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

2. METHODS

2.1. PDT

PDT was performed as described previously (Zacks et al., 2002) on adult BrownNorway rats. Laser parameters used were modified from the standard protocol used to treat humans as follows. First, the 689 nm laser fluence was reduced from 50 J/cm2 to 10 J/cm2 because preliminary experiments demonstrated severe retinal damage to the retina after PDT laser using a fluence greater than 10 J/cm2. Second, to permit interocular comparison between control and BDNF-treated eyes, the first eye of each rat received laser 3 minutes, and the second eye 4 minutes after intravenous verteporfin injection. All rats received 6 mg/m2 verteporfin. The PDT laser spot size (3.0 mm) was determined by the size of the dilated rat pupil. Because there is no macula in the rat fundus, all eyes were treated superior to the optic disk for reproducible localization using fundus imaging and histology.

2.2. Intravitreal Injections

Rats were anesthetized with xylazine (13 mg/kg) and ketamine (87 mg/kg), and pupils were dilated using phenylephrine 2.5% and atropine 1%. One eye of each rat was injected with 2 ml BDNF (2 mg/ml). The contralateral eye received 2 ml phosphate buffered saline (PBS) or remained uninjected to serve as a control. Intravitreal injections were performed transsclerally using a 0.5-inch, 32-gauge beveled needle 2 days prior to PDT.

2.3. Retinal Analysis

Retinal function was evaluated using both full-field and multifocal electroretinography (ERG). Fundus photographs and fluorescein angiograms were performed 7 days after PDT. Rats were perfused, and eyes were embedded in plastic and sectioned for study using light microscopy as described previously (LaVail and Battelle, 1975).

3. RETINAL TOXICITY OF PDT IS REDUCED BY BDNF

3.1. Fundus Appearance

As described elsewhere, (Paskowitz et al., 2004) one week after PDT, a discrete circle of choroidal and RPE hypopigmentation with RPE clumping was visible superior to the optic disk in all eyes. This region demonstrated early hypofluorescence with delayed filling of choroidal vessels. A small ring of hyperfluorescence around the hypofluorescent region was visible in the late frames of the fluorescein angiogram, consistent with RPE damage surrounding the region of choriocapillaris closure. There was no significant difference in the appearance of the PDT-treated region between eyes that received BDNF and control eyes.

3.2. Histological Characteristics after Verteporfin PDT

Compared to eyes treated with 689-nm laser in the absence of verteporfin dye, eyes that received verteporfin PDT demonstrated severe damage to the retina and RPE when studied

Figure 41.1. Light micrographs 1 week after PDT show the region of laser application in 3 eyes. (A) Retinal and RPE structure is normal after PDT laser without verteporfin dye. (B) PDT laser delivered 3-4 minutes after verteporfin injection produces severe retinal and RPE damage in this control eye treated with PBS 2 days prior to PDT. (C) The contralateral eye of the rat shown in (B) demonstrates greater ONL, IS and presumptive OS layer thickness after receiving BDNF 2 days prior to PDT. Epon-Araldite, 1 mm-thick sections; bar, 20 mm. Reproduced with permission of Investigative Ophthalmology and Visual Science in the format Other Book via Copyright Clearance Center from Paskowitz DM, Nune G, Yasumura D, Yang H, Bhisitkul RB, Sharma S, Matthes MT, Zarbin MA, LaVail MM, Duncan JL. BDNF Reduces the Retinal Toxicity of Verteporfin Photodynamic Therapy, Invest Ophthalmol Vis Sci 2004;45:4190-4196.

histologically 1 week after PDT (Fig. 41.1A and 41.1B). The outer nuclear layer (ONL) thickness in PDT-treated eyes was reduced from 9-10 rows in rats that had not received verteporfin PDT (Fig. 41.1A) to 3-4 irregular rows. In addition, variable photoreceptor outer segment and inner segment loss was apparent, with partial rosette formation in some areas (Fig. 41.1B). Damage to the RPE was present with pigment clumping, RPE cell attenuation and duplication, and migration of pigmented cells into the subretinal space. The retinal damage was limited to the PDT-treated region.

