Dendritic cells play no significant role in the laser-induced choroidal neovascularization model.

Age-related macular degeneration (AMD) is genetically associated with complement. Dendritic cells (DCs) play key roles during innate and adaptive immunity, and express complement components and their receptors. We investigated ocular DC heterogeneity and the role of DCs in the laser-induced choroidal neovascularization (CNV) model. In order to determine the function of DCs, we used two models of DC deficiency: the Flt3-/- and Flt3l-/- mouse. We identified three types of ocular DCs: plasmacytoid DC, classical DC-1, and classical DC-2. At steady-state, classical DCs were found in the iris and choroid but were not detectable in the retina. Plasmacytoid DCs existed at very low levels in iris, choroid, and retina. After laser injury, the number of each DC subset was up-regulated in the choroid and retina. In Flt3-/- mice, we found reduced numbers of classical DCs at steady-state, but each DC subset equally increased after laser injury between wildtype and Flt3-/- mice. In Flt3l-/- mice, each DC subsets was severely reduced after laser injury. Neither Flt3-/- or Flt3l-/- mice demonstrated reduced CNV area compared to wildtype mice. DCs do not play any significant role during the laser-induced CNV model of neovascular AMD.

Associations between the Complement System and Choroidal Neovascularization in Wet Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness affecting the elderly in the Western world. The most severe form of AMD, wet AMD (wAMD), is characterized by choroidal neovascularization (CNV) and acute vision loss. The current treatment for these patients comprises monthly intravitreal injections of anti-vascular endothelial growth factor (VEGF) antibodies, but this treatment is expensive, uncomfortable for the patient, and only effective in some individuals. AMD is a complex disease that has strong associations with the complement system. All three initiating complement pathways may be relevant in CNV formation, but most evidence indicates a major role for the alternative pathway (AP) and for the terminal complement complex, as well as certain complement peptides generated upon complement activation. Since the complement system is associated with AMD and CNV, a complement inhibitor may be a therapeutic option for patients with wAMD. The aim of this review is to (i) reflect on the possible complement targets in the context of wAMD pathology, (ii) investigate the results of prior clinical trials with complement inhibitors for wAMD patients, and (iii) outline important considerations when developing a future strategy for the treatment of wAMD.

Neutrophil-to-lymphocyte ratio in age-related macular degeneration: a systematic review and meta-analysis.

Age-related macular degeneration (AMD) is aetiologically linked to immunological ageing and dysfunction. One aspect of this is the altered neutrophil-to-lymphocyte ratio (NLR), which in other domains have been associated with inflammation and angiogenesis, and therefore investigated in patients with AMD in several papers. In this systematic review and meta-analysis, we summarize findings in patients with AMD in relation to NLR, both qualitatively and quantitatively. We searched PubMed/MEDLINE, EMBASE, Web of Science, and the Cochrane Central and identified six studies from where we extracted data on 1178 individuals (777 patients with AMD and 401 healthy controls). Patients with AMD had a higher NLR (weighted mean difference: 0.37, CI 95% 0.08 to 0.66, p = 0.013) when compared to healthy controls. In subgroup analyses, we did not find a significant difference between patients with dry AMD and healthy controls (weighted mean difference: 0.34, CI 95% -0.03 to 0.69, p = 0.068), but did find a strong significant difference between patients with neovascular AMD and healthy controls (weighted mean difference: 0.54, CI 95% 0.23 to 0.86, p = 0.00068). Hence, we find that the association between AMD and elevated NLR may have stronger relevance to the neovascular subtype of AMD. However, the clinical value of measuring the NLR remains unclear.

Increased levels of circulating CD34+ cells in neovascular age-related macular degeneration: relation with clinical and OCT features.

