Down-expression of the NLRP3 inflammasome delays the progression of diabetic retinopathy.

The investigation aimed to evaluate the effects of Mcc950, an inhibitor of the NLRP3 inflammasome, on diabetic retinopathy (DR) mice. The general physiological condition of each group of mice was recorded. Retinal blood vessels were stained for observation of the density of blood vessels, and retinas were used for further morphological examination and fluorescent staining after the intravitreal injection of Mcc950. Mcc950 partially reversed hyperglycemia-induced vascular damage and had reduced histological changes compared to DR mice. IL-1ß production in mice retinas in the diabetic model (DM) group increased, but pretreatment with Mcc950 significantly reversed these changes. Additionally, Mcc950 engineered reduced FITC dextran extravasation and vascular leakage. Therefore, it played an apparent protective role in DR and could be a new treatment strategy for DR.

Bulk Gene Expression Deconvolution Reveals Infiltration of M2 Macrophages in Retinal Neovascularization.

Purpose: This study interrogated the transcriptional features and immune cellular landscape of the retinae of rats subjected to oxygen-induced retinopathy (OIR). Methods: Bulk RNA sequencing was performed with retinal RNA isolated from control and OIR rats. Gene set enrichment analysis (GSEA) was undertaken to identify gene sets associated with immune responses in retinal neovascularization. Bulk gene expression deconvolution analysis by CIBERSORTx was performed to identify immune cell types involved in retinal neovascularization, followed by functional enrichment analysis of differentially expressed genes (DEGs). Protein-protein interaction analysis was performed to predict the hub genes relevant to identified immune cell types. CIBERSORTx was applied to profile immune cell types in the macula of patients with both proliferative diabetic retinopathy (PDR) and diabetic macular edema using a public RNA-seq dataset. Results: Transcriptome analysis by GSEA revealed that the retina of OIR rats and patients with PDR is characterized by increased immunoregulatory interactions and complement cascade. Deconvolution analysis demonstrated that M2 macrophages infiltrate the retinae of OIR rats and patients with PDR. Functional enrichment analysis of DEGs in OIR rats showed that the dysregulated genes are related to leukocyte-mediated immunity and myeloid leukocyte activation. Downstream protein-protein interaction analysis revealed that several potential hub genes, including Ccl2, Itgam, and Tlr2, contribute to M2 macrophage infiltration in the ischemic retina. Conclusions: This study highlights application of the gene expression deconvolution tool to identify immune cell types in inflammatory ocular diseases with transcriptomes, providing a new approach to assess changes in immune cell types in diseased ocular tissues.

Angiogenesis-Inflammation Cross Talk in Diabetic Retinopathy: Novel Insights From the Chick Embryo Chorioallantoic Membrane/Human Vitreous Platform.

Pathological angiogenesis of the retina is a key component of irreversible causes of blindness, as observed in proliferative diabetic retinopathy (PDR). The pathogenesis of PDR is complex and involves vascular, inflammatory, and neuronal mechanisms. Several structural and molecular alterations associated to PDR are related to the presence of inflammation that appears to play a non-redundant role in the neovascular response that characterizes the retina of PDR patients. Vascular endothelial growth factor (VEGF) blockers have evolved over time for the treatment of retinal neovascularization. However, several limitations to anti-VEGF interventions exist. Indeed, the production of other angiogenic factors and pro-inflammatory mediators may nullify and/or cause resistance to anti-VEGF therapies. Thus, appropriate experimental models are crucial for dissecting the mechanisms leading to retinal neovascularization and for the discovery of more efficacious anti-angiogenic/anti-inflammatory therapies for PDR patients. This review focuses on the tight cross talk between angiogenesis and inflammation during PDR and describe how the chick embryo chorioallantoic membrane (CAM) assay may represent a cost-effective and rapid in vivo tool for the study of the relationship between neovascular and inflammatory responses elicited by the vitreous humor of PDR patients and for the screening of novel therapeutic agents.

Comprehensive expression patterns of inflammatory cytokines in aqueous humor of patients with neovascular age-related macular degeneration.

