Differences in the temporal expression of regulatory growth factors during choroidal neovascular development.

Although the roles of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) in pathologic neovascularization have been well characterized in certain tissues, their particular functions and expression patterns in choroidal neovascularization (CNV) have not been clearly established. After localized laser trauma to Bruch's membrane to induce CNV development, the temporal changes in mRNA and protein expression of these 3 cytokines were documented and compared histologically to areas of immunofluorescence, the proliferation of endothelial cells, neovascular development, and temporal changes in vascular permeability. Changes in mRNA and protein levels of bFGF and HGF occurred quickly and reached peak expression within hours. This activity corresponded in time to intense and localized immunofluorescence for these cytokines within the choriocapillaris within laser lesion sites. During this same initial time period, mRNA upregulation of VEGF occurred, primarily within the neural retina and this expression corresponded to intense immunolabeling of Müller cells immediately adjacent to the lesion sites. By 3 days after lasering, increased VEGF(164) protein expression was measurable, whereas early neovascular development histologically corresponded to HGF and bFGF mRNA expansion into the developing choroidal neovascular membrane (CNVM). At 7 days, CNV expansion, maturation, and increased vascular permeability corresponded to peak VEGF mRNA and protein expression and to immunofluorescence of the CNVM. Differences also occurred in the expression of precursor and activated isoforms of these cytokines in the retinal pigment epithelium/choroid as compared to those in the retina. These molecular and immunocytochemical results suggest that bFGF and HGF may be important as initial regulators neovascularization in this CNV model; whereas VEGF may be important during later phases of angiogenesis and neovascular hyperpermeability.

Comparing pegaptanib and triamcinolone efficacy in the rat choroidal neovascularization model.

OBJECTIVE: To evaluate the prophylactic effect of intravitreal pegaptanib sodium on choroidal neovascularization membrane (CNVM) development and compare its performance with that of triamcinolone acetonide. METHODS: In drug-treated and control groups, CNVMs were induced by laser trauma. Immediately after undergoing the laser procedure, animals received intravitreal injections of pegaptanib sodium, 8 or 17 mug; triamcinolone acetonide, 200 mug; or a vehicle solution. After 21 days, fluorescein angiography was performed. The CNVM mean diameters and radial thicknesses were measured histologically. RESULTS: Mean CNVM diameters were 10% to 13% smaller in pegaptanib-treated eyes and 43% smaller in triamcinolone-treated eyes compared with laser-only control eyes. Late-stage fluorescein angiography leakage scores, on a scale of 0 to 3, suggested a statistical difference between triamcinolone- (0.6) and pegaptanib(8 microg)-treated (1.5) groups compared with the laser-only control group (2.0). The CNVM mean thicknesses were greater in the pegaptanib(8 microg)- (79 microm) and pegaptanib(17 microg)-treated (71 microm) groups and significantly smaller in the triamcinolone-treated group (26 microm) compared with the laser-only control group (67 microm). CONCLUSION: In this animal model of choroidal neovascularization, intravitreal pegaptanib exhibited marginal or no effect on CNVM development; whereas intravitreal triamcinolone evoked robust inhibition of CNVMs. Clinical Relevance Pegaptanib treatment may be insufficient to prevent CNVM formation.

Oral administration of lumiracoxib reduces choroidal neovascular membrane development in the rat laser-trauma model.

PURPOSE: To determine whether lumiracoxib, a highly selective cyclooxygenase-2 (COX-2) inhibitor that exhibits anti-inflammatory and antiangiogenic properties, can inhibit experimental choroidal neovascular membrane (CNVM) development induced by focal laser trauma in a well-characterized Brown Norway rat CNVM model. METHODS: Over a 35-day period, 24 rats received daily oral gavage dosages of 20 mg/kg lumiracoxib in a 0.5% (w/v) suspension of sodium carboxymethylcellulose (CMC), while a control group received the 0.5% CMC suspension only. After 7 days, eight laser photocoagulation sites were placed concentrically around the optic disk to induce CNVMs. Thirty-five days later, fundus photography and fluorescein angiography (FA) were performed and eyes were processed for histopathologic analysis. RESULTS: Masked FA grading of lesion sites revealed a small, but statistically significant difference (P<0.0001) in late stage staining intensity and leakage between the mean group scores of treated (1.4) and control (1.7) eyes. Histopathologic analysis demonstrated that the mean CNVM thickness +/- SD of 38 +/-19 microm (n=24 eyes, 175 photocoagulation sites) in the lumiracoxib-treated animals was reduced by 30% (P<0.001) compared to the CNVM mean thickness+/- SD of 54+/- 20 microm (n=24 eyes, 171 photocoagulation sites) in the control animals. CONCLUSION: Systemic administration of the selective COX-2 inhibitor lumiracoxib results in a partial but significant reduction in CNVM development in the rat laser-trauma model and thus may be clinically beneficial as a potential inhibitor of CNVM formation in exudative age-related macular degeneration.