PURPOSE: To investigate the levels of circulating CD34+ stem cells in patients with neovascular type age-related macular degeneration (AMD) and its relation with clinical and optical coherence tomography (OCT) findings. METHODS: The study consisted of 55 patients: 28 patients (18 male and 10 female) with neovascular type AMD as a study group and 27 patients (12 male and 15 female) scheduled for cataract surgery as a control group. The level of CD34+ stem cells was measured by flow cytometry. Demographic and clinical data were recorded. RESULTS: The mean ages of patients in the study and control groups were 71 ± 8 and 68 ± 6 years, respectively. There was no statistically significant difference in terms of age, sex, or systemic disease association between study and control groups. However, smoking status was significantly higher in the study group (67.9% vs 37.0%; p = 0.02). Stem cell levels were significantly higher in the study group (1.5 ± 0.9 vs 0.5 ± 0.3; p<0.001), but there was no relation between stem cell levels and clinical and OCT findings. CONCLUSIONS: Increased circulating CD34+ stem cell levels were observed in patients with choroidal neovascular membrane associated with AMD, but no significant relation was found between cell levels and clinical and OCT findings.

Inhibition of Placenta Growth Factor Reduces Subretinal Mononuclear Phagocyte Accumulation in Choroidal Neovascularization.

Purpose: The cellular immune response driven by mononuclear phagocytes (MPs) is crucial for choroidal neovascularization (CNV) progression. Case reports show that a switch from pure anti-vascular endothelial growth factor-A (VEGF-A) intravitreal treatment to aflibercept, a drug with combined anti-VEGF-A and anti-placenta growth factor (PlGF) activity, can be beneficial for patients who do not respond to anti-VEGF-A alone. Since MPs harbor VEGFR1, we hypothesize that the interplay of P1GF/vascular endothelial growth factor receptor 1 (VEGFR1) in immune cells plays a pivotal role for CNV. Methods: CNV was induced with laser, and immune cells and neovascularization were analyzed in vivo and ex vivo. Immunohistochemistry was employed for protein detection. Differential expression of angiogenic factors and macrophage polarization markers were assessed by quantitative PCR (qPCR). One day after laser, intravitreal injection of aflibercept or anti-PlGF was performed. Results: In the early inflammatory phase after laser, Plgf but not Vegfa was significantly upregulated. VEGF-A upregulation is limited to the scar, whereas PlGF shows a wider distribution. M1 (proinflammatory) macrophage markers were upregulated in the early phase of CNV. However, M2 (proangiogenic) markers showed more inconsistent dynamics. We demonstrated that both aflibercept and anti-PlGF treatments decrease the overall amount of activated subretinal MPs, and especially of those expressing PlGF. These data correlated with a reduction in leakage associated to CNV. Aflibercept showed a stronger reduction in both parameters. Conclusions: The results hint at an interplay between PlGF/VEGFR1 and MPs that is important in the early phase of CNV. A combined inhibition of VEGF-A and PlGF is superior to a specific anti-PlGF treatment in terms of subretinal MP recruitment.

[Polysialic Acid for Immunomodulation in an Animal Model for Wet Age-Related Macular Degeneration (AMD)]. / Polysialinsäure zur Immunmodulation im Tiermodell der feuchten altersabhängigen Makuladegeneration (AMD).

Background Chronic activation of the innate immune system is a hallmark of retinal degenerative diseases, including age-related macular degeneration (AMD). Overt microglia and macrophage reactivity, as well as dysregulation of the alternative complement system, trigger and sustain retinal degeneration. It is now accepted that there exists a vicious cycle of mononuclear phagocyte reactivity, abnormal intrinsic complement activation, damage of Bruch's membrane, dysfunction of the retinal pigment epithelium, photoreceptor degeneration and choroidal neovascularization (CNV). Targeting the innate immune system may, therefore, be a complementary approach to anti-angiogenic therapy. This article presents data from polysialic acid treatment experiments in the laser-induced mouse model of wet AMD. Material and Methods The laser-CNV mouse model was used to simulate events of neovascular AMD. Polysialic acid was applied by intravitreal injection and microglia activity was assessed with Iba1 staining of retinal and RPE/choroidal flat mounts. Neovascular leakage was determined by fluorescein angiography. Results Intravitreal injection of polysialic acid reduced retinal and RPE/choroidal lesion-associated microglia activity in the laser-induced mouse model of AMD. Polysialic acid treatment also diminished vascular leakage at laser spots in this model. Conclusion Local retinal immunomodulation with polysialic acid presents a novel concept for treatment of inflammatory conditions related to neovascular AMD.

