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

Baum, C., von Kalle, C., Staal, F. J., Li, Z., Fehse, B., Schmidt, M., Weerkamp, F., Karlsson, S., Wagemaker, G., Williams, D. A., 2004, Chance or necessity? Insertional mutagenesis in gene therapy and its consequences.

Mol Ther, 9:5-13.

Berdugo, M., Valamanesh, F., Andrieu, C., Klein, C., Benezra, D., Courtois, Y., Behar-Cohen, F., 2003, Delivery of antisense oligonucleotide to the cornea by iontophoresis. Antisense Nucleic Acid Drug Dev, 13:107-114.

Boulton, M., Foreman, D., Williams, G., McLeod, D., 1998, VEGF localisation in diabetic retinopathy. Br J Ophthalmol, 82:561-568.

Coles, L. S., Bartley, M. A., Bert, A., Hunter, J., Polyak, S., Diamond, P., Vadas, M. A., Goodall, G. J., 2004, A multi-protein complex containing cold shock domain (Y-box) and polypyrimidine tract binding proteins forms on the vascular endothelial growth factor mRNA. Potential role in mRNA stabilization. Eur J Biochem, 271:648-660.

de Boer, R. A., Siebelink, H. J., Tio, R. A., Boomsma, F., van Veldhuisen, D. J., 2001, Carvedilol increases plasma vascular endothelial growth factor (VEGF) in patients with chronic heart failure. Eur J Heart Fail, 3:331333.

Dibbens, J. A., Miller, D. L., Damert, A., Risau, W., Vadas, M. A., Goodall, G. J., 1999, Hypoxic regulation of vascular endothelial growth factor mRNA stability requires the cooperation of multiple RNA elements. Mol Biol Cell, 10:907-919.

Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., Semenza, G. L., 1996, Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol, 16:46044613.

Garrett, K. L., Shen, W. Y., Rakoczy, P. E., 2001, In vivo use of oligonucleotides to inhibit choroidal neovascularisation in the eye. J Gene Med, 3:373-383.

Iida, K., Kawakami, Y., Sone, H., Suzuki, H., Yatoh, S., Isobe, K., Takekoshi, K., Yamada, N., 2002, Vascular endothelial growth factor gene expression in a retinal pigmented cell is up-regulated by glucose deprivation through 3¢ UTR. Life Sci, 71:1607-1614.

Lai, C. M., Brankov, M., Zaknich, T., Lai, Y. K., Shen, W. Y., Constable, I. J., Kovesdi, I., Rakoczy, P. E., 2001, Inhibition of angiogenesis by adenovirus-mediated sFlt-1 expression in a rat model of corneal neovascularization. Hum Gene Ther, 12:1299-1310.

Lai, Y. K., Shen, W. Y., Brankov, M., Lai, C. M., Constable, I. J., Rakoczy, P. E., 2002, Potential long-term inhibition of ocular neovascularisation by recombinant adeno-associated virus-mediated secretion gene therapy.

Gene Ther, 9:804-813.

Levy, N. S., Goldberg, M. A., Levy, A. P., 1997, Sequencing of the human vascular endothelial growth factor (VEGF) 3¢ untranslated region (UTR): conservation of five hypoxia-inducible RNA-protein binding sites.

Biochim Biophys Acta, 1352:167-173.

Marano, R. J., Wimmer, N., Kearns, P. S., Thomas, B. G., Toth, I., Brankov, M., Rakoczy, P. E., 2003, Inhibition of in vitro VEGF expression and choroidal neovascularization by synthetic dendrimer peptide mediated delivery of a sense oligonucleotide. Experimental Eye Research, Accepted.

Marano, R. J., Brankov, M., Rakoczy, P. E., 2004, Discovery of a novel control element within the 5¢UTR of VEGF: Regulation of expression using sense oligonucleotides. J Biol Chem, 279(36):37808-37814.

Ohno-Matsui, K., Morita, I., Tombran-Tink, J., Mrazek, D., Onodera, M., Uetama, T., Hayano, M., Murota, S. I., Mochizuki, M., 2001, Novel mechanism for age-related macular degeneration: an equilibrium shift between the angiogenesis factors VEGF and PEDF. J Cell Physiol, 189:323-333.

Reich, S. J., Fosnot, J., Kuroki, A., Tang, W., Yang, X., Maguire, A. M., Bennett, J., Tolentino, M. J., 2003, Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model. Mol Vis, 9:210-216.

