Ординатура / Офтальмология / Английские материалы / Recent Advances in Retinal Degeneration_LaVail, Hollyfield, Anderson _2008
.pdfCEP Adducts, AMD and Neovascularization |
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A Possible Impaired Signaling Mechanism in Human Retinal Pigment Epithelial Cells from Patients with Macular Degeneration
Piyush C. Kothary and Monte A. Del Monte
1 Introduction
The human retinal pigment epithelial (hRPE) cells form a monolayer of polarized cells in the posterior segments of human eyes. They play an important role in the flow of nutrients from the choroid to the neural retina. They normally are mitotically inactive in adult human eyes. Pathological proliferation in later life has been implicated in the pathogenesis of age related macular degeneration (AMD) (Hamdi and Kenney, 2003; Tezel et al., 2004; Zarbin, 2006)
AMD is a frequent cause of reduced vision among the patients with diabetes (Voutilainen-Kaunisto et al., 2000). Voutilainen-Kaunisto et al. (2000) has also shown that visual acuity deteriorates rapidly in AMD patients with diabetes than non-diabetic patients. Insulin is a mitogen for hRPE cells (Campochiaro et al., 1991). Exogenous insulin is required to treat patients with diabetes. This insulin therapy may result in transient increase in diabetic retinopathy (Lu et al., 1999). This led us to examine the effect of insulin on the proliferation and signaling mechanism in hRPE cells obtained from patients with AMD.
2 Methods
2.1 Chemicals
Insulin was purchased from Sigma Chemicals, St. Louise, MO. Anti-pERK1-2 was purchased from R & D Systems, Minneapolis, MN. 3H-thymidine and 14CMethionine were purchased from Amersham Corporation, Arlington Heights, IL. Ham’s F-12 nutrient medium, Dulbecco’s minimum essential media (DMEM), Hank’s balanced salt solution, fetal bovine serum (FBS), penicillin and streptomycin
P.C. Kothary
Kellogg Eye Center, University of Michigan, Ann Arbor, MI 48105-0714, Tel: 734-936-9254, Fax: 734-647-0228 e-mail: kotha@umich.edu
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and trypsin were purchased from GIBCO BRL, Gaithersburg, MD. PD98059 was purchased from Cell Signaling Technology, Beverly, MA.
2.2 Establishment and Maintenance of hRPE Cell Cultures
Primary cultures of hRPE cells were established from human eyes obtained from a patient with AMD and three without AMD as described previously (Kusaka et al., 1998). Briefly, the anterior segment, vitreous and the retina of human eyes were surgically removed. The posterior segments were then washed with balanced salt solution, filled with papain (0.623 mg/ml in cystein/EDTA) and incubated for one hour at 37◦C. The Papain was aspirated and replaced with Ham’s F-12 nutrient medium containing 15% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin and 0.075% (wt/vol) sodium bicarbonate (medium-1). 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◦C in a 95% air/5% CO2 incubator. The medium was changed every three days until the cells were confluent. Primary cultures were then washed with Hank’s balanced salt solution and subcultured by trypsinization with 0.5g/100 ml trypsin and 0.2g/100 ml EDTA in Hank’s normal salt solution (Sigma T-3924) at 37◦C for 10 minutes. The cell suspension was centrifuged at 500 xg and replated. The morphology of cells was examined daily by phase-contrast microscopy. For maintenance of cell lines, cells were plated in 75-mm flask at density of 50,000 cells/flask. The medium was changed every three days until the cells were ready for trypsinization. Cells were counted by hemocytometer and viability was assessed by trypan blue exclusion test.
2.3 Cellular Proliferation
Cellular proliferation of cultured hRPE cells was measured by tritiated thymidine incorporation (3H-thy) and direct cell counting by hemocytometer. Briefly, hRPE cells at passage 4–8 were trypsinized and plated in 16-mm wells of 24-well plates at 1 x 10,000 cells per well in medium-1. Experimental reagents were added for 48 hours when the cells became confluent. Sixteen hours prior to the termination time of experiment, cells were pulsed with 2 mCi/ml of 3H-thymidine (specific activity 25 Ci/mmol, Amesham Life Science, Arlington Heights, IL). The cells were washed three times with PBS (pH 7.4) and two times with ice-cold 5% trichloroacetic acid. One milliliter of 0.1 M NaOH, containing 0.1% SDS was added to 9.5 ml of scintillation fluid and counted in a Beckman scintillation counter.