3.3. BDNF Improves Retinal Structure after PDT

Eyes that received BDNF 2 days prior to PDT showed less damage than contralateral control eyes. Although not normal, BDNF-treated eyes showed greater ONL thickness than controls (Fig. 41.1C). The number of surviving photoreceptors was quantified both by average ONL thickness in microns and by counting rows of nuclei in the ONL at the 5 most severely damaged regions in each eye. Significant photoreceptor rescue was observed in BDNF-treated eyes compared to control when measured both of these ways. The mean ONL thickness increased from 26.8 ± 1.1 mm (control) to 34 ± 1.1 mm (BDNF) (P < 10-7), and the mean number of rows of ONL nuclei increased from 3.6 ± 0.6 rows (control) to 5.0

± 0.3 rows (BDNF) (P < 10-6, n = 17).

3.4. BDNF Improves Retinal Function after PDT

Bilateral full-field ERG recording demonstrated no significant difference between eyes treated with BDNF and control eyes, suggesting BDNF injection had no effect on global retinal function. To assess retinal function in the region that received PDT, multifocal ERG testing was performed. An infrared fundus camera imaged the retina being tested, and these images were aligned with fundus photos demonstrating RPE hypopigmentation in the PDTtreated area to identify the multifocal ERG responses produced by the PDT-treated retina.

Figure 41.2A shows averaged multifocal ERG responses from 6 normal rat eyes in a topographic pseudocolor plot. Each eye was tested with the optic disk at the same location (white circle). Figure 41.2B shows a representative multifocal ERG performed 1 week after PDT in a PBS-injected eye, demonstrating a large focal region of reduced retinal function superior to the optic disk in the PDT-treated area. The contralateral, BDNF-treated eye shows improvement in retinal function in the PDT-treated region (Fig. 41.2C). When superimposed, the traces used to generate the topographic plots demonstrate similar improved retinal function centrally in the BDNF-treated eye (red traces) compared to control (black traces) (Fig. 41.2D). Among 7 rats tested, BDNF-treated eyes showed significantly less abnormal multifocal ERG responses centrally than contralateral control eyes (P = 0.03).

4. CONCLUSIONS

Intravitreal BDNF injection 2 days prior to verteporfin PDT reduced retinal damage in normal rats. Future studies will investigate the effect of BDNF prior to PDT in rats with experimental CNV. Adjunctive therapy with BDNF or other neuroprotective agents may reduce undesired side effects of visual disturbances and severe vision loss among patients treated with PDT.

5. ACKNOWLEDGMENTS

The authors thank Jose Velarde, Kate Donohue-Rolfe, Shivani Sharma, Kamran Hosseini, Robert J. Lowe, Nancy Lawson and Dean Cruz for assistance, and Dr. Erich Sutter for advice in carrying out the multifocal ERG analyses. This work was supported by a Career Development Award from Research to Prevent Blindness (JLD), National Institutes of

Figure 41.2. Multifocal ERG performed 1 week after PDT demonstrates reduced local retinal function in areas of PDT-treated retina. (A) Averaged responses from 6 normal rat eyes shown topographically demonstrate slightly reduced retinal function at the location of the optic disk (white circle). (B) Responses are reduced centrally in the PDT-treated area of an eye treated with PBS 2 days prior to PDT. (C) The contralateral, BDNF-treated eye shows improved retinal function in the PDT-treated area. (D) Superimposed multifocal ERG responses from the eyes shown in (B) (black traces) and (C) (red traces) show improved function centrally in the BDNF-treated eye. Reproduced with permission of Investigative Ophthalmology and Visual Science in the format Other Book via Copyright Clearance Center from Paskowitz et al., Invest Ophthalmol Vis Sci 2004;45:4190-4196. See also color insert.