Characterization of a transgenic mouse line lacking photoreceptor development within the ventral retina.

A unique transgenic mouse line was generated by incorporating a minigene that contained a cone-specific human cone transducin alpha-subunit (GNAT2) promoter, an attenuated diphtheria toxin A (DTA) gene, and an enhancer element from human interphotoreceptor retinoid-binding protein (IRBP) gene. This transgenic mouse line is designated h-GNAT2pro-DTA. During postnatal retinal development, both transgenic and non-transgenic retinas showed similar morphology and thickness at P1. Between ages P8 and P30, all retinal layers became recognizable in non-transgenic and also in transgenic dorsal retinas. However, in the ventral retina of the transgenic mice the photoreceptor layers did not develop. This aberration occurred as a result of abnormal cellular development, rather than as a consequence of retinal degeneration. In adult transgenic animals, approximately 44% of the retina located dorsally appeared morphologically normal, whereas 32% of the retina located ventrally was completely lacking photoreceptor development. The 24% mid-retinal region exhibited transitional morphology containing malformed photoreceptors. At P360 or older, the thickness of retina layers was reduced in both dorsal and ventral regions. The abnormality observed in transgenic retinas involved mainly the photoreceptors; the other retinal cell types were all present in both dorsal and ventral retinas. Since the DTA gene was only expressed in cone cells, the absence of cone photoreceptors in the transgenic retina was to be expected. However, what was unexpected was the concomitant absence of rod photoreceptors in the ventral retina, suggesting that the presence of cones may be important for the development of rods. This new transgenic line can lead to better understanding of photoreceptor development, and may serve as a new animal model for studying photoreceptor-related retinal diseases.

Anastomotic vessels remain viable after photodynamic therapy in primate models of choroidal neovascularization.

PURPOSE: Anastomotic vessels in exudative age-related macular degeneration (AMD) represent a serious clinical feature that reportedly does not respond well to either photocoagulation or photodynamic therapy (PDT). Anastomoses also occur in various animal models of choroidal neovascularization (CNV). In the present study, anastomotic vessels and their patency were evaluated in two primate CNV laser-trauma models after PDT, by using two novel photosensitizers. METHODS: In cynomolgus (Macaca fascicularis) and squirrel (Saimiri sciureus) monkey eyes (n = 20), matrix placement of laser photocoagulation sites elicited CNV as a component of the development of fibrovascular tissue (FVT). FVT sites received PDT according to specific drug infusion and laser light treatment parameters. FVTs and anastomoses were evaluated by fundus photography, fluorescein angiography, and histologic examination. RESULTS: Anastomoses averaged approximately 48% of FVT sites, with greatest occurrence in the macaque. Although PDT with each photosensitizer effectively produced FVT closure, both retinal vessels and anastomoses remained patent. CONCLUSIONS: Although PDT is effective in closing the choroidal neovascularization in FVT, this technique was ineffective in occluding anastomotic vessels and their associated tributaries within the mid- to proximal retina. Various factors (vascular diameter, composition, blood flow, orientation) may contribute to continued anastomotic patency. By convention, such vessels would typically be defined as chorioretinal anastomoses (CRAs); however, continuing studies suggest the possibility that these neovessels constitute dual-origin hybrids. Regardless of origin, viable anastomoses provide one potential mechanism for revascularization to occur after PDT and may help to explain why CRAs are considered a poor prognostic sign in patients with AMD.

Future pharmacological treatment options for nonexudative and exudative age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in the industrialised world. Within the past decade, researchers have introduced many promising prevention and treatment options in an attempt to minimise the central vision loss imparted from AMD. Based on large-scale, randomised, prospective, placebo-controlled trials, a specially formulated combination of the antioxidants vitamin C, vitamin E, beta-carotene, copper and zinc is the only proven means of AMD prophylaxis. Thermal laser photocoagulation and photodynamic therapy with verteporfin are the only standard treatment options. However, efficacy is limited and treatment is only applicable to a minority of AMD patients. Thus, alternative pharmacological interventions are in all phases of clinical development. Researchers are guardedly optimistic that these advances may change the entire approach to AMD management in the near future. This review article will detail the currently accepted treatment options, as well as describe several of the more promising investigational pharmacological approaches to AMD.