2.4 Immunoprecipitation Assay
To measure intracellular pERK1/2 synthesis, hRPE cells were labeled by 14- C-Methionine and then treated with insulin in the presence and absence of the
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MAP kinase inhibitor, PD98059, using the method described previously (Bitar et al., 1996; Kothary et al., 2006). hRPE cells were then lysed with zwittergent 3–12 and precipitated with phosphorylated ERK1/2 (pERK 1/2) specific antibody. PD98059 is a specific inhibitor of mitogen-activated protein kinase (Davis et al., 2000; Alessi et al., 1995; Kothary et al., 2005).
2.5 Statistical Analysis
All values represent the % mean of control. Differences between two groups of data were tested by Student’s ‘t’ test. A p<0.05 was used to assess significant differences between two groups.
3 Results
Insulin (0–5 g/ml) stimulated proliferation of hRPE cells from normal human eyes as determined by the trypan exclusion method as well as 3H-thy incorporation. However, no stimulation of proliferation was noted in hRPE obtained from a patient with AMD (Fig. 1 and 2).
As shown in Fig. 3 and 4, PD98059 significantly inhibited insulin stimulated 3H-thy incorporation and cell proliferation in hRPE from normal counts. However, PD98059 had no significant effect on hRPE cell growth in the presence of insulin in hRPE from the AMD patient as determined by 3H-thymidine incorporation or cell number.
Insulin (5 g/ml) stimulated 14C-pERK 1/2 synthesis in hRPE cells from eyes obtained of non-AMD patients. However, no stimulation of 14C-pERK 1/2 Synthesis was noted in hRPE obtained from the AMD patient (Fig. 5).
As shown in Fig. 6, PD98059 significantly inhibited 14C-pERK 1/2 synthesis (1654.3±498 vs. 1269.3±339, CPM±SEM, n=6, p<0.05) in hRPE cells obtained from non-AMD patients. However, PD98059 had no effect (stimulation or inhibition) on 14C-pERK 1/2 synthesis in presence of insulin in the AMD patient.
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Fig. 1 Effect of insulin on 3H-thymidine incorporation of hRPE cells
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Fig. 2 Effect of insulin on the growth of hRPE cells
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Fig. 3 Effect of PD 98059 on insulin-stimulated 3H-thydimine incorporation of hRPE cells
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Fig. 6 Effect of PD 98059 on insulin-stimulated 14C-methionine-pERK 1/2 synthesis in hRPE cells
4 Discussion
The biochemical studies presented here demonstrate that cell proliferation and signal protein synthesis in hRPE cells obtained from the eyes of an AMD patient are abnormal when compared with hRPE obtained from the eyes of non-AMD patients. Unlike hRPE obtained from non-AMD normal control eyes, insulin did not stimulate cell proliferation, measured by direct cell counting using the trypan blue exclusion method, in hRPE cells obtained from the eyes of a patient with AMD. This insulin stimulated increase in hRPE proliferation in normal eyes is consistent with previously published data (Campochiaro et al., 1991). In addition, we have shown that increasing concentrations of insulin did not stimulate 3H-thymidine incorporation in hRPE cells obtained from the eyes of a patient with AMD when compared with hRPE obtained from the eyes from non-AMD normal controls. This suggests that insulin does not induce DNA synthesis in hRPE cells from patients with AMD.
The involvement of growth factors in hRPE cell proliferation has been extensively studied (Kothary and Del Monte, 2004; Kothary and Del Monte, 2005; Mascarelli et al., 2001). Insulin is a mitogen for hRPE cells (Campochiaro et al., 1991). However, very little is known about the signaling and molecular mechanism of action of insulin and other growth factors in hRPE cells from patients with AMD. Recently, we documented the role of phosphorylated ERK 1/2 as a signaling molecule in the proliferation of hRPE cells obtained from the eyes of non-AMD patients (Kothary et al., 2005). Since the neural retina in the macular area degenerates in AMD and the hRPE plays an important role in maintenance of the neural retina, we compared the pERK 1/2 synthesis in hRPE cells obtained from the eyes of AMD and non-AMD patients.
Insulin did not significantly change pERK 1/2 synthesis in hRPE cells obtained from the eyes of the AMD patient. However, there was an insulin dose-dependent increase in pERK 1/2 synthesis in hRPE cells obtained from non-AMD controls. The participation of pERK 1/2 in insulin action is suggested by Khoo et al. (2003), who demonstrated regulation of insulin gene transcription by ERK1 and ERK2 in pancreatic beta cells.
