Pigment Epithelium-Derived Factor, a Protective Factor for Photoreceptors in Vivo

Abstract

Pigment epithelium-derived factor (PEDF) is a natural protein of the retina with demonstrable neurotrophic properties, found in the interphotoreceptor matrix in intimate contact with photoreceptors. This review summarizes the effects of PEDF on photoreceptors in several animal models of retinal degeneration.

93.1. Introduction

Pathological photoreceptor cell death leads to visual loss. Therefore natural inhibitors of cell death can prevent this pathology. PEDF is a natural ocular protein, secreted by the retinal pigment epithelium (RPE). The RPE expresses the SERPINF1 gene at higher levels compared to the other tissues in the eye, and releases the gene product in a directional fashion into the interphotoreceptor matrix (Becerra et al. 2004). In this extracellular matrix, the protein associates with glycosaminoglycans and becomes available to interact with receptors on the surface of the photoreceptors. PEDF is a member of the serpin superfamily formed by a group of proteins that share common conformation. Although most of the serpin members are serine protease inhibitors, PEDF is grouped with non-inhibitory serpins (Becerra 2006). Its homologous reactive center loop peptide, located towards its carboxy-end, is not used to block protease activity. However, a peptide region from its amino terminal sequence is responsible for neurotrophic effects, which in the 3D structure is distinct from the homologous serpin reactive loop. The neurotrophic effects of PEDF are independent of its capacity to inhibit serine proteases and depend on interactions with cell-surface receptors. PEDF-R is a cytoprotective receptor for PEDF encoded by the PNPLA2 gene (patatin like phospholipase A2 family member) that is expressed in the retina and distributed in the inner segments of photoreceptors (Notari et al. 2006; Becerra and Notario 2013; Subramanian et al. 2013). Interactions with PEDF-R are likely to mediate the cytoprotective effects of PEDF in photoreceptors. The efficacy of PEDF in protecting photoreceptor cells against degeneration and apoptosis in vivo is reviewed here.

93.2. Biological Function

The PEDF protein exhibits neurotrophic activity and acts on photoreceptor morphogenesis, retinal neuroprotection and neurite outgrowth (Barnstable and Tombran-Tink 2004). The capacity of PEDF to delay photoreceptor cell degeneration and apoptosis is demonstrated in genetic and light-induced damage animal models. PEDF can protect cells of the inner retina and retinal ganglion cell layer from death induced by ischemia and cytotoxic agents. It is also protective of CNS neurons, such as motoneurons, cerebellar granule cells, hippocampal neurons, and cortical neurons, and has demonstrable neurite-outgrowth activities. Here we summarize the effects of PEDF on photoreceptor cells in vivo (see also Table 93.1).

Table 93.1.