Serum anti-endothelial cell antibodies in patients with age-related macular degeneration treated with intravitreal bevacizumab.

PURPOSE: To analyse the prevalence and changes in circulating anti-endothelial cell antibodies (AECA) during anti-vascular endothelial growth factor (anti-VEGF) therapy. METHODS: Ninety-eight patients with exudative age-related macular degeneration (AMD) were treated with intravitreal bevacizumab. Fifty sex- and age-matched healthy subjects were used as controls. Serum AECA were detected using indirect immunofluorescence on primate skeletal muscle and cultivated human umbilical vein endothelial cells (HUVEC). These investigations were repeated at 4-week intervals within 8 months of follow-up. RESULTS: At baseline examination, 30 of the 98 patients (30.6%) were positive for AECA. The titres of AECA ranged from 1:10 to 1:320. In the control group, AECA were present in only nine sera (18%) with titres ranging between 1:20 and 1:80 (p = 0.0000). The greatest rates of reduction of AECA titres were observed during the 'loading' phase of therapy. During the 'maintenance' phase, the rates of changes in serum AECA levels were less significant and remained constant. In follow-up period in 13 patients (13.3%), serum AECA were detected de novo in titres of 1:10 to 1:80. Statistical analysis did not show any significant correlation between the presence of AECA and activity of the disease. CONCLUSIONS: There is growing evidence that AMD is an immune-mediated disease, and thus it cannot be excluded that AECA may be involved in its pathogenesis and progression. We also speculate that AECA develop in response to retinal damage and anti-VEGF therapy.

Prevalence of anti-retinal autoantibodies in different stages of Age-related macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of central vision loss in older adults. Anti-retinal autoantibodies (AAbs) have been found in individuals with AMD. The goal of the study was to determine the AAb specificity in different stages of AMD, and determine whether there is a prevalent AAb signature. METHODS: Sera of 134 participants in the Age-related Eye Disease Study were analyzed for anti-retinal AAbs by western blotting. The subjects were classified by diagnostic subgroups based upon their clinical classification: No AMD, Intermediate AMD, and Late AMD - geographic atrophy (GA) and Late AMD - neovascular (NV). RESULTS: The presence of anti-retinal AAb was detected in 58% patients with Intermediate and Late AMD, and 54% of those with no AMD. AAbs bound to fifteen different retinal antigens. Most individuals had 1 specific AAbs (67%), with the remainder having 2 to 4 different AAbs. Over 40% of patients with Intermediate AMD, and 46% of those with GA had anti-enolase AAbs, compared with 29% of individuals with NV and 29% with no AMD. Different AAbs signatures related to NV as compared to GA and/or Intermediate AMD were distinguished. Anti-40-kDa (10%) and 42-kDa (16%) autoantibodies were associated with Intermediate AMD, while anti-30-kDa AAbs (23%) were primarily present in GA. Anti-32-kDa (12%), 35-kDa (21%), and 60-kDa (8%) AAbs were more frequent in NV AMD. CONCLUSIONS: A unique AAb pattern for each of the disease subgroups was present when AMD progressed from the intermediate to the late forms of severity. Differences in the frequency of specific AAbs between AMD subgroups suggested that they may participate in pathogenicity of AMD. Further studies are necessary to confirm these observations in the larger cohort and individual AMD patients over time.

Detection of antiranibizumab antibodies among patients with exudative age-related macular degeneration.