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Hecquet et al. (2002) has shown that (1) fetal bovine serum initially activates the downstream Kinase: MAP kinase/ERK kinase (MEK), which in turn activates pERK 1/2 and (2) PD98059 inhibits MEK in hRPE cells. In our studies, PD98059 inhibited insulin stimulated 3H-thymidine incorporation as well as pERK 1/2 synthesis in hRPE cells obtained from the eyes of non-AMD patients, but had no effect on 3H-thymidine incorporation or pERK 1/2 synthesis in the presence of insulin in hRPE cells obtained from AMD eyes. This suggests that the ERK signaling mechanism may be impaired in hRPE from AMD patients thereby affecting DNA synthesis and cell function. Further studies of the exact kinase signaling abnormality in hRPE cells from patients with AMD and strategies to restore these signals may result in the development of new ways to treat age-related macular degeneration.
Acknowledgments The authors thank Sohil Patel and Jeeyong Kim for their assistance in preparing this chapter. This research was funded by the Skillman Foundation.
References
Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., Saltiel, A. R., 1995, PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo, J Biol Chem 270(46):27489–94.
Bitar, K. N., Kothary, S., Kothary, P. C., 1996, Somatostatin inhibits bombesin-stimulated Gi-protein via its own receptor in rabbit colonic smooth muscle cells, J Pharm Exp Therapeutics 276:714–19.
Campochiaro P. A., Hackett S. F., and Conway, B. P., 1991, Retinoic acid promotes densitydependent growth arrest in human retinal pigment epithelial cells, IOVS 32:65–72.
Davis, S. P., Reddy, H., Caivano, M., et al., 2000, Specificity and mechanism of action of some commonly used protein kinase inhibitors, Biochem J 351:95–105.
Hamdi, H. K., Kenney, C., 2003, Age-related macular degeneration: a new viewpoint, Front Biosci 8:e305–14. Review.
Hecquet, C., Lefevre, G., Valtink, M., Engelmann, K., Mascarelli, F., 2002, Activation and role of MAP kinase-dependent pathways in retinal pigment epithelial cells: ERK and RPE cell proliferation, Invest Ophthalmol Vis Sci 43(9):3091–98.
Khoo, S., Griffen, S. C., Xia, Y., Baer, R. J., German, M. S., Cobb, M. H., 2003, Regulation of insulin gene transcription by ERK1 and ERK2 in pancreatic beta cells, J Biol Chem 278(35):32969–7.
Kothary, P. C., Britton, A., Lewis, N., Patel, V., Rivers, D., and Del Monte, M. A., 2005, ERKpathway inhibitor and proliferative eye disease. Presented at International Drug Discovery Science and Technology, Shanghai, China.
Kothary, P. C., Del Monte, M. A., 2004. Growth factors, and their receptors and inhibitors with a special focus on human retinal pigment epithelial cells and the eye, In: Recent Res Devel Biochem, Edited by S. G. Pandalai, Transworld Research Network, Trivandrum, Kerala, India, pp. 99–116.
Kothary, P. C., Del Monte, M. A., 2005, High glucose modulates growth factor action in human retinal pigment epithelial cells, In: Recent Res Devel, Edited by S. G. Pandalai, Transworld Research Network, Trivandrum, Kerala, India, pp. 1–16.
Kothary, P. C., Lahiri, R., Kee, L., Sharma, N., Chun, E., Kuznia, A., and Del Monte, M. A., 2006, In: Retinal Degenerative Diseases, Edited by J. G. Hollyfield, R. E. Anderson and M. M. LaVail. Springer, New York, pp. 513–518.
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Kusaka, K., Kothary, P. C., Del Monte, M. A., 1998, Modulation of basic fibroblast growth factor effect by retinoic acid in cultured retinal pigment epithelium, Current Eye Research 17(5): 524–30.
Lu, M., Amano, S., Miyamoto, K., Garland, R., Keough, K., Qin, W., Adamis A. P., 1999, Insulininduced vascular endothelial growth factor expression in retina, IOVS 40:3281–86.
Mascarelli, F., Hecquet, C., Guillonneau, X., Courtois, Y., 2001, Control of the intracellular signaling induced by fibroblast growth factors (FGF) over the proliferation and survival of retinal pigment epithelium cells: example of the signaling regulation of growth factors endogenous to the retina, Journal de la Societe de Biologie 195(2):101–6.
