Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

Thus, the differentiation and survival of cells in vivo may be regulated not only by the expression of PEDF and its receptor but also by the temporal and spatial expression of glycosaminoglycans.

8.2.4Interactions with collagens

PEDF has binding affinity also for collagens, and these interactions are

sensitive to pH changes.17,18 At the molecular level, PEDF has two distinct binding sites, one for collagen I (Asp256, Asp258, and Asp300) and another for heparin (Arg146, Lys147, and Arg149)85, which are separated from the

33-mer (anti-angiogenic), 44-mer (neurotrophic) and the homologous serpin reactive site. PEDF may also be regulated by the temporal and spatial expression of collagens and/or play a role in cell adhesion. Qualitative and quantitative changes of extracellular matrix molecules (e.g. collagen and glycosaminoglycans) as well as pH changes in the extracellular matrix, which occur with aging and in certain pathologic conditions (e.g., corneal dystrophies, diabetic retinopathy, glaucoma and wound healing), may alter the molecular assembly and the location of the anti-angiogenic and neurotrophic activities of PEDF (see discussion in Meyer et al.18). Thus, the glycosaminoglycan-PEDF and collagen-PEDF interactions may play important roles in vivo in regulating the local availability of PEDF and/or in modulating its biological activities.

8.2.5Interactions with proteinases

PEDF and proteinases coexist in the extracellular matrix, and they interact in vitro. The majority of proteinases cleave PEDF at its homologous serpin

reactive loop, leaving a core polypeptide that retains the biological activities and affinities for extracellular matrix of the intact protein.16,18,23 However,

we have recently shown that matrix metalloproteinases (MMP) type-2 and type-9 fully degrade PEDF in a calcium dependent-fashion, abolishing its neurotrophic and anti-angiogenic activities.86 Moreover, hypoxia and VEGF can decrease PEDF protein levels by stimulating the MMP-mediated proteolytic degradation of PEDF. Interestingly, the expression and secretion of MMP-2, MMP-9 and VEGF correlate directly with the progression of neovascular ocular diseases87 and inversely with PEDF. Most importantly, these results reveal a novel post-translational mechanism for downregulating PEDF that provides a model for molecular players that control the hypoxiaprovoked increases in VEGF/PEDF ratio, angiogenesis and neuronal death.

9.ANTI-ANGIOGENIC ASSAYS FOR PEDF

PEDF’s anti-angiogenic effects have been studied extensively in vitro,

proving its effectiveness in inhibiting endothelial cell proliferation, migration, apoptosis, and tube formation.1,3,40,43,47,88 Table 5 provides a

summary of assays used by different research groups for testing the antiangiogenic activity of PEDF. An ex vivo assay using chick aortic rings has been used successfully to show the inhibitory activity of PEDF on vessel sprouting.86 However, it is the in vivo models that have given importance to this protein as a potent anti-angiogenic factor. The anti-angiogenic activity of PEDF has been satisfactorily evaluated in several animal models, both locally and systemically. The first demonstrations of its anti-angiogenic effects were made by Dawson et al. using protein-coated hydron pellets in a rat model of corneal neovascularization.1 Later, other researchers used the oxygen-induced retinopathy model to test the effects of systemic43 or intravitreal injections40 of PEDF, showing dose-response patterns and

Table 17-5. Effects of PEDF on endothelial cells in vitro

effectiveness with doses as low as 4 µg/day for systemic administration or 2 µg/eye for intravitreal injections. In our hands, using subconjunctival injections of recombinant PEDF protein in a rat model of laser-induced CNV, the maximal inhibitory effects were obtained with daily doses of 5 ng/eye; lower doses were less effective, while daily doses of 50 µg/eye had no inhibitory effect.89 In mouse models of non-proliferative diabetic

retinopathy, angiogenic molecules like VEGF increase vascular permeability via breakdown of the blood-retina barrier, and the 44-mer derived from PEDF blocks this increase in permeability as viewed by fluorescein angiography extravasation.90 Interestingly, even though the 34-mer region has the anti-angiogenic activity of PEDF, increased vascular permeability precedes neovessel formation, and it is the 44-mer that inhibits vascular permeability. These observations suggest that the 44-mer region might have both antipermeability and anti-angiogenic effects at higher doses.