Health Grants EY01919, EY02162 (MML) and EY00415 (JLD); the Bernard A. Newcomb Macular Degeneration Foundation (JLD, MML); the Foundation Fighting Blindness, Inc.; the Macular Vision Research Foundation (MML); and That Man May See, Inc. MML is a Research to Prevent Blindness Senior Scientist Investigator.

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directly (de Boer et al., 2001). However, in most cases, the temporal control in addition to tissue localization remains a limiting factor for this method of therapy.

In this chapter we will discuss several methods used in controlling VEGF expression at both the transcriptional and translational levels in addition to post translational inhibition of VEGF activity and how these may be exploited as possible therapies for neovascular diseases of the eye.

2. OLIGONUCLEOTIDE THERAPY

Oligonucleotides consist of short sequences of nucleotides generally between 15 and 30 base pairs in lengths and possess a phosphodiester (P) backbone in the native form. Many chemical modifications can be made to improve the biological half-life of oligonucleotides in vivo and include substituting the P backbone for phophorothioate (PS), which results in the oligonucleotide being less susceptible to the effects of nucleases. Oligonucleotides used in expressional control of genes consist of three main forms; sense oligonucleotides, which as the name suggests resembles a protion of the sense stand of duplex DNA; antisense oligonucleotides, which resemble a protion of the complimentary stand of duplex DNA; and aptamers, whose activity relies on the secondary structure formed by folded oligonucleotides. Depending on which form is used, oligonucleotides are able to control gene expression at either the transcriptional or translational level in addition to affecting downstream, post translation activities.

All three oligonucleotide types have been explored as a possible control for increased VEGF expression associated with neovascularization in aged related macular degeneration (AMD). In our laboratory, we have examined the 5¢-UTR sequence of the VEGF gene for possible oligonucleotide target sites. From this study, a sense oligonucleotide (DS-085) was found to significantly down-regulate VEGF expression in cultured cells (Garrett et al., 2001). Subsequent experiments later demonstrated that in vivo, intravitreal administration of DS085 resulted in retinal cells being transfected, in addition, DS-085 mediated a reduced angiogenic response in a laser photocoagulation induced choroidal neovascularisation (CNV) rodent model (Marano et al., 2003). The most likely mechanism of downregulation in this case was thought to occur through hybridization of the oligonucleotide in the major groove of duplex DNA forming of a triple helix. This would result in polymerase arrest and lead to a reduction in the quantity of transcript available for translation.

In addition to sense oligonucleotides, an antisense oligonucleotide has been developed to target the VEGF-R2 receptor (KDR/Flk) to control neovascularization of the cornea (Berdugo et al., 2003). Antisense oligonucleotides bind to their compliment sequence on the single stranded mRNA. From this point, regulation of the gene may occur in one of three ways; through polymerase arrest; through occupational inhibition, whereby the bound oligonucleotide occupies a functional site such as the 5¢ end capping sequence; and finally through RNAase recruitment caused by the presence of the bound oligonucleotide, which results in the rapid degradation of the mRNA strand.

While these results are promising, it was an aptamer (EYE001) that first progressed to the clinical trial stage as a means of controlling neovascularization of retina (The Eyetech Study Group, 2002; The Eyetech Study Group, 2003). Aptamers are produced by exploiting an inherent natural phenomena of nucleic acids i.e. depending on their sequence, they are able to fold and form complex secondary structures. The folded oligonucleotide then acts similar to an antibody in recognizing and binding to specific elements on a protein. In

the case of EYE001, the aptamer binds to VEGF and inhibits its interaction with its cell surface receptor molecules, preventing any further downstream activity.

