Photodynamic therapy combined with intravitreal bevacizumab (Avastin) in treatment of choroidal neovascularization secondary to age-related macular degeneration

To evaluate photodynamic therapy with verteporfin combined with intravitreal bevacizumab in minimally classic and occult choroidal neovascularization secondary to age-related macular degeneration. 46 eyes of 46 patients (mean age 74.5) included in this prospective, noncomparative, interventional case series. Median follow-up was 24 weeks (12-36). Verteporfin photodynamic therapy (PDT) was followed by 0.05 mL (1.25 mg) of bevacizumab injected intravitreally within 24 hours and again after 6 weeks. Whole procedure was repeated in 3-month intervals in case of leakage. Visual acuity (VA) improved in majority of patients (baseline VA 1.041 log MAR) by mean increase of 1.45 lines (last follow-up) (p = 0.001). Central foveal thickness (CFT) and total macular volume (TMV) decreased by 53 microm (p = 0.03) and 1.04 mm3 (p < 0.001) respectively. No serious complications were observed. Combined treatment may improve outcome of monotherapy. Significant improvement in VA, CFT and TMA was noted in majority of patients and maintained during follow-up.     

Metabolic Syndrome Triggered by High-Fructose Diet Favors Choroidal Neovascularization and Impairs Retinal Light Sensitivity in the Rat

Diabetic retinopathy and age-related macular degeneration are the leading causes of blindness in Western populations. Although it is a matter of controversy, large-scale population-based studies have reported increased prevalence of age-related macular degeneration in patients with diabetes or diabetic retinopathy. We hypothesized that metabolic syndrome, one of the major risk factors for type 2 diabetes, would represent a favorable environment for the development of choroidal neovascularization, the main complication of age-related macular degeneration. The fructose-fed rat was used as a model for metabolic syndrome in which choroidal neovascularization was induced by laser photocoagulation. Male Brown Norway rats were fed for 1, 3, and 6 months with a standard equilibrated chow diet or a 60%-rich fructose diet (n = 24 per time point). The animals expectedly developed significant body adiposity (+17%), liver steatosis at 3 and 6 months, hyperleptinemia at 1 and 3 months (two-fold increase) and hyperinsulinemia at 3 and 6 months (up to two-fold increase), but remained normoglycemic and normolipemic. The fructose-fed animals exhibited partial loss of rod sensitivity to light stimulus and reduced amplitude of oscillatory potentials at 6 months. Fructose-fed rats developed significantly more choroidal neovascularization at 14 and 21 days post-laser photocoagulation after 1 and 3 months of diet compared to animals fed the control diet. These results were consistent with infiltration/activation of phagocytic cells and up-regulation of pro-angiogenic gene expression such as Vegf and Leptin in the retina. Our data therefore suggested that metabolic syndrome would exacerbate the development of choroidal neovascularization in our experimental model.     

A new Chlorin formulation promotes efficient photodynamic action in choriocapillaris of rabbit’s eyes

Age-related macular degeneration (AMD) as well as other choroidal diseases, demand novel therapeutic methods. Photodynamic therapy (PDT), which uses light and photosensitizer (PS) to cause specific vascular occlusion in the macula, is an interesting alternative. The only drug approved for the PDT treatment of AMD (Verteporfin) has a natural tendency to aggregate, demanding an expensive separation procedure during purification. We report a novel and affordable PS that is intrinsically protected against aggregation, the Monomeric Chlorin at High Concentration (MCHC-Chlorin), whose liposomal formulation was developed to provoke effective photodynamic action on the choroidal vasculature. Our report starts by stablishing the conditions to allow the efficient synthesis of MCHC-Chlorin in high yields (92%). We then tested the light stimulated occlusion of choriocapillary vessels in rabbit’s eyes induced by the two MCHC-Chlorin isomers, which are directly obtained from the synthetic route. The PS formulation was infused in the rabbit’s ear vein and eyes were immediately irradiated at 650?nm. Indirect ophthalmoscopy, fundus photography, fluorescein angiography and histopathological evaluations were used to evaluate levels of photo-thrombosis and collateral damage. Choriocapillary occlusion was achieved in all treated rabbits’ eyes, while retina and sclera were completely preserved. There was no photochemical reaction in none of the eyes that received LASER without PS. Both MCHC-Chlorin isomers were separately tested and exhibited similar positive results with no systemic toxicity. Therefore, PDT occurred equally well in all treated eyes and none of the controls showed any effect in the ophthalmological exams. MCHC-Chlorin offers great potential and should be further studied as an alternative drug for choroidal diseases.     