PURPOSE: The aim of this study was to detect immune responses induced by intravitreal injection (IVT) of ranibizumab in patients with exudative age-related macular degeneration (AMD) in real life conditions. METHODS: An ELISA protocol from blood samples, following 2 different steps, was used to detect antibodies directed against the variable regions of ranibizumab. RESULTS: Among 91 patients included, 46 received more than 10 IVTs, 36 had received 10 IVTs or fewer, and 9 were treatment naïve. Specific antiranibizumab immunoglobulins G were detected in 14/82 treated patients (17.1%). No immunization was detected among naïve patients. For patients with 10 or fewer previous IVTs, immunization against ranibizumab was detected in 4/36 patients (11.1%) whereas immunization was observed in 10/46 patients (21.7%) with more IVTs (p = 0.20). CONCLUSIONS: Immunization against ranibizumab can be detected in 17% of treated patients. Further clinical studies are needed to investigate the relationship between specific immunization to anti-vascular endothelial growth factor antibodies and response or resistance to ranibizumab treatments.

Plasma antiphospholipid antibody levels in age-related macular degeneration.

PURPOSE: To investigate the association of age-related macular degeneration (AMD) with plasma antiphospholipid antibody levels. METHODS: This prospective study included 19 patients diagnosed as having dry-type AMD, 23 patients with exudative-type AMD, and 25 control subjects. Venous blood samples of the participants were obtained. Anticardiolipin antibodies (aCL) isotypes IgG and IgM were measured by means of an enzyme-linked immunosorbent assay. Lupus anticoagulant (LA) antibodies were measured by the dilute Russell viper venom time screen test. RESULTS: The mean aCL IgG concentration in patients with exudative-type AMD was significantly higher than in patients with dry-type AMD and control subjects. The mean ± SE of aCL IgG levels in patients with exudative-type AMD and dry-type AMD and control subjects was 5.46 ± 1.26; 2.55 ± 0.78; and 0.32 ± 0.1, respectively. The mean aCL IgM levels and LA levels in the 3 groups were not statistically different. CONCLUSIONS: Our findings suggest that elevated levels of serum aCL, a risk factor for cardiovascular and cerebrovascular diseases, may be associated with exudative-type AMD.

Identification of anti-retinal antibodies in patients with age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrial counties. Recent findings indicate that the autoimmunity is involved in the pathogenesis of the disease. However, there is no autoantibody biomarker applied in a clinical setting for diagnosis and prognosis of AMD. In order to reveal retinal antigens targeted by serum IgG from AMD patients, mouse retinal tissue proteins were separated by 2-dimensional electrophoresis and the proteins in the immunoblots that were specific for dry and wet AMD patients IgG were identified by LC-MS/MS. Retinol-binding protein 3 and aldolase C (ALDOC) were mainly recognized by IgG form wet AMD patients. Pyruvate kinase M2 (PKM2) was targeted by both dry and wet AMD and level of anti-PKM2 IgG antibody was correlated best with the stage of AMD. Expression of ALDOC and PKM2 was decreased in mouse retina from aging whereas PKM2 deposit on RPE was increased in aged mice. Our data demonstrate that sera of AMD patients contain autoantibodies against retinal proteins and anti-PKM2 IgG serves as a biomarker for diagnosis and prognosis of AMD. Further investigation of the association of anti-retinal antibody level with expression level of antigens in retina will be needed to reveal the disease pathogenesis.

Safety of ranibizumab therapy in wet AMD and the role of vascular endothelial growth factors in physiological angiogenesis.