Evaluation of photopoint photosensitizer mv6401, indium chloride methyl pyropheophorbide, as a photodynamic therapy agent in primate choriocapillaris and laser-induced choroidal neovascularization.

PURPOSE: To assess the potential of a new photosensitizer, indium chloride methyl pyropheophorbide (PhotoPoint MV6401), for ocular photodynamic therapy (PDT) in normal choriocapillaris vessels and experimentally induced choroidal neovascularization in New-World monkeys (Saimiri sciureus). METHODS: PhotoPoint MV6401 (Miravant Pharmaceuticals, Inc., Santa Barbara, CA) was activated at 664 nm using a DD3-0665 (Miravant Systems, Inc., Santa Barbara, CA) 0.5 W diode laser. The efficacy of MV6401 was evaluated by indirect ophthalmoscopy, fundus photography, fluorescein angiography, and histology. The drug and light doses were 0.10 micromoles/kg to 0.3 micromoles/kg and 10 J/cm to 40 J/cm, respectively, and post-injection activation times ranged from +10 minutes to +120 minutes. RESULTS: Best closure of normal choriocapillaris was achieved at a dosage level of 0.15 micromoles/kg in primates. Histology demonstrated that increased post-injection activation times (+60 minutes to +90 minutes) and low laser light doses (10 J/cm to 20 J/cm) in the primate model resulted in selective closure of the choriocapillaris and medium sized choroidal vessels with minimal effect to the retina. Histology from neovascular lesions PDT-treated with MV6401 revealed significant diminution of vascularity, correlating with diminution of leakage observed on angiography. CONCLUSION: PhotoPoint MV6401, indium chloride methyl pyropheophorbide, is a potent photosensitizer that demonstrates both efficacy and selectivity in primate choriocapillaris and laser-induced choroidal neovascularization occlusion. Maximum selectivity was achieved using a post infusion interval of +60 to +90 minutes.

Corticosteroids in posterior segment disease: an update on new delivery systems and new indications.

PURPOSE OF REVIEW: Corticosteroids are traditionally used for inflammatory disorders because of their ability to diminish neutrophil transmigration, limit access to sites of inflammation, and decrease cytokine production. More recently, however, investigators have focused on the angiostatic and antipermeability properties of corticosteroids for posterior segment diseases such as age-related macular degeneration (AMD), diabetic retinopathy, and macular edema. Both new angiostatic and traditional corticosteroids are currently undergoing evaluation as new delivery techniques such as intravitreal injection and intraocular sustained-release devices facilitate high local angiostatic and antipermeability concentrations while minimizing extraocular toxicity. The purpose of this review is to discuss recent work concerning both the mechanism and effectiveness of these newer treatments. RECENT FINDINGS: Steroids may exert a beneficial effect in AMD-related choroidal neovascular membranes (CNVM) through inhibition of CNVM-promoting macrophages and direct inhibition of angiogenic growth factors. They may also alter extracellular matrix turnover and inhibit matrix metalloproteinases involved in CNVM formation. Intravitreal steroid injections potently inhibit experimental CNVM in primates and rats and have shown promise in some early human pilot trials. In proliferative diabetic retinopathy, steroids may directly inhibit growth factors such as vascular endothelial derived growth factor and inhibit leukocytes that play an important role in early microvascular alterations. Intravitreal steroid injections inhibit experimental preretinal neovascularization in pigs and rats, and rubeosis in some early human studies. In addition, the effect of steroids on vascular permeability has led to their use for macular edema from many causes such as diabetes and venous occlusive disease. SUMMARY: The use of steroids to treat a number of retinal diseases is gaining wide spread acceptance. The apparent short-term success must be balanced by the fact that the long-term safety and efficacy have yet to be determined for any of these approaches. A number of large randomized prospective clinical trials of steroid compounds and new delivery systems are currently under way for AMD, diabetic retinopathy, uveitis, and other retinovascular diseases, and hopefully these studies will provide guidance about the use of these new modalities.

The squirrel monkey: characterization of a new-world primate model of experimental choroidal neovascularization and comparison with the macaque.