New therapies for the treatment of age-related macular degeneration]

Age-related macular degeneration has a natural progression from the precursors (the drusen) towards atrophic or neovascular complications. Choroidal neovascularization is undoubtedly the aspect of the disease that benefits most from new therapeutical approaches. Destructive photocoagulation based on fluorescein angiography has demonstrated since 20 years its efficiency on choroidal neovascularization. The same approach based on indocyanine green (ICG) angiography would increase the number of patients available to therapy. Very recently photodynamic therapy has demonstrated its efficiency to stabilize visual acuity at least at two years in patients with choroidal new vessels predominantly well defined. Other treatment developments are considered, such as refinement of photocoagulation techniques or of surgery. Until now, none has demonstrated its efficiency although they raise justified hopes. The future approaches rely upon the progress of the research both in physiopathology of the disease and on the angiogenic process requiring a constant interaction with all thematics of research. Finally, palliative treatments will be required before heading up to a preventive treatment.     

Fas ligand (CD95 ligand) controls angiogenesis beneath the retina

A principal cause of blindness is subretinal neovascularization associated with age-related macular degeneration. Excised neovascular membranes from patients with age-related macular degeneration demonstrated a pattern of Fas+ new vessels in the center of the vascular complex, surrounded by FasL+ retinal pigment epithelial cells. In a murine model, Fas (CD95)-deficient (Ipr) and FasL-defective (gld) mice had a significantly increased incidence of neovascularization compared with normal mice. Furthermore, in gld mice there is massive subretinal neovascularization with uncontrolled growth of vessels. We found that cultured choroidal endothelial cells were induced to undergo apoptosis by retinal pigment epithelial cells through a Fas-FasL interaction. In addition, antibody against Fas prevented vascular tube formation of choroidal endothelial cells derived from the eye in a three-dimensional in vitro assay. Thus, FasL expressed on retinal pigment epithelial cells may control the growth and development of new subretinal vessels that can damage vision.     

Intraocular microablation of choroidal tissue by a 308 nm AIDA excimer laser for RPE-transplantation in patients with age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the western nations beyond 50 years of age. The most frequent cause for severe visual loss is the growth of neovascular membrances from the choroid into the subretinal space. This usually results in irreversible degeneration of the overlying retina. Surgical removal of the membrane is feasible, however, usually results in functional loss of apposing retinal photoreceptors since retinal pigment epithelial (RPE) cells are removed concurrently due to their tight adherence to the neovascular complex. Therefore, various attempts have been undertaken to fill the resulting RPE cell defect with either heterologous or autologous RPE cell transplants. So far cell survival, function and subsequent visual function has been disappointing. To minimize trauma and resulting dedifferentiation harvesting in the eye and transplantation in whole sheets and without temporary removal from the eyes would be desirable. This may be achieved by isolating grafts consisting of choroid, Bruch's membrance and RPE cells from the peripheral retina and transplantation of this graft under the neurosensory retina after removal of the choroidal neovascularization. However, the choroidal component of such a graft would be expected to interfere with diffusion of metabolites to and from the retina. Therefore, outcome would be expected to be better if the choroidal tissue would be removed before translocation. In preclinical experiments we used a 308 nm UV AIDA excimer laser to microablate choroidal tissue from such a graft in human donor eyes.     

Influence of plasminogen activator inhibitor type 1 on choroidal neovascularization.