Neovascular age-related macular degeneration (nAMD) is a complex and multi-factorial disease, and low-grade inflammation is associated with pathogenesis of nAMD. Aqueous humor could reflect intraocular immune environments in various eye diseases. The research so far used aqueous humor samples and revealed that inflammation is involved in pathophysiology of nAMD, although immunological roles of cytokines were evaluated inadequately with aspect to individual effects. Here we used 27 kinds of cytokines covering general immunologic reactions, examined specific expression patterns of cytokines, and assessed relationships between inflammation and pathophysiology of nAMD by multivariate analyses. In nAMD eyes, principal component analysis showed that IL-7, MCP-1, MIP-1ß and VEGF had high principal component loadings of over 0.6 in the first principal component constituting 32.6% of all variability of the data. In exploratory factor analysis, IL-6, MCP-1 and MIP-1ß had high factor loadings (FL) of over 0.5 in Factor 1 constituting 32.6% of all variability, while VEGF had FL of over 1.0 in Factor 3 constituting 10.7% of all variability. In hierarchical cluster analysis, MCP-1 and VEGF were located in the cluster of first proximate mutual distance to central retinal thickness. These data could suggest that low-grade inflammation is a principal contributor in nAMD.

Mast cell hyperactivity underpins the development of oxygen-induced retinopathy.

Mast cells are classically thought to play an important role in protection against helminth infections and in the induction of allergic diseases; however, recent studies indicate that these cells also contribute to neovascularization, which is critical for tissue remodeling, chronic inflammation, and carcinogenesis. Here, we demonstrate that mast cells are essential for sprouting angiogenesis in a murine model of oxygen-induced retinopathy (OIR). Although mouse strains lacking mast cells did not exhibit retinal neovascularization following hypoxia, these mice developed OIR following infusion of mast cells or after injection of mast cell tryptase (MCT). Relative hypoxia stimulated mast cell degranulation via transient receptor potential ankyrin 1. Subsequent surges in MCT stimulated retinal endothelial cells to produce monocyte chemotactic protein-1 (MCP1) and angiogenic factors, leading to sprouting angiogenesis. Mast cell stabilizers as well as specific tryptase and MCP1 inhibitors prevented the development of OIR in WT mice. Preterm infants with early retinopathy of prematurity had markedly higher plasma MCT levels than age-matched infants without disease, suggesting mast cells contribute to human disease. Together, these results suggest therapies that suppress mast cell activity should be further explored as a potential option for preventing eye diseases and subsequent blindness induced by neovascularization.

Foxp3+ Tregs are recruited to the retina to repair pathological angiogenesis.

Neovascular retinopathies are major causes of vision loss; yet treatments to prevent the condition are inadequate. The role of regulatory T cells in neovascular retinopathy is unknown. Here we show that in retinopathy regulatory T cells are transiently increased in lymphoid organs and the retina, but decline when neovascularization is established. The decline is prevented following regulatory T cells expansion with an IL-2/anti-IL-2 mAb complex or the adoptive transfer of regulatory T cells. Further, both approaches reduce vasculopathy (vaso-obliteration, neovascularization, vascular leakage) and alter the activation of Tmem119+ retinal microglia. Our in vitro studies complement these findings, showing that retinal microglia co-cultured with regulatory T cells exhibit a reduction in co-stimulatory molecules and pro-inflammatory mediators that is attenuated by CTLA-4 blockade. Collectively, we demonstrate that regulatory T cells are recruited to the retina and, when expanded in number, repair the vasculature. Manipulation of regulatory T cell numbers is a previously unrecognized, and promising avenue for therapies to prevent blinding neovascular retinopathies.The local immune responses in the eye are attenuated to preserve sight. Surprisingly, Deliyanti et al. show that regulatory T cells (Tregs) take an active role in protecting the eye from neovascularization in oxygen-induced retinopathy, and that interventions that augment the retinal Treg numbers reduce neovascular retinopathy in mice.