Vascular endothelial growth factor - A (VEGF-A), is a major factor implicated in choroidal neovascularisation (CNV) and therefore a target for therapeutic agents in wet age related macular degeneration (AMD). Ranibiuzumab (Lucentis) blocks all active isoforms of VEGF-A and the products of their degradation. It penetrates through all layers of the retina in order to reach the target tissue. It is quickly removed from the system and it is characterised by low level of immunogenicity. The essence of angiogenesis is formation of new vessels by branching and expansion of already existing ones. Angiogenesis is an important physiological process that takes place during the healing of wounds, reconstruction of hypoxic injury and reproduction. However some diseases such as cancer, arthritis, diabetes and neovascular AMD are associated with persistent unregulated angiogenesis. There is an important question whether binding vascular-endothelial growth factors in wet AMD therapies using ranibizumab is correlated with the increase of the incidence of systematic adverse effects (AEs), such as cardiovascular episodes or thrombosis. The aim of this article is to present ranibizumab as a safe drug in treating wet AMD patients. Even though the concentration of Lucentis administered in a dose of 0.3 or 0.5 mg into the vitreous body in the organism is very low, the incidence of AEs during the anti-VEGF therapy was traced. In MARINA and ANCHOR studies, occurrence of possible AEs was observed. No statistically significant differences were shown in the AEs frequency between the patients treated with ranibizumab and the control group, and in correlation with the general population of patients suffering from wet AMD.

Complement depletion with humanized cobra venom factor in a mouse model of age-related macular degeneration.

The effect of complement depletion with humanized cobra venom factor (CVF) on retinal lesion development/neovascularization was determined in a mouse model of wet age-related macular degeneration (AMD). Mice were treated with the humanized CVF protein HC3-1496 prior to, and once daily for 28 days after laser coagulation surgery of the retina. CVF transgenic mice exhibiting permanently low levels of serum complement activity and PBS-treated mice served as positive and negative controls, respectively. Fluorescein isothiocyanate (FITC)-dextran funduscopy after laser surgery indicated the presence of lesions in all mice that underwent laser surgery. In HC3-1496-treated mice as well as CVF transgenic mice smaller lesions were seen after 8 days. Measurement of lesion sizes by histopathological examination of eyes after 28 days revealed a significant reduction of lesion area and volume in both HC3-1496-treated animals and CVF transgenic animals compared to PBS-treated control animals. Systemic complement depletion with a complement depletor, such as the humanized CVF protein HC3-1496, represents a promising therapeutic concept for patients with wet AMD.

[Serum antiretinal antibodies (ARA) in patients with exudative age-related macular degeneration (AMD)--preliminary findings]. / Wystepowanie przeciwcial przeciwsiatkówkowych (PPS) w surowicy chorych z wysiekowa postacia zwyrodnienia plamki zwiazanego z wiekiem (AMD)--obserwacje wstepne.

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. Despite the magnitude of this clinical problem, the pathogenesis of the disease remains still unclear. AIM: To determine the tissue specificity and titer of antiretinal antibodies (ARA) in sera of patients with exudative AMD. MATERIAL AND METHODS: Forty-six patients (92 eyes) 16 males and 30 women with exudative AMD were stratified arbitrary into four groups of AMD activity/severity: group I (n = 19)--patients with active choroidal neovascularization (CNV) in one eye and with drusen in the other eye; group II (n = 14)--patients with bilateral CNV; group III (n = 10)--patients with CNV in one eye and with disciform scar in the contralateral eye; group IV (n = 3)--patients with bilateral disciform scars. In all patients serum ARA were determined using indirect immunofluorescence detection on normal monkey retina as antigens substrate and FITC--labelled goat's anti-human IgA, G, M serum as the secondary antibody. RESULTS: In all patients' serum ARA were present in the range of titres from 1:40 to 1:5120. Control sera (n = 28, 28 males) demonstrated the presence of ARA in titres 1:10 to 1:40 in 46.4% cases. High serum titres of ARA characterized AMD patients with bilateral CNV and CNV in one eye and drusen in the contralateral eye. Low titres of ARA were detected in serum of patients with bilateral disciform scars. Indirect immunofluorescence (IIF) showed eight types histological patterns of patients' sera reactivity against retinal tissue. No correlation was found between serum ARA type and macular lesion activity. CONCLUSIONS: Our preliminary observations indicate a common presence of various ARA in serum of patients with AMD. Thus, it cannot exclude that ARA are involved in the pathogenesis or progression of AMD.