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Expression and Cell Compartmentalization
of EFEMP1, a Protein Associated with Malattia Leventinese
Adam Kundzewicz, Francis Munier, and Jean-Marc Matter
1 Introduction
Malattia Leventinese holds its name from the beautiful Leventine Valley in Ticino, Southern Switzerland, from which all the Swiss families touched by this disease come from. It is an autosomal, dominant retinal dystrophy, first described by ophthalmologists in 1925 (Vogt, 1925). A characteristic hallmark of Malattia Leventinese, extracellular, amorphous deposits known as drusen, between the retinal pigment epithelium (RPE) and Bruch’s membrane (Doyne, 1899), are also an early hallmark of age related macular degeneration (AMD), which accounts for approximately 50% of registered blindness in the developed world (Bressler et al., 1988). Malattia Leventinese exhibits features more consistent with AMD than any other heritable macular disorder so it is easy to understand why it should be considered as a burning issue, specially when taking into consideration the fact that the population affected by AMD is expected to nearly double in the next 25 years. What distinguishes Malattia Leventinese from other types of AMD is a radial pattern of drusen (Stone et al., 1999). In few cases of Malattia Leventinese drusen were only observed around the optic nerve head (Forni et al., 1962). Patients are usually asymptomatic until the age of 30 to 40 years and there is a high variability of the disease phenotype. At a later stage of the disease Malattia Leventinese exhibits a variety of clinical and histopathological features, including decreased visual acuity, geographic athropy, pigmentary changes and choroidal neovascularisation (Piguet et al., 1995).
Malattia Leventinese has been mapped to chromosome 2 (Heon et al., 1996) and associated with a single missense mutation (R345W) in a widely expressed gene of rather unknown function called EFEMP1 (S1-5, fibulin 3, FBNL3) for EGF-containing fibrillin-like extracellular matrix protein 1 (Stone et al., 1999), first
A. Kundzewicz
Department of Ophthalmology, School of Medicine, University of Geneva, 22 Rue Alcide Jentzer, 1211 Geneva 14, Switzerland, Tel: 41763472742, Fax: 41216268888
e-mail: Adam.Kundzewicz@unil.ch
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isolated from fibroblasts of a patient with Werner syndrome, a premature ageing disease (Lecka-Czernik et al., 1995).
This mutation is believed to interfere with the secretion of EFEMP1, resulting in the accumulation of misfolded protein between RPE and Bruch’s membrane (Marmorstein et al., 2002). Although mutated protein accumulates in a region directly overlaying drusen, it doesn’t appear to be its major component (Marmorstein et al., 2002).
Human EFEMP1 cDNA encodes a putative protein of 387 to 493 amino acids, with a predicted molecular mass of 43 to 55 kDa, depending on a splice variant. There are five predicted splice variants (Fig. 1), but only the shortest (43.1 kDa) and the longest (54.6 kDa) are expressed in substantial amounts at the protein level (Lecka-Czernik et al., 1995). EFEMP1 contains 5 to 6 EGF-like, calcium binding domains and is highly conserved among humans and rodents, but this homology is restricted to the EGF-like domains. Single Arg-Trp mutation associated with Malattia Leventinese alters the last, calcium binding EGF-like domain of EFEMP1 and it is very similar to number of fibrillin mutations. Fibrillins are found throughout the connective tissue as integral components of extended fibrils, which occur both isolated or in conjunction with elastin. Out of over 500 published fibrillin mutations leading to disease phenotypes many are missense mutations affecting one of the conserved cysteine residues of the EGF domains. Many of these mutations result in Marfan syndrome, a heritable disorder of the connective tissue that affects different organ systems, including the skeleton, lungs, eyes, heart and blood vessels (Dietz et al., 1991, 1992). EFEMP1 shows a strong homology (around 35% identity) to members of the fibrillin family (Ikegawa et al., 1996). Single mutation associated with Malattia Leventinese is thought to cause an abnormal accumulation of this protein between the RPE and Bruch membrane, what might create a physical barrier between the RPE and the choroidal blood vessels, which might result in accumulation of all set of other molecules, leading to a drusen formation (Marmorstein, 2004).
EFEMP1 mutation, present in Malattia Leventinese, affects an EGF-like, calcium binding domain of the protein. Many mutations known to result in Marfan
493aa (54.6kDa)
485aa (53.7kDa)
470aa (52.1kDa)
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387aa (43.1kDa)
Secretory signal peptide
EGF-like domain
Fig. 1 Modular structure of the putative EFEMP1 proteins. Only the longest (54.6kDa) and the shortest (43.1kDa) variants are predicted to be expressed in substantial amounts at the protein level (Lecka-Czernik et al., 1995)