Neurotrophic Factors

The neurotrophic approach to treating retinal diseases is of therapeutic relevance in ophthalmology because trophic factors target apoptotic mechanisms that are independent of the genetic mutation(s) for the disease. Treatment with soluble neurotrophic factors has been shown to prevent the death of retinal neurons in complex or difficult-to-treat ocular diseases where the etiologies are not completely defined or where mutations in several genes are associated with the progression of the disease. The method of delivering highly concentrated amounts of trophic factors to the eye is straightforward and relatively simple to perform and bypasses the need for complex viral or non viral delivery systems. Subretinal or intravitreal injections are common routes of delivery to the affected area. Preparative amounts of neurotrophic proteins can be easily purified from recombinant expression systems, and combinations of several therapeutic proteins can be administered simultaneously to the area of pathology. Another clinically appealing feature of this approach is that the therapeutic efficacy of soluble trophic factors is not hindered by the immunologic and toxic limitations that are usually associated with vector-mediated delivery of DNA.

Designing an effective treatment protocol, however, is based on adequate knowledge of the pharmacokinetics of the trophic factor in a biological system and establishing its ability to function in a physiological environment. A limitation associated with the use of trophic factors in retinal diseases is the need for multiple treatments to significantly effect reversal of the pathology. Unless these agents are administered topically to the eye or packaged in a slow-release system, the method, while safe, is not convenient for long-term management of retinal diseases.

One endogenous neurotrophic factor, which we have isolated and characterized in our laboratory, is a 50 kDa protein, pigment epithelum-derived factor (PEDF) (103,213), so named because it was initially isolated from the retinal pigment epithelium. Functionally, there is a striking association between PEDF, or the lack thereof, and biological processes involving survival and death of retinal cells as well as angiogenic mechanisms in the eye (35,215-219,229). The PEDF gene is well characterized and is classified as a serine protease inhibitor because of its structural and sequence homology with members of this group of genes (104,227). In addition, PEDF maps to human chromosome 17p13.3 and is tightly linked to an autosomal dominant retinitis pigmentosa locus in that region of the chromosome. Several polymorphisms have been identified in the gene, but none has shown a direct correlation between PEDF and specific retinal pathologies (220-224). However, in vivo and in vitro studies with the soluble protein consistently demonstrate the neuroprotective and antiangiogenic activities of PEDF, suggesting a promising future for this protein as a therapeutic that can circumvent the effects of specific mutations or chemical stimulators that cause the death of visual cells.

We first identified the PEDF protein in the conditioned medium of primary cultures of fetal human RPE cell and in the interphotoreceptor matrix (IPM) located between the RPE and neural retina (103,213,225,226). The protein is expressed in high concentration in fetal and young adult RPE cells but appears to be severely downregulated in senescing RPE cultures, a finding that suggests that it may play a role in age-related retinal dysfunctions. In one of the first studies, we showed that PEDF inhibits the growth of a human retinoblastoma cell line (Y79) by inducing differentiation of the tumor cells into a phenotype that is reminiscent of matured neurons. In nontreated cultures, the Y79 cells grow as clusters in suspension and do not spontaneously attach or differentiate. Treatment of these cells with a small dose of PEDF is effective in promoting extensive neurite outgrowths from the tumor cells, upregulating neurofilament proteins and neuron-specific enolase, and promoting connections between the growing neurites of newly differentiated cells (Fig. 8). Approximately 90% of the cultures attach and differentiate on poly-D-

Figure 8 PEDF induces differentiation in human Y79 retinoblastoma cells. (Left) Proliferating nontreated cells are rounded in appearance. (Right) Cells treated with 50 ng/mL PEDF. Treated cells are nonproliferating, project long neurites, and stain positive for the neurofilament protein.

lysine-coated surfaces and maintain their differentiated phenotype for longer than 4 weeks in culture after a single PEDF treatment. This period is extended by additional doses of PEDF to the cultures. From these initial results, PEDF is implicated as a useful therapeutic for reducing ocular tumor progression and prevent degeneration, associated with the growing tumor, in the surrounding retinal tissue.

A role for PEDF in angiogenesis emerged when Dawson and colleagues provided convincing evidence that PEDF, in addition to its neurotrophic activity, functions as an angiogenic inhibitor (214). They showed that PEDF inhibited the formation of new blood vessels in a rat model of corneal neovascularization and also prevented the migration of endothelial cells in a dose-dependant manner. The group also reported that PEDF inhibited endothelial cell migration even in the presence of such angiogenic stimulators as bFGF, VEGF, interleukin-8, aFGF, and lyopohosphatic acid. Furthermore, when tested against other antiangiogenic agents, such as angiostatin and endostatin, its efficacy was slightly more potent than those inhibitors. In support of their study, we showed higher concentrations of PEDF in the vitreous of patients with avascular proliferative vitreal retinopathy and diabetic retinopathy when compared to patients with retinal pathologies associated with increased angiogenic activity (228). Based on the clinical data, as well as vivo studies using animal models, it appears that the concentration of PEDF in the eye is important to the vascular state of ocular tissues. These results have stirred much interest in the ophthalmic field and have encouraged several groups to exploit the therapeutic potential of PEDF in ocular diseases, such as age-related macular degeneration, where both cell death and increased angiogenesis contribute to severe visual loss.

