differences between two groups of data were tested by students’ ‘t’ test. A p < 0.05 was used to assess significant differences between two groups and are indicated by single or double or triple asterisks in each figure.

Figure 71.5. The effect of PEDF (0.1 mg/ml) on FBS (10%) stimulated VEGF synthesis in cultured hRPE cells by immunohistochemical localization of VEGF (left vertical panel) and DIC microscopy (right vertical panel).

methionine VEGF. These findings were confirmed by immunohistochemical studies which showed less VEGF positive immunoreactivity in hRPE cells exposed to PEDF and FBS together than FBS alone (Figure 71.5).

4. DISCUSSION

In this study, we have demonstrated that PEDF inhibits FBS stimulated proliferation of hRPE cells. Our data also indicate that the inhibitory effect of PEDF is not caused by cytotoxicity, as remaining cells appear healthy and viable. Instead, the effect of PEDF on FBS induced hRPE cell proliferation was inhibitory. The concentrations of PEDF (10-100 ng/ml) that inhibited hRPE cell number and titrated thymidine incorporation are in the same range as that of Duh et al. (2002) who showed that PEDF at the concentration of 2-20 nM partially suppressed the work of VEGF-induced retinal endothelial cell proliferation.

To characterize the biochemical events affected by PEDF, we examined the effects of PEDF on well known growth inducing factor, VEGF. We demonstrated that FBS stimulates VEGF synthesis in a dose dependent manner and PEDF (0.1 mg/ml) inhibits it in our hRPE cell cultures. The inhibitory effect of PEDF on FBS stimulated hRPE cell number and VEGF syntheses were almost equivalent on a percentage basis. This suggests that VEGF plays an important role in FBS mediated hRPE cell proliferation.

We have shown that PEDF inhibits FBS stimulated hRPE cell proliferation as well as intracellular VEGF synthesis. If we extend our observation, it is reasonable to postulate that PEDF may inhibit hRPE cell proliferation in vivo. Intravenous injection of PEDF has shown to inhibit retinal neovascularization in a mouse model (Duh et al., 2002). This suggests that PEDF may be of therapeutic value in proliferative eye disease.

518 P.C. KOTHARY ET AL.

5. REFERENCES

Bitar, K. N., Kothary, S., and Kothary, P. C., 1996, Somatostatin inhibits bombesin-stimulated Gi-protein via its own receptor in rabbit colonic smooth muscle cells, J. of Pharm & Exp. Therap. 276:714-9.

Del Monte, M. A., Rabbani, R., Diaz, T. C., Lattimer, S. A., Nakamura, J., Brennan, M. C., and Greene, D. A., 1991, Sorbitol, myoinositol, and rod outer segment phagocytosis in cultured hRPE cells exposed to glucose. In vitro model of myoinositol depletion hypothesis of diabetic complications, Diabetes. 40:1335-45.

Duh, E. J., Yang, H. S., Suzuma, I., Miyagi, M., Youngman, E., Mori, K., Katai, M., Yan, L., Suzuma, K., West, K., Davarya, S., Yong, P., Gehlbach, P., Pearlman, J., Crabb, J. W., Aieloo, L. P., Campochario, P., and Zack, D. J., 2002, Pigment epithelium-derived factor suppresses ischemia-induced retinal neovascularization and VEGFinduced migration and growth, Invest Ophthalmol Vis Sci. 43:823-9.

Kothary, P. C., and Del Monte, M. A., 2003, in: Recent Res. Devel. Cell Biochem, edited by S. G. Pandalai, Transworld Research Network, Trivandrum (India), pp. 99-116.

Kothary, P. C., Singal, P., Patel, P., and Del Monte, M. A., 2001, in: Proceedings of World CongressNeuroinformatics, Part II, edited by Frank Rattay, (ARGESIN/ASIMVerlag, Vienna (Austria), pp. 341-8.

Kothary, P. C., Paauw, J. D., Bansal, A. K., Grace, C. C., and Del Monte, M. A., 2004, in: Proceedings of 5th International Symposium on Ocular Pharmacology and Therapeutics, Medimond S.r.l., Bologna (Italy), pp.237-41.

Kusaka, K., Kothary, P. C., and Del Monte, M. A., 1998, Modulation of basic fibroblast growth factor effect by retinoic acid in cultured retinal pigment epithelium, Current Eye Research. 17: 524-30.

Mori, K., Gehlbach, P., Ando, A., Mcvey, D., Wei, L., and Campochiaro, P. A., 2002, Regression of ocular neovascularization in response to increased expression of pigment epithelium-derived factor, Invest Ophthalmol Vis Sci. 43:2428-3.

Lahiri, R., Kothary, P., and Del Monte, M. A., 2004, The effect of pigment epithelium derived factor (PEDF) on human retinal pigment epithelial cell proliferation and intracellular growth factor synthesis. Poster presentation at the ARVO Meeting, Ft. Lauderdale, Florida.

2. RPE MICROVILLI STRUCTURE

The RPE microvillar structure has not been extensively studied. However, available information indicates that RPE microvilli possess an internal core bundle of densely packed actin filaments.9 Myosin VIIa has been detected at the base of apical processes10 while villin, fimbrin and myosin I have not been detected.11,12 The entire length of the RPE microvilli has been shown to contain ezrin and EBP50.11,13-15 The mouse RPE microvilli-enriched fraction, described below, contained several cytoskeletal components, among them various types of actin and tubulin, b-spectrin, ezrin, moesin, EBP50, and profilin.

A more complete definition of the protein composition of the RPE apical microvilli should provide insights into other biochemical processes occurring at this critical interface that are important for the support and maintenance of vision.