10.IN VIVO DELIVERY OF PEDF

Due to the multifunctional properties and potential therapeutic capabilities of PEDF, several delivery routes and systems have been tested in order to optimize its effects. The most common methods for delivering PEDF are the use of viral vectors, transplantation of genetically modified cells, and the use of recombinant PEDF protein. Although systemic delivery has been tested, local delivery seems the most logical way for targeting the eye given that the eye is a closed system with a vascular barrier that limits the diffusion of molecules to and from it.

Adenoviral (Ad) vectors are the most extensive platforms used for the delivery of PEDF. Among the major concerns about their use are (1) that adenoviral vectors can infect a variety of cells without specificity91 and

(2) that they induce an inflammatory response that reduces transgene

expression by destroying transduced cells.92 Systemic,93 periocular,94-96 and intraocular42,97 routes of PEDF delivery have been used in animal models of

both retinal and choroidal neovascularization. All of them have shown that PEDF blocks neovascularization. Takita and co-workers used intravitreal Ad-PEDF in an ischemia-reperfusion model that also showed the protective effects of PEDF in the neural retina.32 A phase I clinical trial for testing the safety, tolerability and potential activity of intravitreal delivery of Ad-PEDF in neovascular AMD has been conducted.98 So far there have been no reports on toxicity from this trial, implying that PEDF was generally well tolerated with no dose-limiting toxicities.

Although adeno-associated viral (AAV) vectors are more difficult to produce, they seem to invoke lower immune responses and mediate a longer transgene expression than adenoviral ones. Mori et al. showed that intravitreal or subretinal injections of AAV-PEDF effectively reduce the amount of CNV using laser-induced photocoagulation99.

Autologous transplantation of genetically modified iris pigment epithelium cells that overexpress PEDF is another approach used for the delivery of PEDF. Using this delivery system, PEDF blocked both choroidal

and retinal neovascularization in a laser model of CNV and an ischemic model for induction of retinal neovascularization, respectively. This delivery system was also used to show that PEDF increases the survival and preserves rhodopsin expression of photoreceptor cells in the RCS rat, a model of retinal degeneration.100

The use of recombinant PEDF protein is also effective both in systemic and local delivery routes. This delivery method presents several advantages over the ones described above, especially when used locally: higher efficacy is achieved with lower doses and minimal undesirable side effects. Stellmach et al., using intraperitoneal injections in an ROP model, showed regression of neovessels in the retina,43 hence proving the diffusion of recombinant PEDF through the blood-retina barrier, a fact that has been corroborated by our group using the subconjunctival route.101 In our hands, subconjunctival PEDF injections effectively reduced choroidal neovessels in a rat laserinduced CNV model.70 Tumor therapy is one of the most challenging fields of research. Given its anti-angiogenic properties, PEDF has also been administered in animal models for tumor growth and metastasis. The delivery systems used in these studies have varied. Purified recombinant protein has been administered systemically or injected intratumorally. The PEDF gene has been overexpressed using viral or plasmid DNA vectors as

well as PEDF-transduced cells delivered either systemically or injected locally. In all cases, PEDF induced tumor regression.45-50,102 PEDF’s effects

in tumors might be due to its capacity for inhibiting tumor cell mitosis and tumor vasculature, the latter due to apoptotic cell death.50 These observations make PEDF a promising agent for cancer therapy.

11.PEDF THERAPEUTIC IMPLICATIONS

The ideal anti-angiogenic molecule should have specificity for its vascular target with minimal or no side effects. PEDF seems to be a perfect candidate, since it is effective against multiple inducers of angiogenesis, has no side effects on mature vessels, and is well tolerated.1 Furthermore, its neurotrophic and neuroprotective properties confer upon PEDF additional benefits in a complex neurovascular system like the eye.