3. GENE THERAPY

A primary drawback in the use of oligonucleotides as a therapeutic has been the relative biological instability of the compound. Oligonucleotides are generally short lived in vivo, and while the use of modified chemistries has led to improved half lives, longevity remains a limiting issue and re-administration is required on a regular basis. In this respect, gene therapy, whereby a modified, recombinant gene is inserted into the genome, is a superior method of controlling gene function. Once inserted, the new gene remains functional for an indefinite period, producing the therapeutic agent for the life of the cell. In addition to longevity, specific cell types may be targeted for gene expression through the use of cell and tissue specific promoters, which control gene expression. This approach may be useful for systemic delivery of gene constructs as it ensures that the gene is not expressed in tissues where its presence may prove detrimental. For the control of VEGF associated with ocular angiogenesis, this approach of secretion gene therapy has been explored on several occasions. Constructs containing the von Hippel-Lindau gene, (Akiyama et al., 2004) pigment epithelium-derived factor gene, (Auricchio et al., 2002) and genes expressing the soluble VEGF receptor sFlt-1, (Bainbridge et al., 2002; Lai et al., 2001; Lai et al., 2002) have been used to transform retinal cells in vivo to produced recombinant proteins and control the expression and/or the downstream effects of VEGF. In these cases, the gene construct was delivered to cells using a modified virus, which has been engineered to be unable to replicate. The virus genomes containing the recombinant gene constructs are packaged into virus particles in vitro, which are then injected into the eye. The virus particles attaches to the cell surface and injects the nucleic acid material into the host cell, where it is incorporated into the genome and expressed using the hosts metabolic processes.

Several limitations exist in this system of gene therapy. Using viral vectors to insert genes into the host’s genome runs the risk of causing insertional inactivation of other genes within the cell, which can be highly damaging depending on the gene that is inactivated (Baum et al., 2004). In addition, while it is possible to exert some crude control over the level of expression through the use of weak or strong promoters, a more refined, temporal control system is more difficult to achieve. In the case of angiogenic diseases of the eye, VEGF concentrations may fluctuate over time; therefore, maintaining high levels of an antiVEGF compound may prove detrimental in the long term. Attempts have been made to address this issue, and involve incorporating the same elements that control the expression of endogenous VEGF (such as the hypoxia control element), into the anti-VEGF transgene construct (Bainbridge et al., 2003). In addition to these logistical issues involved in the science and ‘workability’ of gene therapy, the use of viruses as a delivery vehicle for modified genetic material has not received wide public acceptance at this point in time.

4. RNA INTERFERENCE (RNAi)

RNAi through the use of short interfering RNA (siRNA) molecules has also proven efficient at controlling laser induced CNV in mouse and primate models (Reich et al., 2003; Tolentino et al., 2004). The mechanism of siRNA occurs through the activity of a multi-

component ribonuclease-protein complex termed RISC (RNA-induced silencing complex), which recruits siRNA and guides them to the homologous portion on the mRNA transcript. This triggers the degradation of the mRNA strand, once again reducing the quantity of transcript available for translation. Several advantages of RNAi include high specificity for the target gene and as it utilizes a naturally occurring pathway for mRNA degradation, there are relatively few side effects. In addition, siRNA may be administered directly as duplex RNA strands for immediate recruitment by the RISC, alternatively, siRNA can be introduced as a gene therapy style construct whereby the sense sequence of the siRNA is cloned into a vector upstream of the reverse compliment sequence separated by a short spacer sequence. When the construct is transcribed, the resulting mRNA strand will fold on itself forming a duplex strand with a hairpin loop at one end. This structure can then be processed by the dicer protein, which removes the hairpin and facilitates recruitment by the RISC (Arenz and Schepers, 2003).

5. CONCLUSION

The use of anti-VEGF therapies are becoming a popular method for controlling diseases where angiogenesis is one of the major pathologies. For angiogenic diseases affecting the eye, several such methods are currently being explored, each with their own merits and limitations. At present, efficient delivery and longevity of the biological effect continue to be the major issues facing novel therapeutics. However, with the dearth of techniques and methods aimed at controlling VEGF expression currently being developed, a decision will need to be made on which is the most suitable as a clinical treatment. Future research may eventually indicate that the most effective treatment is a combination of two or more methods i.e. one treatment to achieve a rapid early response followed by a second, alternative treatment which provides a longer effect.

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