High levels of the plasminogen activators, but also their inhibitor, plasminogen activator inhibitor 1 (PAI-1), have been documented in neovascularization of severe ocular pathologies such as diabetic retinopathy or age-related macular degeneration (AMD). AMD is the primary cause of irreversible photoreceptors loss, and current therapies are limited. PAI-1 has recently been shown to be essential for tumoral angiogenesis. We report here that deficient PAI-1 expression in mice prevented the development of subretinal choroidal angiogenesis induced by laser photocoagulation. When systemic and local PAI-1 expression was achieved by intravenous injection of a replication-defective adenoviral vector expressing human PAI-1 cDNA, the wild-type pattern of choroidal angiogenesis was restored. These observations demonstrate the proangiogenic activity of PAI-1 not only in tumoral models, but also in choroidal experimental neovascularization sharing similarities with human AMD. They identify therefore PAI-1 as a potential target for therapeutic ocular anti-angiogenic strategies.     

Photosensitizers incorporation in SOPC films at different hydration levels

In the present work, two photosensitizing drugs, Temoporfin and Verteporfin have been studied. Both have regular approval in Europe, Temoporfin for the treatment of head and neck cancers and Verteporfin for the treatment of age-related macular degeneration (AMD). The treatment modality, known as “Photodynamic Therapy” (PDT), involves drug activation with visible light in the presence of oxygen and production of reactive oxygen species (ROS) to destroy the pathological tissues. Both drugs are inactive in the absence of light, presenting only few side effects.The incorporation of the two drugs into a SOPC bilayer -used as a model membrane- was studied by ATR-FTIR. An original approach was applied, involving lyotropic transitions and a very slow dehydration rate of the sample. In low water content and dry film, Temoporfin highly affects stretching vibrations of SOPC chains and polar groups, showing that Temoporfin is inserted into the bilayer in both apolar and polar regions. In fully hydrated layers, Temoporfin - SOPC interactions still take place but only impact Temoporfin vibration bands. Verteporfin shows smaller effect on both chain and polar groups' vibrations of SOPC, with the exception of choline group, suggesting that Verteporfin is inserted into the bilayer to a lesser extent and remains at the bilayer polar interface. These results can be used to better understand drugs behavior in biological media.     

The role of PIWIL4 and piRNAs in the development of choroidal neovascularization

Wet age-related macular degeneration (wAMD) is the leading cause of blindness among the elderly in industrialized nations. Anti-vascular epidermal growth factor (VEGF) therapy via intravitreal injection is the most effective clinical treatment for wAMD due to high concentrations of VEGF that promote choroidal neovascularization. While PIWI proteins participate in various biological processes, their function in AMD remains unclear. In this study, we discovered that PIWIL4 expression is elevated in a laser-induced choroidal neovascularization (CNV) model and that it regulates angiogenesis in vitro and in vivo. Differentially expressed piwi-interacting RNAs (piRNAs) were identified in a CNV model and were shown to potentially regulate angiogenesis via bioinformatics analysis. PIWIL4 knockdown inhibits VEGF secretion and VEGFR2 phosphorylation. Overall, PIWIL4 may serve as a novel target to block pathological choroidal neovascularization, and the study of the PIWI-piRNAs pathway in wAMD highlights its broad function in somatic cells.     

Topical administration of a multi-targeted kinase inhibitor suppresses choroidal neovascularization and retinal edema

Age-related macular degeneration, diabetic retinopathy, and retinal vein occlusions are complicated by neovascularization and macular edema. Multi-targeted kinase inhibitors that inhibit select growth factor receptor tyrosine kinases and/or components of their down-stream signaling cascades (such as Src kinases) are rationale treatment strategies for these disease processes. We describe the discovery and characterization of two such agents. TG100572, which inhibits Src kinases and selected receptor tyrosine kinases, induced apoptosis of proliferating endothelial cells in vitro. Systemic delivery of TG100572 in a murine model of laser-induced choroidal neovascularization (CNV) caused significant suppression of CNV, but with an associated weight loss suggestive of systemic toxicity. To minimize systemic exposure, topical delivery of TG100572 to the cornea was explored, and while substantial levels of TG100572 were achieved in the retina and choroid, superior exposure levels were achieved using TG100801, an inactive prodrug that generates TG100572 by de-esterification. Neither TG100801 nor TG100572 were detectable in plasma following topical delivery of TG100801, and adverse safety signals (such as weight loss) were not observed even with prolonged dosing schedules. Topical TG100801 significantly suppressed laser-induced CNV in mice, and reduced fluorescein leakage from the vasculature and retinal thickening measured by optical coherence tomography in a rat model of retinal vein occlusion. These data suggest that TG100801 may provide a new topically applied treatment approach for ocular neovascularization and retinal edema.     