3. RPE MICROVILLI PROTEINS AND FUNCTION

Recently, we have improved a method to isolate RPE apical microvilli. The procedure relies on the binding of N-acetylglucosamine and sialic acid-containing glycoconjugates present in abundance on the RPE apical surface16 to the WGA lectin conjugated to agarose beads. Mass interactions of the surface glycoconjugates with the immobilized lectin on the bead allow for the detachment of the RPE microvilli upon physical removal of the WGA beads. The RPE isolated microvilli are resolved by SDS-PAGE, in gel digested with trypsin, and peptides extracted and analyzed by mass spectrometry.3,17 This procedure was done in mice eyecups with the RPE exposed and it has resulted in the identification of over 283 proteins, distributed over functional categories such as retinoid-metabolizing, cytoskeletal, enzymes, extracellular matrix components, membrane proteins and transporters, among others. A summary of selected proteins identified by this method is presented in Table 72.1 and has been recently described.3,17

The beads with the isolated RPE microvilli on their surface can be used for immunolabeling experiments, morphological (light and electron microscopy) as well as in biochemical experiments. In Figure 72.1 beads with isolated mouse RPE were fixed in 4% paraformaldehyde, permeabilized in triton X100, reacted with both a rabbit antibody to (A) protein kinase A regulatory subunit II (PKARII) and (B) a mouse antibody to protein kinase A regulatory subunit I (PKARI). Parallel samples were processed for transmission electron microscopy and RPE microvilli are observed on the surface of the agarose beads (C and D).

Examples of proteins identified in the RPE microvilli by both mass spectrometry and other methods include Na,K-ATPase, Glut-1, monocarboxylate transporter, carbonic anhydrase, basigin, and the chloride intracellular channel 6.17 The cone and rod-associated matrix, present on top of the RPE apical surface, are firmly attached to the RPE apical surface. This is one of the reasons for the mass spectrometric identification of several novel extracellular matrix components such as fibromodulin, lumican, undulin 1, and neuroglycan C in the RPE isolated microvilli. The proteomic method therefore provides an unbiased account of proteins present in the RPE apical microvilli.

RPE apical microvilli play important roles in retinal attachment. Ensheathment of the outer segment tips by apical projections may contribute to adhesion by providing frictional or electrostatic interactions.18 Any disruption of the relationship between cone and rod photoreceptors and the RPE will result in pathological consequences. A retinal detachment, for

example, is a separation of the photoreceptor outer segments from its apical RPE microvilli. After clinical reattachment, return of normal vision depends, upon the restoration of a functional relationship between proteins present in the RPE apical surface and the photoreceptors outer segments.

Alterations in the proteins present in the RPE apical microvilli will likely impair vision as a consequence of disrupting the structural and functional nurturing of the photoreceptors by the RPE. Some of the RPE apical proteins identified in the RPE microvilli fraction have already been shown to be involved in retinal degenerations. The list of RPE apical proteins involved in retinal diseases is likely to grow as we learn more about the RPE proteome.

An interaction between cellular retinaldehyde-binding protein (CRALBP) and ERMbinding phosphoprotein 50 (EBP50) in RPE microsomes was recently described.15 Our proteomic analyses was highly enriched in several retinoid processing proteins such as cellular retinaldehyde-binding protein, 11-cis-retinol dehydrogenase, cellular retinol-binding protein 1, interphotoreceptor retinoid-binding protein, EBP50, and ezrin. These results support the existence of a visual cycle protein complex in the RPE apical microvilli.3 Several forms of retinitis pigmentosa are known to be caused by mutations in visual-cycle protein genes such as RPE65, CRALBP, IRBP.19,20

Macular edema resulting from pathologies such as uveitis, postoperative period following cataract extraction,21 retinitis pigmentosa,22 serpiginous choroiditis23 and epiretinal membranes,24 has been widely treated with carbonic anhydrase inhibitors.25 Polarized dis-

Table 72.1. Selected proteins identified on WGA-beads after incubation with apical RPE.

a Swiss Protein database and NCBI (in italics) accession numbers are shown. b Results from three independent experiments.

Figure 72.1. Morphological analysis of isolated WGA-beads with mouse RPE microvilli on their surface.

WGA beads scraped off the mouse eyecups were reacted with antibodies to protein kinase A regulatory subunit alpha II (PKARII) (A) and protein kinase A regulatory subunit I (PKARI) (B). Alternatively, the isolated beads were fixed in 2.5% glutaraldehyde, and processed for transmission electron microscopy (TEM). Low (C) and high (D) magnification of these beads revealed extensive surface areas covered by the RPE microvilli. Bars = 100 mm (A, B), 1 mm (C) and 0.5 mm (D).

tribution of carbonic anhydrase activity in the RPE apical surface has been reported. Carbonic anhydrase XIV was one of the proteins we identified by proteomic analysis in isolated RPE microvilli.17

Aging studies have shown a decrease in both the number and the length of epithelial microvilli, and a declined function of plasma membrane enzymes and receptors.26-29 Specifically, a decrease in the activity of some of the enzymes detected in RPE microvilli like Na,K-ATPase, LDH, glutathione S-transferase, phosphoglycerate kinase, adenylate kinase30 and catalase has been established in various epithelia.31-33 Future studies involving these and other proteins may help to improve our understanding of aging diseases such as macular degeneration.

Most recently we have pursued proteomic analysis of rat RPE microvilli. One of the proteins consistently found in rat RPE microvilli is ceruloplasmin. The localization of ceruloplasmin in the RPE has been previously described.34,35 Retinal degeneration has been reported in patients with the autosomal recessive disease called aceruloplasminemia, a deficiency in ceruloplasmin.36