The effects of PEDF on photoreceptor degeneration in vivo

Animal model	Genetic or induced damage	Photoreceptor course of degeneration	Method and time of PEDF delivery	Effect of treatment method and timing for assay	References
rd1 (m)	Loss of function mutation of photoreceptor β-Pde6B gene	Rods: start at P10, peak at P14, end at ∼P21. Cones: start at P15, end by 6 months	Intravitreal injection of human rPEDF (1 μg) protein at P14	Thicker ONL; reduced number of TUNEL positive cells at 3 days p.i. (P17)	(Cayouette et al. 1999)
Rds (m)	Null mutation of Prph2 (Peripherin 2)	Photoreceptors: start at P14, peak after 3 weeks. Complete loss by 12 months	Intravitreal injection of human rPEDF (1 μg) protein at P17	Reduced number of TUNEL positive cells at 3 days p.i. (P20)	(Cayouette et al. 1999)
DKO rd8 (m) Cc12−/−Cx3cr1−/− Crb1rd8	Constitutive deletion of the Ccl2 chemokine and Cx3cr1 chemokine receptor in Cbr1rd8 background mice	Photoreceptor degeneration at 6 post-natal weeks	Intravitreal injection of human rPEDF (1 μg) protein at 6 weeks; subconjuctival injection of rPEDF (3 μg) 4 weeks after	Thicker ONL; reduced number of TUNEL positive cells; decreased expression of FasL and Bax; increased expression of Bcl-2 (2 months after the last injection)	(Wang et al. 2013)
RCS (r)	Loss of function mutation of RPE Mertk gene	Photoreceptor degeneration between P20 and P60	Subretinal injection of lentiviral SIV-PEDF (human) in 3 weeks old rat	Thicker ONL; reduced loss of photoreceptors (at 2, 8–12 weeks p.i.); decreased TUNEL-positive cells at 2 and 4 weeks p.i.; rescue of functionality (ERG at 4 and 8 weeks p.i.)	(Miyazaki et al. 2003; Murakami et al. 2008)
RCS (r)	Loss of function mutation of RPE Mertk gene	Photoreceptor degeneration between P20 and P60	Intravitreal injection of PEDF (2.5 μg)-NP at P21	Thicker ONL; reduced loss of photoreceptors and TUNEL-positive cells (at 4–8 weeks p.i.); rod-opsin expression preserved and rescue of functionality by ERG (at 8 weeks p.i.)	(Akiyama et al. 2012)
Sprague-Dawley rats (r)	Light damage (LD): constant light (CL) exposure (1200–1500 lux) for 3, 7, 10, 14 days	Reduction of ONE thickness (∼70%) and null ERG response after 7 days of CL exposure	Intravitreal injection of PEDF (2 μg) or PEDF/bFGF (1 μg/each) in 2–5 month-old rats, at 1 and 2 days pre LD and 0, 1 and 2 days post LD	Thicker ONL; improved ERG response (CL for 3, 7, 10 days)	(Cao et al. 1999)
Lewis rats (r)	Light damage (LD): constant light (CL) exposure (2500 lux) for 24, 96, 168 h	Progressive reduction of ONL thickness and number of photoreceptors after 24, 96 and 168 h of CL exposure	Intravitreal injection of PEDF-expressing or null adenoviral vector (AdPEDF.11 or AdNull.11) 3 days before FD	Thicker ONL; reduced loss of photoreceptors (LD for 96 h) and TUNEL-positive cells (LD for 12 h); improved functionality by ERG (LD for 48 h, recovery 7–28 days)	(Imai et al. 2005)

93.2.1. The rd1/rd1 Mouse

The rd1 mouse is an animal model for one variant of recessive human Retinitis Pigmentosa (RP) that carries a homozygous loss of function mutation of the gene encoding the β-subunit of rod photoreceptor cGMP phosphodiesterase 6 (PDE6). The mutation induces cell death of rod photoreceptors, which starts around postnatal day 10 (P10), peaks at P14, and ends almost completely by P21. Cone photoreceptor death starts around P15, with complete degeneration within 6 months (Sancho-Pelluz et al. 2008). Cayouette et al. (1999) evaluated the effects of human recombinant PEDF (rPEDF) in this animal model. rd1 mice were intravitreally injected with 1 μg rPEDF in one eye at P14. Their contralateral eyes were similarly injected with rβ-galactosidase (1 μg) or left uninjected and used as controls. Photoreceptor degeneration was evaluated 3 or 9 days after administration. At 3 days post-injection (p.i.), the effect of a single PEDF injection on the outer nuclear layer (ONL) height was significant at 120–161 % that of controls. However, the authors did not observe an effect at 9 days p.i. They found that biotin-conjugated PEDF injected in the vitreous of wild type mice cleared from the eye within 24 h, suggesting a transient effect of the injected protein to maintain photoreceptor morphology in rd1 mice.

93.2.2. The rds/rds Mouse

The antiapoptotic effects of rPEDF on photoreceptors were tested in another model for RP, the rds mouse, which carries a null mutation in the Prph2 (peripherin 2) gene (Sancho-Pelluz et al. 2008). In rds homozygous mutant mice, retinal degeneration starts at P7 and peaks 3 weeks after birth. Photoreceptors degenerate slowly as compared to rd1 mice and retinal rod and cone cells are completely lost by 12 months of age (Sanyal et al. 1980). Rds mice were intravitreally injected in one eye with human rPEDF (1 μg) at P17 while the contralateral eye, used as a control, was left untreated or administered with rβ-galactosidase (1 μg). The eyes were then collected 3 days p.i. (i.e., P20) to evaluate apoptosis in the ONL. The protective effect of PEDF on photoreceptor apoptosis was significant, with less TUNEL-positive nuclei in the ONL, between 61.5–79.8 % of control eyes, confirming the antiapoptotic activity of PEDF on photoreceptors in vivo (Cayouette et al. 1999).