The Eyetech Study Group, 2002, Preclinical and phase 1A clinical evaluation of an anti-VEGF pegylated aptamer (EYE001) for the treatment of exudative age-related macular degeneration, Retina, 22:143-152.

The Eyetech Study Group, 2003, Anti-vascular endothelial growth factor therapy for subfoveal choroidal neovascularization secondary to age-related macular degeneration: phase II study results. Ophthalmology, 110:979-986.

Tolentino, M. J., Brucker, A. J., Fosnot, J., Ying, G. S., Wu, I. H., Malik, G., Wan, S., Reich, S. J., 2004, Intravitreal injection of vascular endothelial growth factor small interfering RNA inhibits growth and leakage in a nonhuman primate, laser-induced model of choroidal neovascularization. Retina, 24:132-138.

with diffuse macular edema refractory to laser treatment. Intravitreal injection of TA has been shown to reduce retinal thickening, improve blood retinal barrier function and improve visual acuity in patients with diffuse macular edema. (Jonas et al., 2003; Martidis et al., 2002) TA has also been shown to reduce vitreal levels of Vascular Endothelial Growth Factor (VEGF) and Stromal-Derived Factor 1 (SDF1), angiogenic growth factors that potentially play an important role in the pathogenesis of diffuse macular edema. (Brooks et al., 2004; Funatsu et al., 2003)

Central retinal vein occlusion (CRVO) is another common retinal vascular disorder that has shown promising results following treatment with TA. Experimental studies in monkeys have shown that a hypoxic environment is produced in the retina following venous occlusion. (Hockley et al., 1979) This hypoxia causes functional structural changes in the retinal capillaries resulting in increased permeability and retinal edema, potentially through factors including VEGF and SDF1. Although both ischemic and nonischemic types of CRVO show anatomical improvement following treatment with TA, visual acuity does not improve as well in ischemic CRVO. (Ip et al., 2004)

Cystoid macular edema (CME) can result from cases of long standing retinitis pigmentosa (RP), uveitis, or Irvine-Gass Syndrome. The exact mechanism of CME occurrence in patients with RP is not known. The high prevalence of antiretinal autoantibodies in those with CME associated with RP suggests an inflammatory, autoimmune process. (Heckenlively et al., 1999) There has been one previous case report of a patient with CME and RP treated with intravitreal injections of TA. (Sallum et al., 2003; Saraiva et al., 2003) Intravitreal TA not only allows improvement in visual acuity with minimal systemic sideeffects, but has also been shown to be effective in reducing visual loss in those with longterm refractory inflammatory CME. (Antcliff et al., 2001)

Complications of corticosteroids administered ocularly include intraocular pressure elevation, retinal detachment, vitreous hemorrhage, cataractogenesis, endophthalmitis, and potential cytotoxicity to photoreceptors and the retinal pigment epithelium. TA is a minimally water-soluble steroid that is injected in a suspension form. The decreased water solubility contributes to its prolonged duration of action. After intravitreal injection, the duration of effect has been reported to last between 4 weeks and 9 months. (Beer et al., 2003; Jonas et al., 2004)

2. METHODS

A retrospective chart review was performed for the historical period of June through November 2003. During this period, 51 eyes of 50 patients were treated for macular edema with intravitreal injections of TA at the Moran Eye Center. This includes 18 patients with chronic diffuse macular edema due to the following conditions: retinitis pigmentosa (two patients), CSME (seven patients), Irvine-Gass Syndrome (four patients), and central retinal vein occlusion (five patients). Of the eyes treated for macular edema, 20 eyes were measured before and after TA injection with optical coherence tomography (OCT) on a Zeiss-Humphrey Stratus OCT version 3.0. The data for these eyes were analyzed for this study. Pre-injection OCT measurements were taken between 47 days and zero days before TA injection, with a mean of seven days and a median of zero days. The time of the first post-injection measurement ranged from 21 days to 62 days with a mean of 35 days and a median of 33 days.

One-time injections of triamcinolone acetonide were performed as follows: eyes were anesthetized with proparacaine or tetracaine and sterilized with topical 5% povidone-iodine solution. Four mg of TA was injected through a 27-gauge needle 3.5 mm temporal to the limbus in phakic patients and 3.0 mm temporal from the limbus in pseudophakic patients.

Three parameters were analyzed between the pre-injection and first post-injection visit: LogMAR best-corrected visual acuity, OCT foveal thickness in microns, and Goldmann or Tono-Pen XL applanation pressure in mm Hg. The differences in these values were analyzed with a one-tailed Wilcoxon Signed-Rank analysis for statistical significance.