In several modes of induced retinal degeneration, convincing evidence that photoreceptor cells survive in the presence of PEDF has been provided. In an in vitro model of retinal damage, a large percentage of retinal neurons undergo apoptosis and die after exposure to hydrogen peroxide (H2O2) (215-217). Hydrogen peroxide is a reactive oxygen species (ROS) found in elevated concentration in light-damaged retinas. It is believed that ROS contribute to degenerative and aging processes in the eye. To test the protective effects of PEDF in H2O2-damaged eyes, rat retinal cultures were treated with PEDF before they were exposed to H2O2. In the presence of PEDF, apoptotic mechanisms that led to cell death were inhibited, and approximately 60% of the cells that would have otherwise degenerated survived. Furthermore, a high percentage of the treated cells were rhodopsin positive and, therefore, highly likely to be rod photoreceptors. In vivo, the retina can also be damaged by exposure to constant light, in part because of the generation of reactive oxygen species in a high-lipid-content region of the retina. Photoreceptor degeneration is visible as early as the third day of light exposure in the rat. In a study aimed at testing the effect of PEDF in light-damaged rat eyes, we found that a single intravitreal injection of PEDF, prior to chronic light exposure, was potent enough to inhibit the light damage effects on photoreceptor. This was clearly seen in histological preparations of the treated retina and electrophysical measurements of the nuclei in the outer nuclear layer (ONL) (Fig. 9).

In a similar study, photoreceptor survival with PEDF treatment was examined in two mutant mice types, homozygous retinal degeneration (rd/ rd) and retinal degeneration slow (rds/rds), in which photoreceptor loss is a hallmark of the mutations. Intravitreal injections of PEDF resulted in a transient but significant delay in the death of photoreceptors in both mutants (229). The efficacy of PEDF was also assessed in an embryonic Xenopus model of retinal degeneration (218). In this model, mechanical removal of the RPE cells from the Xenopus retina results in a distortion of photoreceptor ultrastructure and disruption of outersegment formation,

Figure 9 Neuroprotection of rat photoreceptor cells (ONL) by PEDF after 7 days exposure to constant light. Severe damage to photoreceptor cells (ONL) is seen after light exposure. (A) Unexposed normal rat retina; (B) retinal exposed to 7 days constant light. (C-F) Cross section of rat retina exposed to 7 days of constant light after injections with PEDF, bFGF, or both: (C) PBS; (D) 1 ^g PEDF; (E) 1 ^g bFGF; (F) 1 ^g bFGF + 1 ^g PEDF; (inset) electroretinograms (ERG) B-waves amplitudes of the retina with each treatment. RPE, Retinal pigment epithelial cells; OS, photoreceptor outersegment; IS photoreceptor inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Courtesy of Dr. James McGinnis and Wei Cao, Dean McGee Eye, Oklahoma, USA.)

Figure 9 Neuroprotection of rat photoreceptor cells (ONL) by PEDF after 7 days exposure to constant light. Severe damage to photoreceptor cells (ONL) is seen after light exposure. (A) Unexposed normal rat retina; (B) retinal exposed to 7 days constant light. (C-F) Cross section of rat retina exposed to 7 days of constant light after injections with PEDF, bFGF, or both: (C) PBS; (D) 1 ^g PEDF; (E) 1 ^g bFGF; (F) 1 ^g bFGF + 1 ^g PEDF; (inset) electroretinograms (ERG) B-waves amplitudes of the retina with each treatment. RPE, Retinal pigment epithelial cells; OS, photoreceptor outersegment; IS photoreceptor inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Courtesy of Dr. James McGinnis and Wei Cao, Dean McGee Eye, Oklahoma, USA.)

a condition that eventually leads to the death of photoreceptors. In this study it was found that administration of PEDF to the retina after RPE detachment promoted features such as reorganization of the outersegments, increased opsin synthesis by the photoreceptors, and a general correction of the ONL ultrastructure. Moreover, the activity of PEDF in RPE detached retinas was blocked by a neutralizing polyclonal antibody to the 50 kDa native protein, suggesting that the rescuing effect was specific.

From these findings it appears that the course of photoreceptor degeneration can be altered by neuroprotective agents, like PEDF, which can prevent pathomorphological and apoptotic effects of neurodegenerative promoters. Other trophic factors, such as bFGF, CNTF, and BDNF have shown similar results in promoting photoreceptor survival in naturally occurring inherited retinal degeneration models with genetic defects similar to those in human inherited retinal degeneration (230). Survival factors are, therefore, particularly attractive therapeutic tools that may prove to be increasingly important in treating retinal degenerations if long-term, sustained delivery to the affected area is to be maintained.

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