93.2.3. The DKO rd8 Mouse

The Ccl2/Cx3cr1 double knockout mouse on Crb1^rd8 background (DKO rd8) represents a model for progressive focal retinal degeneration, recapitulating some of the features of age-related macular degeneration (AMD), such as RPE alteration, photoreceptor degeneration, immune activation and A2E elevation in the RPE. The mouse was generated by knocking out genes for a chemokine ( Ccl2) and a chemokine receptor ( Cx3cr1) created on the C57BL/6N background carrying the Crb1^rd8 mutation (Chu et al. 2013). By 6 weeks of age, all DKO mice show AMD-like retinal lesions, including RPE alteration and photoreceptor degeneration (Chu et al. 2013). Wang et al. (2013) reported that the concentration of PEDF secreted in the conditioned media of primary DKO rd8 RPE was 84 % decreased relative to wild type (WT). Recombinant human PEDF protein (1 μg) was exogenously administered to 6 week-old DKO rd8 by intravitreous injection in the right eye, with a subsequent subconjuctival rPEDF (3 μg) injection 4 weeks later. Contralateral eyes were left untreated and used as controls. Four weeks after the last injection, PEDF-mediated protection was observed in the ONL with more than 2-fold reduction of the number of TUNEL-positive nuclei along with increased ONL thickness and significantly lower levels of A2E in the retina in the rPEDF-treated compared to the contralateral eye. The authors also reported reduction in the expression of pro-apoptotic factors such as FasL and Bax, and increased expression of the anti-apoptotic factor Bcl-2 in the retina (Wang et al. 2013).

93.2.4. The RCS Rat

The Royal College of Surgeons (RCS) rat is the first known model of inherited retinal degeneration. Similar to the human disease (Gal et al. 2000), the cause of retinal degeneration in the RCS rats is a mutation in the receptor tyrosine kinase Mertk, (D’Cruz et al. 2000), a gene that is expressed in the RPE. The mutation leads to defective RPE phagocytosis of photoreceptor outer segments followed by progressive loss of photoreceptor cells, which degenerate between P20 and P60 (Mullen and LaVail 1976). Miyazaki et al. (2003) evaluated the effects of PEDF in the retina of RCS rats. Exogenous PEDF was delivered by gene transfer, via subretinal injection of the simian lentiviral vector (SIV) containing the human SERPINF1 gene in 3-week-old RCS rats. Control animals were left untreated or injected with either SIV-LacZ or vehicle solution. The expression of transduced genes was observed in the RPE at 4 weeks p.i. and persisted at later time points (8, 12, 24 weeks p.i.). Similarly, the number of photoreceptors was preserved in the PEDF-injected eyes, only 4 weeks after gene transduction. The protection was significant compared to control eyes at 8 and 12 weeks. However, the ONL regions furthest from the PEDF injection sites displayed the least protection. The antiapoptotic effect of PEDF is likely responsible for protection from photoreceptor loss in this model, as evidenced by diminished numbers of TUNEL positive nuclei in the ONL of PEDF-transduced eyes relative to controls. PEDF-mediated rescue of the photoreceptors was evaluated by TUNEL assay 4 weeks after the injection, showing that PEDF-treated eyes had reduced numbers of apoptotic photoreceptors compared to controls. Retinal function was also assayed by ERG at 4 and 8 weeks p.i. The authors showed that 8 week-old RCS rats had almost no ERG response; however, PEDF treatment significantly improved the retinal functionality at 4 and 8 weeks after the injection. In conclusion, the gene transfer of human SERPINF1 in the RPE via lentiviral vectors results in the protection of photoreceptors from death and delayed degeneration in RCS rats (Miyazaki et al. 2003).

The same group (Murakami et al. 2008) showed that the lentivirus-mediated retinal gene transfer of PEDF in RCS rats prevented the nuclear translocation of apoptosis-inducing factor (AIF), resulting in reduced apoptotic loss of their photoreceptors and up-regulated Bcl-2 expression. They claimed that inhibiting the nuclear translocation of AIF is an essential mechanism of the protective activity of PEDF in this rat model.