In ophthalmology as well as in other fields of medicine, VEGF is a major target to inhibit for treating neovascularization. Although there have been promising results, anti-VEGF therapy alone is unlikely to be sufficient to counteract angiogenesis in the same way as VEGF alone is insufficient to induce a full neovascular response.103 Since multiple inducers have a synergistic effect on endothelial cells,104 and several molecules have proved to

be effective in reducing the angiogenic response, an alternative approach could be to augment the effect of natural anti-angiogenic molecules like PEDF.

Several delivery alternatives are available for the delivery of PEDF to the eye. Systemic delivery has been tested and works in animal models,43 but the high doses needed and the possibility of systemic side effects make this route far from ideal. The local route, periocular or intraocular, is a more logical approach for delivering PEDF in a closed system like the eye. Protein can be delivered with injections or delivery systems, through gene therapy, or with cell systems.

The most widely used route for the delivery of PEDF has been with intraocular injections into the vitreous cavity or subretinal space. Even though these routes allow the delivery of PEDF close to its target and have already proved its efficacy in animal models, we think that the potential complications of intraocular injections (vitreous or subretinal hemorrhage, cataract, glaucoma, endophthalmitis) preclude this delivery route from being ideal. This same route (intravitreal) has been used with PEDF gene therapy, and a clinical trial using Ad-PEDF is on its way. This is encouraging for researchers in the PEDF field, because it demonstrates that PEDF research is sufficiently mature to be tested in patients. However, gene therapy has its own risks, and adenoviral vectors have a short period of effectiveness,92 which could mean several injections, which in turn will increase the possibility of complications.

The ideal delivery system is one that can administer pure agent as locally as possible with minimal invasiveness. In this regard, local delivery of native PEDF, such as the periocular route, is preferred. The effectiveness of systemic administration of PEDF in a model of oxygen-induced retinopathy by Stellmach and coworkers43 suggested that PEDF can reach the retina if administered periocularly. We have shown that PEDF injected into the subconjunctiva can cross the sclera and outer blood-retina barrier in animal models. The use of diffusible PEDF peptides with discrete anti-angiogenic activity should be explored. Preparation of delivery devices for PEDF polypeptides and peptides with acceptable release rates will provide testable systems for the clinic.

12.CONCLUSIONS

Understanding the biochemistry and molecular biology of PEDF, as well as its distribution and regulation in the eye through development, aging and disease plays an important role in taking this interesting protein to the clinic. During the last ten years, there have been great advances in the understanding of these aspects of PEDF. We have outlined some relevant

aspects of the mechanisms of action and regulation of PEDF. Much of what has been reported is aimed toward the development of new approaches for the treatment of both angiogenic and neurotrophic eye diseases. The development of a useful PEDF delivery system is promising for the retinochoroidal field.

REFERENCES

1.D. W. Dawson, O. V. Volpert, P. Gillis, S. E. Crawford, H. Xu H, W. Benedict, and

N.P. Bouck, Pigment epithelium-derived factor: a potent inhibitor of angiogenesis, Science 285, 245-248, (1999).

2.N. Ogata, J. Tombran-Tink, M. Nishikawa, T. Nishimura, Y. Mitsuma, T. Sakamoto, and

M.Matsumura, Pigment epithelium-derived factor in the vitreous is low in diabetic retinopathy and high in rhegmatogenous retinal detachment, Am. J. Ophthalmol. 1 (32), 378-382, (2001).

3.N. M. Holekamp, N. Bouck, and O. Volpert, Pigment epithelium-derived factor is deficient in the vitreous of patients with choroidal neovascularization due to age-related macular degeneration. Am. J. Ophthalmol. 134, 220-227, (2002).

4.E. J. Duh, H. S. Yang, J. A. Haller, E. De Juan, M. S. Humayun, P. Gehlbach, M. Melia,

D.Pieramici, J. B. Harlan, P. A. Campochiaro, and D. J. Zack, Vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor: implications for ocular angiogenesis. Am. J. Ophthalmol. 137, 668-674, (2004).

5.B. O. Boehm, G. Lang, O. Volpert, P. M. Jehle, A. Kurkhaus, S. Rosinger, G. K. Lang, and N. Bouck, Low content of the natural ocular anti-angiogenic agent pigment epithelium-derived factor (PEDF) in aqueous humor predicts progression of diabetic

retinopathy. Diabetologia 46, 394-400, (2003).