Effects of Phenotype of Retinal Macrophages on the Features of Angiogenesis of Murine Retina.

The period of forming of superficial vascular plexus during physiological retinal angiogenesis was shorter in C57Bl/6 mice. Experiments on the model of oxygen-induced retinopathy showed that avascular and vascularized zones in BALB/c mice on day 17 are smaller than in C57Bl/6 mice are by 5 and 1.5 times, respectively. The obtained results confirmed the importance of phenotype of retinal macrophages in the regulation of processes of both physiological and pathological retinal angiogenesis.

Inhibition of the Nuclear Receptor RORγ and Interleukin-17A Suppresses Neovascular Retinopathy: Involvement of Immunocompetent Microglia.

OBJECTIVE: Although inhibitors of vascular endothelial growth factor (VEGF) provide benefit for the management of neovascular retinopathies, their use is limited to end-stage disease and some eyes are resistant. We hypothesized that retinoic acid-related orphan nuclear receptor γ (RORγ) and its downstream effector, interleukin (IL)-17A, upregulate VEGF and hence are important treatment targets for neovascular retinopathies. APPROACH AND RESULTS: Utilizing a model of oxygen-induced retinopathy, confocal microscopy and flow cytometry, we identified that retinal immunocompetent cells, microglia, express IL-17A. This was confirmed in primary cultures of rat retinal microglia, where hypoxia increased IL-17A protein as well as IL-17A, RORγ, and tumor necrosis factor-α mRNA, which were reduced by the RORγ inhibitor, digoxin, and the RORα/RORγ inverse agonist, SR1001. By contrast, retinal macroglial Müller cells and ganglion cells, key sources of VEGF in oxygen-induced retinopathy, did not produce IL-17A when exposed to hypoxia and IL-1ß. However, they expressed IL-17 receptors, and in response to IL-17A, secreted VEGF. This suggested that RORγ and IL-17A inhibition might attenuate neovascular retinopathy. Indeed, digoxin and SR1001 reduced retinal vaso-obliteration, neovascularization, and vascular leakage as well as VEGF and VEGF-related placental growth factor. Digoxin and SR1001 reduced microglial-derived IL-17A and Müller cell and ganglion cell damage. The importance of IL-17A in oxygen-induced retinopathy was confirmed by IL-17A neutralization reducing vasculopathy, VEGF, placental growth factor, tumor necrosis factor-α, microglial density and Müller cell, and ganglion cell injury. CONCLUSIONS: Our findings indicate that an RORγ/IL-17A axis influences VEGF production and neovascular retinopathy by mechanisms involving neuroglia. Inhibition of RORγ and IL-17A may have potential for the improved treatment of neovascular retinopathies.

The alternative complement pathway aids in vascular regression during the early stages of a murine model of proliferative retinopathy.

Proliferative retinopathic diseases often progress in 2 phases: initial regression of retinal vasculature (phase 1) followed by subsequent neovascularization (NV) (phase 2). The immune system has been shown to aid in vascular pruning in such retinopathies; however, little is known about the role of the alternative complement pathway in the initial vascular regression phase. Using a mouse model of oxygen-induced retinopathy (OIR), we observed that alternative complement pathway-deficient mice (Fb(-/-)) exhibited a mild decrease in vascular loss at postnatal day (P)8 compared with age- and strain-matched controls (P = 0.035). Laser capture microdissection was used to isolate the retinal blood vessels. Expression of the complement inhibitors Cd55 and Cd59 was significantly decreased in blood vessels isolated from hyperoxic retinas compared with those from normoxic control mice. Vegf expression was measured at P8 and found to be significantly lower in OIR mice than in normoxic control mice (P = 0.0048). Further examination of specific Vegf isoform expression revealed a significant decrease in Vegf120 (P = 0.00032) and Vegf188 (P = 0.0092). In conjunction with the major modulating effects of Vegf during early retinal vascular development, our data suggest a modest involvement of the alternative complement pathway in targeting vessels for regression in the initial vaso-obliteration stage of OIR.