Doxycycline Inhibits Polarization of Macrophages to the Proangiogenic M2-type and Subsequent Neovascularization

Macrophages occur along a continuum of functional states between M1-type polarized macrophages with antiangiogenic and antitumor activity and M2-type polarized macrophages, which have been implicated to promote angiogenesis and tumor growth. Proangiogenic M2-type macrophages promote various pathologic conditions, including choroidal neovascularization in models of neovascular age-related macular degeneration, or certain cancers, such as glioblastoma multiforme. Thus, a potential novel therapeutic approach to target pathological angiogenesis in these conditions would be to inhibit the polarization of macrophages toward the proangiogenic M2-type. However, no pharmacological inhibitors of M2-type macrophage polarization have been identified yet. Here we performed an unbiased pharmacological and small chemical screen to identify drugs that inhibit proangiogenic M2-type macrophage polarization and block pathologic macrophage-driven neovascularization. We identified the well tolerated and commonly used antibiotic doxycycline as a potent inhibitor of M2-type polarization of macrophages. Doxycycline inhibited, in a dose-dependent manner, M2-type polarization of human and bone marrow-derived mouse macrophages without affecting cell viability. Furthermore, doxycycline inhibited M2-type macrophage polarization and subsequent neovascularization in vivo in a laser injury model of choroidal neovascularization. Thus, doxycycline could be used to enhance current antiangiogenic treatment approaches in various conditions that are promoted by proangiogenic M2-type macrophages, including neovascular age-related macular degeneration and certain cancers.     

Genotoxic potential of porphyrin type photosensitizers with particular emphasis on 5-aminolevulinic acid: implications for clinical photodynamic therapy

Photodynamic therapy (PDT) uses exogenously administered photosensitizers activated by light to induce cell death or modulation of immunological cascades, presumably via formation of reactive oxygen species (ROS). 5-Aminolevulinic acid (ALA) mediated photosensitization is increasingly used for the treatment of nonmelanoma skin cancer and other indications including benign skin disorders. Long-term side effects of this investigational modality are presently unknown. Just as tumor treatments such as ionizing radiation and chemotherapy can cause secondary tumor induction, PDT may potentially have a carcinogenic risk. Evaluation of the biological effects of ALA in absence of activating light and analysis of the mechanism of ALA-PDT and porphyrin-type photosensitizers mediated photosensitization indicate that this therapy has a pro-oxidant and genotoxic potential. However, porphyrin type molecules also possess antioxidant and antimutagenic properties. ALA-PDT delays photocarcinogenesis in mice, and topical ALA alone does not increase skin cancer incidence in these animals. Patients with increased tissue levels of ALA have an increased incidence of internal carcinoma, however, it is not clear whether this relationship is casual or causal. There is no evidence indicating higher rates of skin cancer in patients with photosensitivity diseases due to presence of high protoporphyrin IX (PP) levels in skin. Overall, the presently available data indicate that the risk for secondary skin carcinoma after topical ALA-PDT seems to be low, but further studies must be carried out to evaluate the carcinogenic risk of ALA-PDT in conditions predisposed to skin cancer.     

Sterculic acid antagonizes 7-ketocholesterol-mediated inflammation and inhibits choroidal neovascularization

Sterculic acid is a cyclopropene fatty acid with numerous biological activities. In this study we demonstrate that sterculic acid is a potent inhibitor of endoplasmic reticulum (ER) stress and related inflammation caused by 7-ketocholesterol (7KCh). 7KCh is a highly toxic oxysterol suspected in the pathogenesis of various age-related diseases such as atherosclerosis, Alzheimer's disease and age-related macular degeneration. Sterculic acid demonstrated to be 5-10 times more effective than other anti-inflammatory fatty acids at inhibiting 7KCh-mediated inflammatory responses in cultured cells. In vivo, sterculic acid was effective at inhibiting the formation of choroidal neovascularization (CNV) in the laser-injury rat model. Our data suggests that sterculic acid may be useful in treating CNV in certain forms of age-related macular degeneration.     