3. RESULTS

Patients treated for macular edema were classified into four categories: RP, diabetic, Irvine-Gass, and vascular. Foveal thickness, measured by OCT, improved proportionately in all categories, but was greatest in RP patients (Figure 43.1). Visual acuity also showed improvement in all categories, but was greatest in Irvine-Gass and vascular patients and least in diabetics (Figure 43.2). Intraocular pressures increased in all categories, as expected, but the overall magnitude of that change was only 2.5 mm Hg. Long-term OCT data are available on too few patients to achieve statistical relevance, but the overall trend showed fluid reaccumulation starting 60 days post-injection without return to pre-injection levels.

Figure 43.1. Mean Foveal Thickness, measured by OCT, is broken down into RP, diabetic, Irvine-Gass, and vascular categories. Thickness improved in all categories, although most improvement was seen in RP patients.

Figure 43.2. Visual Acuity improved in all categories. Improvement was greatest in Irvine-Gass and vascular patients and least in diabetics.

Four eyes of two RP patients were treated for CME. In all cases, there was a marked reduction of CME on OCT measurements (Figure 43.1 & 43.3) and some improvement of visual acuity (Figure 43.2).

4. DISCUSSION

Intravitreal TA injection appears to be a promising treatment option for macular edema from various retinal diseases including RP. Our OCT foveal thickness data show definite, and sometimes dramatic, reductions in macular thickness during the expected duration of action of TA. Pressure increases were relatively modest, 2.5 mm Hg on average and were reversed by topical medications (data not shown). Certainly, there is a greater pressure rise in the subset of steroid-responding patients. Even so, the highest IOP response at 3-9 week follow-up was 8 mm Hg. Unfortunately, visual acuity did not consistently increase with decreased foveal thickness. The improvement in visual acuity was most impressive in the Irving-Gass class of patients, perhaps in keeping with their shorter duration of edema. The diabetic patients in our series showed only a modest improvement in visual acuity. Perhaps this reflects the fact that the diabetic patients selected for intravitreal TA injections at our center predominantly had long-standing chronic diffuse macular edema. One limitation to intravitreal TA treatment is apparent even in this limited study: the long-term follow-up OCT

Figure 43.3a. OCT scans of right eye of patient with CME resulting from RP. Top image is before TA injection, notice large intraretinal cysts; bottom image is 10 weeks after injection. Foveal thickness improved from 672 mM to 192 mM. Foveal contour was restored.

Figure 43.3b. OCT scans of left eye of patient with CME resulting from RP. Top image is before TA injection, notice large intraretinal cysts; bottom image is 14 weeks after injection. Foveal thickness improved from 661 mM to 246 mM. Notice foveal contour was restored, yet there were still intraretinal cysts.

numbers seem to reach their minimum around 60 days, and the effect was gone by 150 days in our longest follow-up. This study indicates further investigation with more patients and longer duration are warranted.

5. REFERENCES

Antcliff, R.J., Spalton, D.J., Stanford, M.R., Graham, E.M., Ffytche, T.J. & Marshall, J. (2001). Intravitreal triamcinolone for uveitic cystoid macular edema: an optical coherence tomography study. Ophthalmology, 108:765-72.

Beer, P.M., Bakri, S.J., Singh, R.J., Liu, W., Peters, G.B., 3rd & Miller, M. (2003). Intraocular concentration and pharmacokinetics of triamcinolone acetonide after a single intravitreal injection. Ophthalmology, 110:681-6.

Bresnick, G.H. (1983). Diabetic maculopathy. A critical review highlighting diffuse macular edema. Ophthalmology, 90:1301-17.

Bresnick, G.H. (1986). Diabetic macular edema. A review. Ophthalmology, 93:989-97.

Brooks, H.L., Jr., Caballero, S., Jr., Newell, C.K., Steinmetz, R.L., Watson, D., Segal, M.S., Harrison, J.K., Scott, E.W. & Grant, M.B. (2004). Vitreous levels of vascular endothelial growth factor and stromal-derived factor 1 in patients with diabetic retinopathy and cystoid macular edema before and after intraocular injection of triamcinolone. Arch Ophthalmol, 122:1801-7.

Funatsu, H., Yamashita, H., Ikeda, T., Mimura, T., Eguchi, S. & Hori, S. (2003). Vitreous levels of interleukin-6 and vascular endothelial growth factor are related to diabetic macular edema. Ophthalmology, 110:1690-6.