Interleukin-17A neutralization alleviated ocular neovascularization by promoting M2 and mitigating M1 macrophage polarization.

Neovascularization (NV), as a cardinal complication of several ocular diseases, has been intensively studied, and research has shown its close association with inflammation and immune cells. In the present study, the role of interleukin-17A (IL-17A) in angiogenesis in the process of ocular NV both in vivo and in vitro was investigated. Also, a paracrine role of IL-17A was demonstrated in the crosstalk between endothelial cells and macrophages in angiogenesis. In the retinas of mice with retinopathy of prematurity, the IL-17A expression increased significantly at postnatal day 15 (P15) and P18 during retinal NV. Mice given IL-17A neutralizing antibody (NAb) developed significantly reduced choroidal NV and retinal NV. Studies on vascular endothelial growth factor (VEGF) over-expressing mice suggested that IL-17A modulated NV through the VEGF pathway. Furthermore, IL-17A deficiency shifted macrophage polarization toward an M2 phenotype during retinal NV with significantly reduced M1 cytokine expression compared with wild-type controls. In vitro assays revealed that IL-17A treated macrophage supernatant gave rise to elevated human umbilical vascular endothelial cell proliferation, tube formation and VEGF receptor 1 and receptor 2 expression. Therefore, IL-17A could potentially serve as a novel target for treating ocular NV diseases. The limitation of this study involved the potential mechanisms, such as which transcription accounted for macrophage polarization and how the subsequent cytokines were modulated when macrophages were polarized. Further studies need to be undertaken to definitively determine the extent to which IL-17A neutralizing anti-angiogenic activity depends on macrophage modulation compared with anti-VEGF treatment.

Functional Characterization of AAV-Expressed Recombinant Anti-VEGF Single-Chain Variable Fragments In Vitro.

PURPOSE: Most retinal neovascular disorders are caused by upregulation of vascular endothelial growth factor (VEGF) expression. These disorders are treated with repeated injections of anti-VEGF molecules, which may have severe side effects. The expression of anti-VEGF molecules by the retina itself in a controlled manner following adeno-associated viral (AAV) gene transfer could be a replacement of this therapy. METHODS: The open reading frames (orf) of the light and the heavy chain of ranibizumab were cloned into an expression plasmid separated by an internal ribosomal entry site (IRES). The construct was mutated to generate ranibizumab single-chain variable fragments (scFv). Expression was verified by western blotting and the concentrations were measured with a custom-made ranibizumab ELISA. Biological activity, VEGF-binding properties, and the doxycycline-dependent induction of anti-VEGF expression were tested. An AAV2/5 vector was generated containing the optimal variant Ra02. RESULTS: Ra01-Ra05 molecules were detected in the cell culture medium. While the VEGF-binding affinity was significantly lower for Ra01 and Ra02 compared to Lucentis(®), the inhibition of cell migration was comparable and the maximum inhibition of Ra01 and Ra02 was reached at lower doses. The expression of Ra01 and Ra02 was shown to be regulable with the TetOn-system(®) as plasmid (Ra01, Ra02) and AAV vector construct (Ra02). CONCLUSION: Ra01 and Ra02 can be produced in eukaryotic cells after AAV-mediated gene transfer in a regulable manner in vitro and display comparable biological activity as Lucentis. These results are the basis for in vivo studies in human VEGF-overexpressing mice, a model for human neovascular disorders.

Angiogenesis mediated by toll-like receptor 4 in ischemic neural tissue.