Retinal pigment epithelial cell proliferation

The human retinal pigment epithelium forms early in development and subsequently remains dormant, undergoing minimal proliferation throughout normal life. Retinal pigment epithelium proliferation, however, can be activated in disease states or by removing retinal pigment epithelial cells into culture. We review the conditions that control retinal pigment epithelial proliferation in culture, in animal models and in human disease and interpret retinal pigment epithelium proliferation in context of the recently discovered retinal pigment epithelium stem cell that is responsible for most in vitro retinal pigment epithelial proliferation. Retinal pigment epithelial proliferation-mediated wound repair that occurs in selected macular diseases is contrasted with retinal pigment epithelial proliferation-mediated fibroblastic scar formation that underlies proliferative vitreoretinopathy. We discuss the role of retinal pigment epithelial proliferation in age-related macular degeneration which is reparative in some cases and destructive in others. Macular retinal pigment epithelium wound repair and regression of choroidal neovascularization are more pronounced in younger than older patients. We discuss the possibility that the limited retinal pigment epithelial proliferation and latent wound repair in older age-related macular degeneration patients can be stimulated to promote disease regression in age-related macular degeneration.     

Fluorescence characterisation of multiply-loaded anti-HER2 single chain Fv-photosensitizer conjugates suitable for photodynamic therapy.

We report the synthesis, spectroscopic properties and intracellular imaging of recombinant antibody single chain fragment (scFv) conjugates with photosensitizers used for photodynamic therapy of cancer (PDT). Two widely-studied photosensitizers have been selected: preclinical pyropheophorbide-a (PPa) and verteporfin (VP), which has been clinically approved for the treatment of acute macular degeneration (Visudyne). Pyropheophorbide-a and verteporfin have been conjugated to an anti-HER2 scFv containing on average ten photosensitizer molecules per scFv with a small contribution (相似文献   

Heat treatment of retinal pigment epithelium induces production of elastic lamina components and antiangiogenic activity

Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. In advanced AMD, new vessels from choriocapillaris (CC) invade through the Bruch's membrane (BrM) into the retina, forming choroidal neovascularization (CNV). BrM, an elastic lamina that is located between the retinal pigment epithelium (RPE) and CC, is thought to act as a physical and functional barrier against CNV. The BrM of patients with early AMD are characterized by decreased levels of antiangiogenic factors, including endostatin, thrombospondin-1 (TSP-1), and pigment epithelium-derived factor (PEDF), as well as by degeneration of the elastic layer. Motivated by a previous report that heat increases elastin expression in human skin, we examined the effect of heat on human ARPE-19 cell production of BrM components. Heat treatment stimulated the production of BrM components, including TSP-1, PEDF, and tropoelastin in vitro and increased the antiangiogenic activity of RPE measured in a mouse corneal pocket assay. The effect of heat on experimental CNV was investigated by pretreating the retina with heat via infrared diode laser prior to the induction of CNV. Heat treatment blocked the development of experimental CNV in vivo. These findings suggest that heat treatment may restore BrM integrity and barrier function against new vessel growth.     

VEGF-TRAP(R1R2) suppresses choroidal neovascularization and VEGF-induced breakdown of the blood-retinal barrier

Vascular endothelial growth factor (VEGF) plays a central role in the development of retinal neovascularization and diabetic macular edema. There is also evidence suggesting that VEGF is an important stimulator for choroidal neovascularization. In this study, we investigated the effect of a specific inhibitor of VEGF, VEGF-TRAP(R1R2), in models for these disease processes. VEGF-TRAP(R1R2) is a fusion protein, which combines ligand binding elements taken from the extracellular domains of VEGF receptors 1 and 2 fused to the Fc portion of IgG1. Subcutaneous injections or a single intravitreous injection of VEGF-TRAP(R1R2) strongly suppressed choroidal neovascularization in mice with laser-induced rupture of Bruch's membrane. Subcutaneous injection of VEGF-TRAP(R1R2) also significantly inhibited subretinal neovascularization in transgenic mice that express VEGF in photoreceptors. In two models of VEGF-induced breakdown of the blood-retinal barrier (BRB), one in which recombinant VEGF is injected into the vitreous cavity and one in which VEGF expression is induced in the retina in transgenic mice, VEGF-TRAP(R1R2) significantly reduced breakdown of the BRB. These data confirm that VEGF is a critical stimulus for the development of choroidal neovascularization and indicate that VEGF-TRAP(R1R2) may provide a new agent for consideration for treatment of patients with choroidal neovascularization and diabetic macular edema.     