6. J. Spranger, M. Osterhoff, M. Reimann, M. Mohlig, M. Ristw, M. K. Francis, V. Cristofalo, H. P. Hammes, G. Smith, M. Boulton, and A. F. Pfeiffer, Loss of the antiangiogenic pigment epithelium-derived factor in patients with angiogenic eye disease. Diabetes 50, 2641-2645, (2001).

7.B. O. Boehm, G. Lang, B. Feldmann, A. Kurkhaus, S. Rosinger, O. Volpert, G. K. Lang, and N. Bouck, Proliferative diabetic retinopathy is associated with a low level of the natural ocular anti-angiogenic agent pigment epithelium-derived factor (PEDF) in aqueous humor. A pilot study. Horm. Metab. Res. 35, 382-386, (2003).

8.N. Ogata, M. Nishikawa, T. Nishimura, Y. Mitsuma, and M. Matsumura, Unbalanced vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor in diabetic retinopathy. Am. J. Ophthalmol. 134, 348-353, (2002).

9.M. Matsuoka, N. Ogata, T. Otsuji, T. Nishimura, K. Takahashi, and M. Matsumura, Expression of pigment epithelium derived factor and vascular endothelial growth factor in choroidal neovascular membranes and polypoidal choroidal vasculopathy.

Br. J. Ophthalmol. 88, 809-815, (2004).

10.S. Y. Kim, C. Mocanu, D. S. McLeod, I. A. Bhutto, M. Eid, P. Tong, and G. A. Lutty, Expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in sickle cell retina and choroid. Exp. Eye Res. 77, 433-445, (2003).

11.F. R. Steele, G. J. Chader, L. V. Johnson, and J. Tombran-Tink, Pigment epitheliumderived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family. Proc. Natl. Acad. Sci. USA 90, 1526-1530, (1993).

12.S. P. Becerra, R. N. Fariss, Y. Q. Wu, L. M. Montuenga, P. Wong, and B. A. Pfeffer, Pigment epithelium-derived factor in the monkey retinal pigment epithelium and interphotoreceptor matrix: apical secretion and distribution. Exp. Eye Res. 78, 223-234, (2004).

13.G. Maik-Rachline, S. Shaltiel, and R. Seger, Extracellular phosphorylation converts pigment epithelium-derived factor from a neurotrophic to an antiangiogenic factor. Blood 105 (2), 670-678, (2005).

14.E. Stratikos, E. Alberdi, P. G. Gettins, and S. P. Becerra, Recombinant human pigment epithelium-derived factor (PEDF): characterization of PEDF overexpressed and secreted by eukaryotic cells. Protein Sci. 5, 2575-2582, (1996).

15.Y. Q. Wu, V. Notario, G. J. Chader, and S. P. Becerra, Identification of pigment epithelium-derived factor in the interphotoreceptor matrix of bovine eyes. Protein Expr. Purif. 6, 447-456, (1995).

16.E. Alberdi, C. C. Hyde, and S. P. Becerra, Pigment epithelium-derived factor (PEDF) binds to glycosaminoglycans: analysis of the binding site. Biochemistry 37, 10643-10652, (1998).

17.K. Kozaki, O. Miyaishi, O. Koiwai, Y. Yasui, A. Kashiwai, Y. Nishikawa, S. Shimizu, and S. Saga, Isolation, purification, and characterization of a collagen-associated serpin, caspin, produced by murine colon adenocarcinoma cells. J. Biol. Chem. 273, 1512515130, (1998).

18.C. Meyer, L. Notari, and S. P. Becerra, Mapping the type I collagen-binding site on pigment epithelium-derived factor. Implications for its antiangiogenic activity. J. Biol. Chem. 277, 45400-45407, (2002).

19.H. Loebermann, R. Tokuoka, J. Deisenhofer, and R. Huber, Human alpha 1-proteinase inhibitor. Crystal structure analysis of two crystal modifications, molecular model and preliminary analysis of the implications for function. J. Mol. Biol. 177, 531-557, (1984).