OBJECTIVE: Activation of the immune system via toll-like receptors (TLRs) is implicated in atherosclerosis, microvascular complications, and angiogenesis. However, the involvement of TLRs in inflammation-associated angiogenesis in ischemic neural tissue has not been investigated. The goal of this study is to determine the role of TLR4 signaling in oxygen-induced neovascularization in retina, a neural tissue. METHODS AND RESULTS: In oxygen-induced retinopathy model, we found that retinal neovascularization was significantly attenuated in TLR4(-/-) mice. The further study revealed that the absence of TLR4 led to downregulation of proinflammatory factors in association with the attenuated activation of glia in the ischemic retina, which was also associated with reduced expression of high-mobility group box-1, an endogenous ligand for TLR4. The application of high-mobility group box-1 to the ischemic retina promoted the production of proinflammatory factors in wild-type but not TLR4(-/-) mice. High-mobility group box-1 treatment in vitro also significantly promoted the production of proinflammatory factors in retinal glial cells from wild-type mice, but much less from TLR4(-/-) mice. CONCLUSIONS: Our results suggest that the release of high-mobility group box-1 in ischemic neural tissue initiates TLR4-dependent responses that contribute to neovascularization. These findings represented a previously unrecognized effect of TLR4 on angiogenesis in association with the activation of glia in ischemic neural tissue.

MEDI3617, a human anti-angiopoietin 2 monoclonal antibody, inhibits angiogenesis and tumor growth in human tumor xenograft models.

Angiopoietin 2 (Ang2) is an important regulator of angiogenesis, blood vessel maturation and integrity of the vascular endothelium. The correlation between the dynamic expression of Ang2 in tumors with regions of high angiogenic activity and a poor prognosis in many tumor types makes Ang2 an ideal drug target. We have generated MEDI3617, a human anti-Ang2 monoclonal antibody that neutralizes Ang2 by preventing its binding to the Tie2 receptor in vitro, and inhibits angiogenesis and tumor growth in vivo. Treatment of mice with MEDI3617 resulted in inhibition of angiogenesis in several mouse models including: FGF2-induced angiogenesis in a basement extract plug model, tumor and retinal angiogenesis. In xenograft tumor models, treatment with MEDI3617 resulted in a reduction in tumor angiogenesis and an increase in tumor hypoxia. The administration of MEDI3617 as a single agent to mice bearing human tumor xenografts resulted in tumor growth inhibition against a broad spectrum of tumor types. Combining MEDI3617 with chemotherapy or bevacizumab resulted in a delay in tumor growth and no body weight loss was observed in the combination groups. These results, combined with pharmacodynamic studies, demonstrate that treatment of tumor-bearing mice with MEDI3617 significantly inhibited tumor growth as a single agent by blocking tumor angiogenesis. Together, these data show that MEDI3617 is a robust antiangiogenic agent and support the clinical evaluation and biomarker development of MEDI3617 in cancer patients.

Serum autoantibody biomarkers for age-related macular degeneration and possible regulators of neovascularization.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrial counties. Its pathogenesis is at least partially mediated by immunological factors, including a possible autoimmune response. To date, only a few antibodies have been identified in sera from patients with AMD. In order to reveal an autoantibody profile for AMD and identify biomarkers for progression of this disease, we have performed an antigen microarray analysis of serum samples from patients with AMD and healthy controls. Sera from the AMD groups contained high levels of IgG and IgM autoantibodies to some systemic antigens when compared to the normal group. Targeted antigens included cyclic nucleotide phosphodiesterase, phosphatidylserine (PS) and proliferating cell nuclear antigen. The IgG/IgM ratio for antibodies to PS was notably elevated in the AMD group compared to the normal group, and this ratio correlated best with the stage of AMD patients with an anti-PS ratio greater than the cut-off value had a 44-fold risk for advanced AMD with choroidal neovascularization. PS immunoreactivity was also elevated in AMD retina. Moreover, IgG autoantibodies purified from sera of AMD patients induced more tube formation on choroidal-retinal endothelial cells compared to those of healthy donors. Hence, sera from patients with AMD contain specific autoantibodies which may be used as biomarkers for AMD, and the IgG/M ratio for autoantibodies to PS might allow better monitoring of AMD progression.

Preclinical safety evaluation of subretinal AAV2.sFlt-1 in non-human primates.