The preventive effect of PEDF from photoreceptor degeneration in the RCS rats was also assayed by intravitreal injection of nanoparticles (NP) carrying 2.5 μg of the human protein (PEDF-NP) in the right eye of P21 rats. To evaluate the effects of PEDF-NP on photoreceptor survival, 2 additional groups of 3-week old RCS rats were injected in the right eye with 2.5 μg of PEDF protein or empty NP. Contralateral eyes were either left untreated or injected with phosphate buffered saline (PBS). The protective effect of each treatment was evaluated 4 and 8 weeks p.i. Eyes treated with PEDF-NP had a significant increase in ONL column height and in the number of photoreceptors but reduced TUNEL-positive cells compared to PEDF, empty NP and contralateral eyes. Moreover, PEDF-NP contributed to the preservation of rod-opsin levels and a- and b-wave amplitudes in ERG studies at 8 weeks p.i. (Akiyama et al. 2012).

93.2.5. Light-Induced Damage of Photoreceptors

Constant white light can induce retinal degeneration and is used in established rodentl models to degenerate photoreceptors. In Sprague-Dawley albino rats, exposure to white light (1200–1500 lux) continuously for 7 days reduces the ONL thickness to 12.5–30 % of that of unexposed eyes and eliminates the ERG response. Cao et al. (1999) tested the PEDF-mediated protective effect on photoreceptors damaged with light by intravitreally injecting human PEDF (2 μg) in one eye, using the PBS-injected contralateral eye as a control in rats of 2–5 month old. Injections were at 1 or 2 days pre-light exposure, or 0, 1 or 2 days after constant light (CL) exposure. ERG and histopathology analysis after 14 days of recovery showed that PEDF injected 1 or 2 days before light-induced damage protected photoreceptors from degeneration. When injected 2 days before light-induced damage, PEDF attenuated the reduction in ONL thickness and improved ERG response in eyes exposed to constant light for 3–10 days relative to controls. However, no protection by PEDF was observed after 14 days of CL exposure. Slightly enhanced protective effects have been reported when the eyes were pre-treated with PEDF combined with basic fibroblast growth factor (bFGF) (1 μg each).

Imai et al. (2005) have also assessed PEDF photoreceptor protection from damage induced by constant white light exposure using Lewis albino rats (females of 4–8 weeks). Progressive retinal degeneration, determined as the reduction of ONL thickness and cell number, was observed at 24, 96 and 168 h of continuous light exposure (2500 lux) in untreated rats. PEDF was delivered 3 days before light damage by intravitreal injection of adenoviral vector (AdPEDF.11), which promotes the expression of the gene under the regulation of the CMV promoter. Photoreceptor morphology was evaluated after 96 h of CL exposure and compared among animals injected with AdPEDF.11, untreated or injected with the empty AdNull.11 vector. PEDF rescued ONL thickness and number of photoreceptors as compared to controls. However, the empty vector itself had some protective effect when compared to the uninjected ones. Similarly, PEDF lowered the number of TUNEL-positive nuclei in the ONL (after 12 h of CL exposure) and improved the ERG response (after 48 h of exposure and 7–28 days of recovery after light damage). Animals injected with the empty vector again exhibited significant protection compared to untreated mice. The authors suggested that PEDF induced protection from apoptosis and loss of functionality in photoreceptors damaged by light exposure.

In summary, exogenous administration of the PEDF protein and the SERPINF1 gene transfer via viral vectors are beneficial in protecting photoreceptors against degeneration and death caused by genetic and/or environmental factors. The mechanisms by which PEDF acts on photoreceptors are beginning to emerge. Overall the data from several groups point to PEDF as an antiapoptotic factor that targets signaling pathways of the Bcl2 family and AIF in degenerating photoreceptors, likely mediated by interactions with PEDF-R (Subramanian et al. 2013) The findings also point to the applicability of the human PEDF sequence in rodent models of retinal degenerations. PEDF holds promise to clinical neuroprotection therapy, and in particular in ocular diseases.