20.P. E. Stein, A. G. Leslie, J. T. Finch, and R. W. Carrell, Crystal structure of uncleaved ovalbumin at 1.95 A resolution. J. Mol. Biol. 221, 941-959, (1991).

21.H. T. Wright, H. X. Qian, and R. Huber, Crystal structure of plakalbumin, a proteolytically nicked form of ovalbumin. Its relationship to the structure of cleaved alpha-1-proteinase inhibitor. J. Mol. Biol. 213, 513-528, (1990).

22.M. Simonovic, P. G. Gettins, and K. Volz, Crystal structure of human PEDF, a potent anti-angiogenic and neurite growth-promoting factor. Proc. Natl. Acad. Sci. USA 98, 11131-11135, (2001).

23.S. P. Becerra, A. Sagasti, P. Spinella, and V. Notario, Pigment epithelium-derived factor behaves like a noninhibitory serpin. Neurotrophic activity does not require the serpin reactive loop. J. Biol. Chem. 270, 25992-25999, (1995).

24.S. P. Becerra, Structure-function studies on PEDF. A noninhibitory serpin with neurotrophic activity. Adv. Exp. Med. Biol. 425, 223-237, (1997).

25.G. M. Seigel, J. Tombran-Tink, S. P. Becerra, G. J. Chader, D. A. Diloreto, Jr., C. del Cerro, E. S. Lazar, and M. del Cerro, Differentiation of Y79 retinoblastoma cells with pigment epithelial-derived factor and interphotoreceptor matrix wash: effects on tumorigenicity. Growth Factors 10, 289-297, (1994).

26.J. Tombran-Tink, G. G. Chader, and L. V. Johnson, PEDF: a pigment epithelium-derived factor with potent neuronal differentiative activity. Exp. Eye Res. 53, 411-414, (1991).

27.W. Cao, J. Tombran-Tink, W. Chen, D. Mrazed, R. Elias, and J. F. McGinnis, Pigment epithelium-derived factor protects cultured retinal neurons against hydrogen peroxideinduced cell death. J. Neurosci. Res. 57, 789-800, (1999).

28.M. M. Jablonski, J. Tombran-Tink, D. A. Mrazek, and A. Iannaccone, Pigment epithelium-derived factor supports normal development of photoreceptor neurons and

opsin expression after retinal pigment epithelium removal. J. Neurosci. 20, 7149-7157, (2000).

29.M. Cayouette, S. B. Smith, S. P. Becerra, and C. Gravel, Pigment epithelium-derived factor delays the death of photoreceptors in mouse models of inherited retinal degenerations. Neurobiol. Dis. 6, 523-532, (1999).

30.W. Cao, J. Tombran-Tink, R. Elias, S. Sezate, D. Mrazek, and J. F. McGinnis, In vivo protection of photoreceptors from light damage by pigment epithelium-derived factor.

Invest. Ophthalmol. Vis. Sci. 42, 1646-1652, (2001).

31.D. Imai, S. Yoneya, P. L. Gehlbach, L. L. Wei, and K. Mori, Intraocular gene transfer of pigment epithelium-derived factor rescues photoreceptors from light-induced cell death. J. Cell. Physiol. 202, 570-578, (2005).

32.H. Takita, S. Yoneya, P. L. Gehlbach, E. J. Duh, L. L. Wei, and K. Mori, Retinal neuroprotection against ischemic injury mediated by intraocular gene transfer of pigment epithelium-derived factor. Invest. Ophthalmol. Vis. Sci. 44, 4497-4504, (2003).

33.T. Taniwaki, S. P. Becerra, G. J. Chader, and J. P. Schwartz, Pigment epithelium-derived factor is a survival factor for cerebellar granule cells in culture. J. Neurochem. 64, 2509-2517, (1995).

34. T. Taniwaki, N. Hirashima, S. P. Becerra, G. J. Chader, R. Etcheberrigaray, and

J.P. Schwartz, Pigment epithelium-derived factor protects cultured cerebellar granule cells against glutamate-induced neurotoxicity. J. Neurochem. 68, 26-32, (1997).