PURPOSE: To evaluate and characterize the New-World squirrel monkey as a primate model for experimental choroidal neovascularization (CNV) studies and to compare it with the current Old-World macaque monkey model. METHODS: Fibrovascular tissues (FVT) were elicited in 12 maculae of seven squirrel monkeys by laser photocoagulation using optimized laser parameters (532 nm, 0.05 second, 75 micro m, 650 mW). Follow-up fundus and fluorescein angiography (FA) examinations were conducted on postlaser days 30 and 35, followed by euthanasia and histologic analysis of tissues. For comparative evaluations, FVT development also was induced and analyzed in eight maculae of four macaque monkeys with laser parameters previously used in this species (514 nm, 0.1 second, 50 micro m, 390 and 455 mW). RESULTS: FVT developed in both primate species, consisting of fibrous tissue that contained vessels that ranged from sparse but identifiable capillaries to well-established neovascular networks. Overall, 65% of the photocoagulation sites in the squirrel monkey and 37% of sites in macaque monkey elicited development of FVT. Localized FVT ranged from modest to extensive thickenings of the choriocapillaris layer. Unexpectedly, 76% of the FVT sites in squirrel monkey eyes and 27% of the sites in macaque eyes showed diffuse FVT that expanded beyond the original photocoagulation sites, accompanied by neovascular infiltration of the retina. CONCLUSIONS: Like the macaque, the squirrel monkey can be considered a useful primate model for experimental CNV investigations, while additionally offering certain species-specific advantages. Diffuse FVT permit studies of antiangiogenic therapies in areas distant from laser photocoagulative trauma sites.

Current and future treatment options for nonexudative and exudative age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in the industrialised world. Although relatively simple to diagnose through direct visualisation augmented with rapid sequence fluorescein angiography, treatment has presented a far greater challenge because the true aetiology of AMD is largely unknown. Within the past decade, researchers have introduced many new, potentially promising treatment and prevention options in an attempt to minimise the damage imparted from AMD. They capitalise on many of the theoretical and known factors contributing to AMD progression. A high-dose of an orally administered combination of the antioxidants ascorbic acid (vitamin C), tocopherol (vitamin E) and beta-carotene, in addition to copper and zinc, is the only widely accepted preventive therapy. Thermal laser photocoagulation and verteporfin photodynamic therapy are the only standard treatment options available based on large scale, randomised, prospective, placebo-controlled trials; however, efficacy is limited and only a minority of patients who present with AMD are eligible for these treatments. Many other preventive and treatment options are in all phases of clinical studies and expected to change the entire approach to AMD management in the near future. For example, alternative antioxidants, drusen ablation, apheresis and HMG-CoA reductase inhibitors have shown promise in some studies by preventing or slowing the progression of certain forms of AMD. In addition, alternative photodynamic therapies, low-intensity laser, antiangiogenic medications, radiation treatment and surgery have demonstrated the ability, albeit to differing degrees, to inhibit or possibly even reverse the severe vision loss often associated with AMD characterised by choroidal neovascularisation.

Squalamine lactate reduces choroidal neovascularization in a laser-injury model in the rat.

PURPOSE: To determine if systemically administered squalamine lactate, a novel aminosterol with antineoplastic and antiangiogenic activity, inhibits the development of experimental choroidal neovascularization membranes (CNVMs) induced by laser trauma in a rat model. METHODS: Twenty anesthetized male Brown-Norway rats received a series of 8 krypton red laser lesions per eye (647 nm, 0.05 second, 50 microm, 150 mW). One half the animals received an intraperitoneal injection of squalamine and the other one half received an injection of 5% dextrose in water, all performed in a masked fashion. Fundus photography and fluorescein angiography were performed at postlaser treatment days 14 and 28, and ocular tissues were processed for light microscopic examination following euthanasia of the rats on postlaser treatment day 28. RESULTS: Although fundus photography and fluorescein angiography yielded no statistically significant quantitative differences between the two groups, histologic analysis of the lesion sites revealed a partial but statistically significant reduction of experimental CNVM development in the squalamine-treated population. In particular, the squalamine-treated eyes (n = 20) demonstrated lesions (n = 149) with a mean CNVM thickness +/- SD of 47 +/- 11 microm, as compared with the control eyes (n = 20) that had lesions (n = 142) with a mean CNVM thickness +/- SD of 63 +/- 14 microm (P < 0.001). CONCLUSION: Systemically administered squalamine lactate partially reduced choroidal neovascular membrane development induced by laser trauma in this animal model. In conjunction with other existing and developing therapies, this agent may have a potential role in the treatment of human CNVM formation. Further study of squalamine lactate for treatment of neovascular eye disease is warranted.

An update on photodynamic therapy in age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible loss of central vision in people aged > 50 years in the western world. Until recently, the only proven treatment to reduce the risk of vision loss from its more severe neovascular form was laser photocoagulation, but this treatment was suitable for only 15% of cases. Photodynamic therapy (PDT) with verteporfin was recently proposed to be effective in reducing the risk of visual loss for an estimated 20 - 30% of neovascular AMD patients. This review covers AMD epidemiology, the mechanism of PDT, the 2-year results of the two major clinical studies of PDT with verteporfin, the cost-effectiveness of PDT and the current research status of other drugs for PDT in AMD.