Heckenlively, J.R., Jordan, B.L. & Aptsiauri, N. (1999). Association of antiretinal antibodies and cystoid macular edema in patients with retinitis pigmentosa. Am J Ophthalmol, 127:565-73.

Hockley, D.J., Tripathi, R.C. & Ashton, N. (1979). Experimental retinal branch vein occlusion in rhesus monkeys. III. Histopathological and electron microscopical studies. Br J Ophthalmol, 63:393-411.

Ip, M.S., Gottlieb, J.L., Kahana, A., Scott, I.U., Altaweel, M.M., Blodi, B.A., Gangnon, R.E. & Puliafito, C.A. (2004). Intravitreal triamcinolone for the treatment of macular edema associated with central retinal vein occlusion. Arch Ophthalmol, 122:1131-6.

Jonas, J.B., Degenring, R.F., Kamppeter, B.A., Kreissig, I. & Akkoyun, I. (2004). Duration of the effect of intravitreal triamcinolone acetonide as treatment for diffuse diabetic macular edema. Am J Ophthalmol, 138:158-60.

Jonas, J.B., Kreissig, I., Sofker, A. & Degenring, R.F. (2003). Intravitreal injection of triamcinolone for diffuse diabetic macular edema. Arch Ophthalmol, 121:57-61.

Klein, R., Klein, B.E., Moss, S.E., Davis, M.D. & DeMets, D.L. (1984). The Wisconsin epidemiologic study of diabetic retinopathy. IV. Diabetic macular edema. Ophthalmology, 91:1464-74.

Martidis, A., Duker, J.S., Greenberg, P.B., Rogers, A.H., Puliafito, C.A., Reichel, E. & Baumal, C. (2002). Intravitreal triamcinolone for refractory diabetic macular edema. Ophthalmology, 109:920-7.

Massin, P., Audren, F., Haouchine, B., Erginay, A., Bergmann, J.F., Benosman, R., Caulin, C. & Gaudric, A. (2004). Intravitreal triamcinolone acetonide for diabetic diffuse macular edema: preliminary results of a prospective controlled trial. Ophthalmology, 111:218-24; discussion 224-5.

Sallum, J.M., Farah, M.E. & Saraiva, V.S. (2003). Treatment of cystoid macular edema related to retinitis pigmentosa with intravitreal triamcinolone acetonide: case report. Adv Exp Med Biol, 533:79-81.

Saraiva, V.S., Sallum, J.M. & Farah, M.E. (2003). Treatment of cystoid macular edema related to retinitis pigmentosa with intravitreal triamcinolone acetonide. Ophthalmic Surg Lasers Imaging, 34:398-400.

Yoshikawa, K., Kotake, S., Ichiishi, A., Sasamoto, Y., Kosaka, S. & Matsuda, H. (1995). Posterior sub-Tenon injections of repository corticosteroids in uveitis patients with cystoid macular edema. Jpn J Ophthalmol, 39:71-6.

Zamir, E., Read, R.W., Smith, R.E., Wang, R.C. & Rao, N.A. (2002). A prospective evaluation of subconjunctival injection of triamcinolone acetonide for resistant anterior scleritis. Ophthalmology, 109:798-805; discussion 805-7.

on selective rod replacement, obtained by use of a vibratome sectioning method. Our first results demonstrated that when pure photoreceptor layers were transplanted to the subretinal space in 5 week old rd1 mice [at this stage very few rods remain (<0.02%) whereas about 30% of the cones are still present] the transplant induces a significant increase in the number of surviving cones (an average increase of 30%, p < 0.001) compared to that of non-treated congenic retina.9 This trophic effect can be detected distant from the transplant, suggesting the existence of a diffusible factor liberated by the transplanted cells. Tests carried out in vitro on rd1 retina and wild type retina co-cultures confirmed this hypothesis.10 This study showed that about 40% of cones normally lost during the sixth postnatal week are saved when cultured in the presence of rod-rich samples. This effect is photoreceptor specific since transplants of inner retina exert no beneficial effects on cone survival.11,12 The neuroprotective activity (40-50% increase in viability) on cones is heat labile and has an apparent molecular weight larger than 15 kDa.13 These experiments indicate that the activity is carried out by protein(s): the Rod-dependent Cone Viability Factors (RdCVFs).