We report on the long-term safety of AAV2.sFlt-1 (a recombinant adeno-associated virus serotype 2 carrying the soluble form of the Flt-1 receptor) injection into the subretinal space of non-human primates. Levels of sFlt-1 protein were significantly higher (P<0.05) in the vitreous of four out of five AAV2.sFlt-1-injected eyes. There was no evidence of damage to the eyes of animals that received subretinal injections of AAV2.sFlt-1; ocular examination showed no anterior chamber flare, normal fundus and electroretinography responses equivalent to those observed before treatment. Notably, immunological analysis demonstrated that gene therapy involving subretinal injection of AAV2.sFlt-1 does not elicit cell-mediated immunity. Biodistribution analysis showed that AAV2.sFlt-1 could be detected only in the eye and not in the other organs tested. These data indicate that gene therapy with subretinal AAV2.sFlt-1 is safe and well tolerated, and therefore promising for the long-term treatment of neovascular diseases of the eye.

rAAV.sFlt-1 gene therapy achieves lasting reversal of retinal neovascularization in the absence of a strong immune response to the viral vector.

PURPOSE: To determine the efficacy of rAAV.sFlt-1-mediated gene therapy in a transgenic mouse model of retinal neovascularization (trVEGF029) and to assess whether rAAV.sFlt-1 administration generated any deleterious, long-lasting immune response that could affect efficacy. METHODS: trVEGF029 mice were injected subretinally with rAAV.sFlt-1 or phosphate-buffered saline. Fluorescein angiography and electroretinography were used to compare the extent of fluorescein leakage from retinal vessels and retinal function, respectively. A group of eyes was enucleated, and the retinal vasculature and morphology were studied by confocal and light microscopy. Cells were isolated from the posterior eyecups and spleens of a further group, and immune cell subset populations were investigated by flow cytometry. sFlt-1 protein levels in the eyes were evaluated by ELISA. RESULTS: After a single rAAV.sFlt-1 injection, sFlt-1 protein levels were upregulated, and there was a reduction in fluorescein leakage from the retinal vessels and an improvement in retinal function. Confocal microscopy of isolectin-IB4-labeled retinal wholemounts showed more normal-appearing capillary beds in rAAV.sFlt-1-injected than in PBS-injected trVEGF029 mouse eyes. Light microscopy demonstrated retinal morphology preservation, with fewer aberrant vessels invading the outer nuclear layer of rAAV.sFlt-1-injected eyes. Furthermore, the immune response to subretinal injection of rAAV.sFlt-1 was limited to a transient increase in CD45(+) leukocytes that disappeared by 4 weeks after injection. This transient increase was localized to the eye and did not affect long-term therapeutic efficacy. CONCLUSIONS: The data support the notion that rAAV.sFlt-1 gene therapy is safe and effective for the long-term inhibition of deleterious blood vessel growth in the eye.

Circulating antipericyte autoantibodies: a novel modifier of risk of progression of diabetic retinopathy?

BACKGROUND: Antipericyte autoantibodies (APAAs) are present in high frequency among diabetic subjects with and without nonproliferative retinopathy. This study aimed to determine whether progression of retinopathy in type 2 diabetes was associated with the same medical risk factors in APAA-positive subjects as in APAA-negative subjects. METHODS: Type 2 diabetic patients with nonproliferative diabetic retinopathy at baseline were followed prospectively for 2 years monitoring progression of retinopathy. Thirty-eight (21.7%) of 175 patients had progression in Early Treatment Diabetic Retinopathy Study grade by > or =2 steps in at least 1 eye. Serum APAAs were detected by immunofluorescence on tissue-cultured bovine retinal pericytes. RESULTS: Progression of retinopathy was associated with HbA(1c) level (P = 0.002), diabetes duration (P = 0.03), and albumin/creatinine ratio (P = 0.02) in APAA-negative subjects but not in APAA-positive subjects. The association between progression and APAAs was strongest in the upper quartile for HbA(1c) level (>8.0%), where 71.4% of patients negative for APAAs had progression of retinopathy while only 24.1% of patients positive for APAAs had progression (P = 0.007). CONCLUSION: The results suggest that APAA presence is a modifier of risk of progression of retinopathy due to hyperglycemia and that it could be useful as a biochemical marker of risk of progression of diabetic retinopathy in type 2 diabetic patients with poor metabolic control.