35.T. Araki, T. Taniwaki, S. P. Becerra, G. J. Chader, and J. P. Schwartz, Pigment epithelium-derived factor (PEDF) differentially protects immature but not mature cerebellar granule cells against apoptotic cell death. J. Neurosci. Res. 53, 7-15, (1998).

36.M. A. DeCoster, E. Schabelman, J. Tombran-Tink, and N. G. Bazan, Neuroprotection by pigment epithelial-derived factor against glutamate toxicity in developing primary hippocampal neurons. J. Neurosci. Res. 56, 604-610, (1999).

37.M. M. Bilak, A. M. Corse, S. R. Bilak, M. Lehar, J. Tombran-Tink, and R. W. Kuncl, Pigment epithelium-derived factor (PEDF) protects motor neurons from chronic glutamatemediated neurodegeneration. J. Neuropathol. Exp. Neurol. 58, 719-728, (1999).

38.L. J. Houenou, A. P. D’Costa, L. Li, V. L. Turgeon, C. Enyadike, E. Alberdi, and

S.P. Becerra, Pigment epithelium-derived factor promotes the survival and differentiation of developing spinal motor neurons. J. Comp. Neurol. 412, 506-514, (1999).

39.Y. Sugita, S. P. Becerra, G. J. Chader, and J. P. Schwartz, Pigment epithelium-derived factor (PEDF) has direct effects on the metabolism and proliferation of microglia and indirect effects on astrocytes. J. Neurosci. Res. 49, 710-718, (1997).

40.E. J. Duh, H. S. Yang, I. Suzuma, M. Miyagi, E. Youngman, K. Mori, M. Katai, L. Yan,

K.Suzuma, K. West, S. Davarya, P. Tong, P. Gehlbach, J. Pearlman, J. W. Crabb,

L.P. Aiello, P. A. Campochiaro, and D. J. Zack, Pigment epithelium-derived factor suppresses ischemia-induced retinal neovascularization and VEGF-induced migration and growth. Invest. Ophthalmol. Vis. Sci. 43, 821-829, (2002).

41.T. A. Zaichuk, E. H. Shroff, R. Emmanuel, S. Filleur, T. Nelius, and O. V. Volpert,

Nuclear factor of activated T cells balances angiogenesis activation and inhibition.

J.Exp. Med. 199, 1513-1522, (2004).

42.K. Mori, E. Duh, P. Gehlbach, A. Ando, K. Takahashi, J. Pearlman, K. Mori, H. S. Yang,

D.J. Zack, D. Ettyeddy, D. E. Brough, L. L. Wei, and P. A. Campochiaro, Pigment epithelium-derived factor inhibits retinal and choroidal neovascularization. J. Cell. Physiol. 188, 253-263, (2001).

43.V. Stellmach, S. E. Crawford, W. Zhou, and N. Bouck, Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor.

Proc. Natl. Acad. Sci. USA 98, 2593-2597, (2001).

44.R. Abe, T. Shimizu, S. Yamagishi, A. Shibaki, S. Amano, Y. Inagaki, H. Watanabe,

H.Sugawara, H. Nakamura, M. Takeuchi, T. Imaizumi, and H. Shimizu, Overexpression of pigment epithelium-derived factor decreases angiogenesis and inhibits the growth of human malignant melanoma cells in vivo. Am. J. Pathol. 164, 1225-1232, (2004).

45.M. Garcia, N. I. Fernandez-Garcia, V. Rivas, M. Carretero, M. J. Escamez, A. GonzalezMartin, E. E. Medrano, O. Volpert, J. L. Jorcano, B. Jiménez, F. Larcher, and M. Del Rio, Inhibition of xenografted human melanoma growth and prevention of metastasis development by dual antiangiogenic/antitumor activities of pigment epithelium-derived factor. Cancer Res. 64, 5632-5642, (2004).

46.K. Matsumoto, H. Ishikawa, D. Nishimura, K. Hamasaki, K. Nakao, and K. Eguchi, Antiangiogenic property of pigment epithelium-derived factor in hepatocellular carcinoma. Hepatology 40, 252-259, (2004).