Age-related macular degeneration: a target for nanotechnology derived medicines

Despite the fact that the retina is a fairly accessible portion of the central nervous system, there are virtually no treatments for early age-related macular degeneration (AMD). AMD is a degenerative retinal disease that causes progressive loss of central vision and is the leading cause of irreversible vision loss and legal blindness in individuals over the age of 50. Both environmental and genetic components play a role in its development. AMD is a multifactorial disease with characteristics that include drusen, hyperpigmentation and/or hypopigmentation of the retinal pigment epithelium (RPE), geographic atrophy and, in a subset of patients, late-stage choroidal neovascularization (CNV). Drugs that inhibit vascular endothelial growth factor (VEGF) have proven effective in treating late-stage CNV, but optimal means of drug delivery remains to be determined. Microscopic particles, whose size is on the nanometer scale, show considerable promise for drug delivery to the retina, for gene therapy, and for powering prosthetic "artificial retinas." This article summarizes the pathophysiology of AMD stressing potential applications from nanotechnology.     

Exosomes from Retinal Astrocytes Contain Antiangiogenic Components That Inhibit Laser-induced Choroidal Neovascularization

Exosomes released from different types of host cells have different biological effects. We report that exosomes released from retinal astroglial cells (RACs) suppress retinal vessel leakage and inhibit choroidal neovascularization (CNV) in a laser-induced CNV model, whereas exosomes released from retinal pigmental epithelium do not. RAC exosomes inhibit the migration of macrophages and the tubule forming of mouse retinal microvascular endothelial cells. Further, we analyzed antiangiogenic components in RAC exosomes using an angiogenesis array kit and detected several endogenous inhibitors of angiogenesis exclusively present in RAC exosomes, such as endostatin. Moreover, blockade of matrix metalloproteinases in the cleavage of collagen XVIII to form endostatin using FN-439 reverses RAC exosome-mediated retinal vessel leakage. This study demonstrates that exosomes released from retinal tissue cells have different angiogenic effects, with exosomes from RACs containing antiangiogenic components that might protect the eye from angiogenesis and maintain its functional integrity. In addition, by identifying additional components and their functions of RAC exosomes, we might improve the antiangiogenic therapy for CNV in age-related macular degeneration and diabetic retinopathy.     

A non membrane-targeted human soluble CD59 attenuates choroidal neovascularization in a model of age related macular degeneration

Age related macular degeneration (AMD) is the most common cause of blindness amongst the elderly. Approximately 10% of AMD patients suffer from an advanced form of AMD characterized by choroidal neovascularization (CNV). Recent evidence implicates a significant role for complement in the pathogenesis of AMD. Activation of complement terminates in the incorporation of the membrane attack complex (MAC) in biological membranes and subsequent cell lysis. Elevated levels of MAC have been documented on choroidal blood vessels and retinal pigment epithelium (RPE) of AMD patients. CD59 is a naturally occurring membrane bound inhibitor of MAC formation. Previously we have shown that membrane bound human CD59 delivered to the RPE cells of mice via an adenovirus vector can protect those cells from human complement mediated lysis ex vivo. However, application of those observations to choroidal blood vessels are limited because protection from MAC- mediated lysis was restricted only to the cells originally transduced by the vector. Here we demonstrate that subretinal delivery of an adenovirus vector expressing a transgene for a soluble non-membrane binding form of human CD59 can attenuate the formation of laser-induced choroidal neovascularization and murine MAC formation in mice even when the region of vector delivery is distal to the site of laser induced CNV. Furthermore, this same recombinant transgene delivered to the intravitreal space of mice by an adeno-associated virus vector (AAV) can also attenuate laser-induced CNV. To our knowledge, this is the first demonstration of a non-membrane targeting CD59 having biological potency in any animal model of disease in vivo. We propose that the above approaches warrant further exploration as potential approaches for alleviating complement mediated damage to ocular tissues in AMD.