Taken together, these assays show the existence of at least one trophic factor secreted by rods that is essential for cone survival. Rod degeneration in rd1 mice and RP patients might hence lead to survival factor deprivation and consequently progressive cone loss. A key implication is that prevention of apoptotic rod death could lengthen cone survival. Such a mechanism, which has never been proposed prior to our work, provides a justification for the numerous strategies aimed at preserving non-functional rods, since these will protect cones.14-19 In humans, diagnosis is often made at late stages, and in the absence of residual rods only PR transplantation (mainly rods) could restore the expression of these factors allowing to block or reduce secondary cone degeneration.19 The potential clinical importance of such factors is obvious, cone loss representing the main cause of visual handicap in RP and AMD. Of importance, since cone loss is a late-onset downstream event and occurs independently of the specific mutation expressed by rods, a potentially broad number of patients could benefit from such therapy.

The observed trophic effect of rod transplants on cone survival suggests that nonfunctional rod protection could be sufficient to preserve cone viability.10,12,19,20

3. IDENTIFICATION OF RdCVF

Our systematic approach to characterize factors involved in cone viability has led to identification of a new gene (RdCVF) encoding a secreted protein for which we will explore its functions.

We postulate that the degeneration of rods of the rd1 mouse retina is leading to the loss of expression of secreted protein factor(s) essential for cone viability. This mechanism of cone degeneration is also likely in human retinas affected with RP.25 The identification of the genes encoding these Rod-dependent Cone Viability Factors is a prerequisite to a therapy aimed at preventing the secondary loss of cones and of vision.

To identify the RdCVF genes, we used a systematic strategy based on a functional assay using cone–enriched cultures. We developed a high throughput cone-enriched culture system using chicken retina.26,27 Contrarily to the mammals, birds have retinas dominated by cones. In these cultures, the primary postmitoting cells (60-80% cones) are degenerating over a period of few days. An increase in cell survival was observed when cultured in the presence

of conditioned media isolated from wild-type mouse13. The viability activity on chicken cone, as for RdCVF, is heat labile has an apparent molecular weight larger than 15 kDa. The chicken embryo retinal culture system is an easy, reproducible and high throughput cone viability assay.27

We constructed an expression library from wild type mouse retina and tested all the genes for their potential to promote chicken cone survival by expression cloning methods. Briefly, pooled by 100 clones from the expression library were transfected into a cell line (COS-1). The conditioned media from the COS-1 tranfected cell were added to primary chicken cone cells seeded into 96 well-plates. After 7 days, viable cell counts from the coneenriched cultures were measured using in house high content screening methods and compared to that of empty library vector. Twenty-one hundred pools, corresponding to 210,000 individual clones, were screened. A Pool (number 939) contained twice as many living cells as the negative controls. By limiting dilution clone 939.09.08 was isolated and shown to contain a 502-bp insert with an open reading frame encoding a putative 109-amino acid polypeptide. We named this gene Rod-derived Cone Viability Factor. The novel gene carries many characteristics of the postulated therapeutic gene:

1)Purified recombinant RdCVF protected chicken cones in a dose dependent manner.

2)RdCVF, when transfected into COS-1 cells exerts its survival activity on cones from rd1 retinal explants, the model of the degenerative disease.

3)Purified recombinant RdCVF protected mouse cones when injected into the subretinal space on the rd1 mouse.

4)RdCVF protein is detected in conditioned media from wild-type retina and COS-transfected cells.

5)RdCVF messenger RNA and protein expression is largely decreased in a rod-less mouse retina (the degenerated rd1 retina).

6)RdCVF is expressed by pure cultures of photoreceptors from mouse (97% rods).

7)Immunohistochemistry demonstrated that RdCVF localized in the photoreceptor layer of the retina with a more intense staining in the extracellular matrix surrounding cone cells.

8)RdCVF antibodies are able to block the neuroprotective effect generated by wildtype conditioned media.

In addition, RdCVF expression was found to be restricted to the retina. RdCVF encodes for two polypetides of 17 and 34 kDa by alternative splice, the longer form being extended in its C-terminal region. Both forms have a limited homology with the thioredoxin family and the gene was named accordingly Txnl6 (Thioredoxin-like-6) in the databases. We could not demonstrate any thiol-oxidoreductase activity for the isolated polypeptide (the 17 kDa form). The founder member of the thioredoxin family, Trx-1 has been isolated originally as the adult T-cell leukemia-derived factor28 a factor secreted by cells by a mechanism that does not involve a signal peptide sequence,29 a signal also absent in RdCVF.

4. CONCLUSION

Mutation-independent therapies offer a means of slowing down or even stopping photoreceptor degeneration process in the medium term. They would be applicable to most patients regardless of the genetic defect and limit their handicap. Such therapies are based