Determination of vitreous interleukin-1 (IL-1) and tumour necrosis factor (TNF) levels in proliferative diabetic retinopathy.

We measured interleukin-1 beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) in the vitreous humour and serum of patients with proliferative diabetic retinopathy (PDR), in order to determine the role of these cytokines in the pathogenesis of the disease. Vitreous and serum samples were collected from 21 patients with PDR who were undergoing pars plana vitrectomy. Control vitreous samples were obtained from cadavers and control serum samples from healthy subjects. The cytokines were measured by enzyme-linked immunosorbent assay. Vitreous IL-1beta and TNF-alpha concentrations in patients with PDR exceeded those of controls (P<0.05), as did serum IL-1beta and TNF-alpha. We suggest that increased vitreous IL-1beta and TNF-alpha levels may play a significant role in the pathogenesis of PDR, which features abnormal cell proliferation and neovascularisation.

Circulating anti-retinal antibodies as immune markers in age-related macular degeneration.

Age-related macular maculopathy (ARM) and age-related macular degeneration (AMD) are the leading causes of blindness in the Western world. Despite the magnitude of this clinical problem, very little is known about the pathogenesis of the disease. In this study, we analysed the sera (using indirect immunohistochemistry and Western blot analysis) from a very large cohort of such patients and normal age-matched controls to detect circulating anti-retinal antibodies. Patients with bilateral drusen (n = 64) and with chorioretinal neovascularization (CNV) (n = 51) were recruited in addition to age-matched control subjects (n = 39). The sera were analysed for anti-retinal immunoglobulins on retinal sections. The data were then correlated with the clinical features graded according to the International Classification and Grading System of ARM and AMD. The sera of patients with drusen (93.75%) and CNV (82.27%) were found to have a significantly (P = 0.02) higher titre of autoantibodies to the retina in comparison with controls (8.69%), indicating significant evidence of involvement of the immune process in early stages of AMD. Subsequent statistical analysis of the drusen group showed significant progressive staining (P = 0.0009) in the nuclei layers from early to late stages of ARM. Western blotting confirmed the presence of anti-retinal immunoglobulins to retinal antigens. As anti-retinal immunoglobulins are present in patients with bilateral drusen and exudative AMD, these antibodies could play a significant role in the pathogenesis of AMD. Whilst we do not have evidence that these antibodies precede disease onset, the possibility that their presence might contribute to disease progression needs to be investigated. Finally, the eventual identification of the target antigens detected by these antibodies may permit the future development of new diagnostic methods for ARM and AMD.

SDF-1 is both necessary and sufficient to promote proliferative retinopathy.

Diabetic retinopathy is the leading cause of blindness in working-age adults. It is caused by oxygen starvation in the retina inducing aberrant formation of blood vessels that destroy retinal architecture. In humans, vitreal stromal cell-derived factor-1 (SDF-1) concentration increases as proliferative diabetic retinopathy progresses. Treatment of patients with triamcinolone decreases SDF-1 levels in the vitreous, with marked disease improvement. SDF-1 induces human retinal endothelial cells to increase expression of VCAM-1, a receptor for very late antigen-4 found on many hematopoietic progenitors, and reduce tight cellular junctions by reducing occludin expression. Both changes would serve to recruit hematopoietic and endothelial progenitor cells along an SDF-1 gradient. We have shown, using a murine model of proliferative adult retinopathy, that the majority of new vessels formed in response to oxygen starvation originate from hematopoietic stem cell-derived endothelial progenitor cells. We now show that the levels of SDF-1 found in patients with proliferative retinopathy induce retinopathy in our murine model. Intravitreal injection of blocking antibodies to SDF-1 prevented retinal neovascularization in our murine model, even in the presence of exogenous VEGF. Together, these data demonstrate that SDF-1 plays a major role in proliferative retinopathy and may be an ideal target for the prevention of proliferative retinopathy.