47.L. Wang, V. Schmitz, A. Perez-Mediavilla, I. Izal, J. Prieto, and C. Qian, Suppression of angiogenesis and tumor growth by adenoviral-mediated gene transfer of pigment epithelium-derived factor. Mol. Ther. 8, 72-79, (2003).

48.A. Mahtabifard, R. E. Merritt, R. E. Yamada, R. G. Crystal, and R. J. Korst, In vivo gene transfer of pigment epithelium-derived factor inhibits tumor growth in syngeneic murine models of thoracic malignancies. J. Thorac. Cardiovasc. Surg. 126, 28-38, (2003).

49.J. A. Doll, V. M. Stellmach, N. P. Bouck, A. R. Bergh, C. Lee, L. P. Abramson,

M.L. Cornwell, M. R. Pins, J. Borensztajn, and S. E. Crawford, Pigment epitheliumderived factor regulates the vasculature and mass of the prostate and pancreas. Nat. Med. 9, 774-780, (2003).

50.L. P. Abramson, V. Stellmach, J. A. Doll, M. Cornwell, R. M. Arensman, and S. E. Crawford, Wilms’ tumor growth is suppressed by antiangiogenic pigment epithelium-derived factor in a xenograft model. J. Pediatr. Surg. 38, 336-342, (2003).

51.J. Tombran-Tink, S. M. Shivaram, G. J. Chader, L. V. Johnson, and D. Bok, Expression, secretion, and age-related downregulation of pigment epithelium-derived factor, a serpin with neurotrophic activity. J. Neurosci. 15, 4992-5003, (1995).

52.L. A. Perez-Mediavilla, C. Chew, P. A. Campochiaro, R. W. Nickells, V. Notario V, D.

J.Zack, and S. P. Becerra, Sequence and expression analysis of bovine pigment epithelium-derived factor. Biochim. Biophys. Acta 1398, 203-214, (1998).

53.N. Ogata, M. Wada, T. Otsuji, N. Jo, J. Tombran-Tink, and M. Matsumura, Expression of pigment epithelium-derived factor in normal adult rat eye and experimental choroidal neovascularization. Invest. Ophthalmol. Vis. Sci. 43, 1168-1175, (2002).

54.Y. Q. Wu and S. P. Becerra, Proteolytic activity directed toward pigment epitheliumderived factor in vitreous of bovine eyes. Implications of proteolytic processing. Invest. Ophthalmol. Vis. Sci. 37, 1984-1993, (1996).

55.J. Ortego, J. Escribano, S. P. Becerra, and M. Coca-Prados, Gene expression of the neurotrophic pigment epithelium-derived factor in the human ciliary epithelium. Synthesis and secretion into the aqueous humor. Invest. Ophthalmol. Vis. Sci. 37, 2759-2767, (1996).

56.N. Bouck, PEDF: anti-angiogenic guardian of ocular function. Trends Mol. Med. 8, 330-334, (2002).

57.W. Eichler, Y. Yafai, T. Keller, P. Wiedemann, and A. Reichenbach, PEDF derived from glial Muller cells: a possible regulator of retinal angiogenesis. Exp. Cell Res. 299, 68-78, (2004).

58.P. C. Karakousis, S. K. John, K. C. Behling, E. M. Surace, J. E. Smith, A. Hendrickson,

W.X. Tang, J. Bennett, and A. H. Milam, Localization of pigment epithelium derived factor (PEDF) in developing and adult human ocular tissues. Mol. Vis. 7, 154-163, (2001).

59.E. M. Perruccio, L.-L. S. Rowlette, N. A. Balko, S. P. Becerra, and T. Borras, Dexamethasone Increases Expression of Pigment-Epithelium Derived Factor (PEDF) in Perfused Human Anterior Segments from Post-Mortem Donor Eyes. Invest. Ophthalmol. Vis. Sci. 44, ARVO E-Abstract 1140, (2003).

60.N. Ogata, J. Tombran-Tink, N. Jo, D. Mrazek, and M. Matsumura, Upregulation of pigment epithelium-derived factor after laser photocoagulation. Am. J. Ophthalmol. 132, 427-429, (2001).

61.N. Ogata, M. Matsuoka, M. Imaizumi, M. Arichi, and M. Matsumura, Decrease of pigment epithelium-derived factor in aqueous humor with increasing age. Am. J. Ophthalmol. 137, 935-936, (2004).

62.R. W. Kuncl, M. M. Bilak, S. R. Bilak, A. M. Corse, W. Royal, and S. P. Becerra, Pigment epithelium-derived factor is elevated in CSF of patients with amyotrophic lateral sclerosis. J. Neurochem. 81, 178-184, (2002).

63.S. V. Petersen, Z. Valnickova, and J. J. Enghild, Pigment-epithelium-derived factor (PEDF) occurs at a physiologically relevant concentration in human blood: purification and characterization. Biochem. J. 374, 199-206, (2003).

64.K. C. Behling, E. M. Surace, and J. Bennett, Pigment epithelium-derived factor expression in the developing mouse eye. Mol. Vis. 8, 449-454, (2002).

65.M. K. Francis, S. Appel, C. Meyer, S. J. Balin, A. K. Balin, and V. J. Cristofalo, Loss of EPC-1/PEDF expression during skin aging in vivo. J. Invest. Dermatol. 122, 1096-1105, (2004).

66.N. Ogata, M. Matsuoka, M. Imaizumi, M. Arichi, and M. Matsumura, Decreased levels of pigment epithelium-derived factor in eyes with neuroretinal dystrophic diseases. Am. J. Ophthalmol. 137, 1129-1130, (2004).

67.R. Z. Renno, A. I. Youssri, N. Michaud, E. S. Gragoudas, and J. W. Miller, Expression of pigment epithelium-derived factor in experimental choroidal neovascularization. Invest. Ophthalmol. Vis. Sci. 43, 1574-1580, (2002).

68.S. Halin, P. Wikstrom, S. H. Rudolfsson, P. Stattin, J. A. Doll, S. E. Crawford, and A. Bergh, Decreased pigment epithelium-derived factor is associated with metastatic phenotype in human and rat prostate tumors. Cancer Res. 64, 5664-5671, (2004).

69.A. W. Stitt, D. Graham, and T. A. Gardiner, Ocular wounding prevents pre-retinal neovascularization and upregulates PEDF expression in the inner retina. Mol. Vis. 10, 432-438, (2004).

70.J. Amaral, B. Burkam, and P. Becerra, Antiangiogenic Effects of PEDF Peptide fragments and Cleaved PEDF. Invest. Ophthalmol. Vis. Sci. 46, ARVO E-Abstract 453, (2005).

71.S. P. Becerra, I. Palmer, A. Kumar, F. Steele, J. Shiloach, V. Notario, and G. J. Chader, Overexpression of fetal human pigment epithelium-derived factor in Escherichia coli. A functionally active neurotrophic factor. J. Biol. Chem. 268, 23148-23156, (1993).

72.E. Alberdi, M. S. Aymerich, and S. P. Becerra, Binding of pigment epithelium-derived factor (PEDF) to retinoblastoma cells and cerebellar granule neurons. Evidence for a PEDF receptor. J. Biol. Chem. 274, 31605-31612, (1999).

73.M. M. Bilak, S. P. Becerra, A. M. Vincent, B. H. Moss, M. S. Aymerich, and

R.W. Kuncl, Identification of the neuroprotective molecular region of pigment epithelium-derived factor and its binding sites on motor neurons. J. Neurosci. 22, 9378-9386, (2002).

74.S. Filleur, K. Volz, T. Nelius, Y. Mirochnik, H. Huang, T. A. Zaichuk, M. S. Aymerich,

S.P. Becerra, R. Yap, D. Veliceasa, E. H. Shoff, and O. V. Volpert, Two functional epitopes of pigment epithelial-derived factor block angiogenesis and induce differentiation in prostate cancer. Cancer Res. 65, 5144-5152, (2005).