APOE Christchurch-mimetic therapeutic antibody reduces APOE-mediated toxicity and tau phosphorylation.

INTRODUCTION: We discovered that the APOE3 Christchurch (APOE3Ch) variant may provide resistance to Alzheimer's disease (AD). This resistance may be due to reduced pathological interactions between ApoE3Ch and heparan sulfate proteoglycans (HSPGs). METHODS: We developed and characterized the binding, structure, and preclinical efficacy of novel antibodies targeting human ApoE-HSPG interactions. RESULTS: We found that one of these antibodies, called 7C11, preferentially bound ApoE4, a major risk factor for sporadic AD, and disrupts heparin-ApoE4 interactions. We also determined the crystal structure of a Fab fragment of 7C11 and used computer modeling to predict how it would bind to ApoE. When we tested 7C11 in mouse models, we found that it reduced recombinant ApoE-induced tau pathology in the retina of MAPT*P301S mice and curbed pTau S396 phosphorylation in brains of systemically treated APOE4 knock-in mice. Targeting ApoE-HSPG interactions using 7C11 antibody may be a promising approach to developing new therapies for AD.

Peripheral monocytes and neutrophils promote photoreceptor cell death in an experimental retinal detachment model.

Photoreceptor cell death and immune cell infiltration are two major events that contribute to retinal degeneration. However, the relationship between these two events has not been well delineated, primarily because of an inadequate understanding of the immunological processes involved in photoreceptor degeneration, especially that of peripheral leukocytes that infiltrate the subretinal space and retinal tissues. In this work, we characterized the role of leukocyte infiltration within the detached retina. We observed that CD45+ CD11b+ Ly6G+ neutrophils and CD45+ CD11b+ Ly6G- Ly6C+ monocytes are the predominant peripheral immune cell populations that infiltrate the retinal and subretinal space after detachment. Selective depletion of monocytes or neutrophils using cell-specific targeting is neuroprotective for photoreceptors. These results indicate that peripheral innate immune cells contribute to photoreceptor degeneration, and targeting these immune cell populations could be therapeutic during retinal detachment.

Resilience to autosomal dominant Alzheimer's disease in a Reelin-COLBOS heterozygous man.

We characterized the world's second case with ascertained extreme resilience to autosomal dominant Alzheimer's disease (ADAD). Side-by-side comparisons of this male case and the previously reported female case with ADAD homozygote for the APOE3 Christchurch (APOECh) variant allowed us to discern common features. The male remained cognitively intact until 67 years of age despite carrying a PSEN1-E280A mutation. Like the APOECh carrier, he had extremely elevated amyloid plaque burden and limited entorhinal Tau tangle burden. He did not carry the APOECh variant but was heterozygous for a rare variant in RELN (H3447R, termed COLBOS after the Colombia-Boston biomarker research study), a ligand that like apolipoprotein E binds to the VLDLr and APOEr2 receptors. RELN-COLBOS is a gain-of-function variant showing stronger ability to activate its canonical protein target Dab1 and reduce human Tau phosphorylation in a knockin mouse. A genetic variant in a case protected from ADAD suggests a role for RELN signaling in resilience to dementia.

RIPK necrotic cell death pathway in both donor photoreceptor and host immune cells synergize to affect photoreceptor graft survival.

Photoreceptor transplant has been put forward as a repair strategy to tackle degenerated retinas. Nonetheless, cell death and immune rejection seriously limit the success of this strategy, with only a small fraction of transplanted cells surviving. Improving the survival of transplanted cells is of critical importance. Recent evidence has identified receptor-interacting protein kinase 3 (RIPK3) as a molecular trigger controlling necroptotic cell death and inflammation. However, its role in photoreceptor transplantation and regenerative medicine has not been studied. We hypothesized that modulation of RIPK3 to address both cell death and immunity could have advantageous effects on photoreceptor survival. In a model of inherited retinal degeneration, deletion of RIPK3 in donor photoreceptor precursors significantly increases the survival of transplanted cells. Simultaneous RIPK3 deletion in donor photoreceptors and recipients maximizes graft survival. Lastly, to discern the role of RIPK3 in the host immune response, bone marrow transplant experiments demonstrated that peripheral immune cell RIPK3 deficiency is protective for both donor and host photoreceptor survival. Interestingly, this finding is independent of photoreceptor transplantation, as the peripheral protective effect is also observed in an additional retinal detachment photoreceptor degeneration model. Altogether, these results indicate that immunomodulatory and neuroprotective strategies targeting the RIPK3 pathway can aid regenerative therapies of photoreceptor transplantation.

Atrophic, Tractional, and Neovascular Grading System in a Dome-Shaped Macula and Ridge-Shaped Macula Highly Myopic Cohort.

INTRODUCTION: The aim of this study was to analyze the atrophic, tractional, and neovascular (ATN) components grading in highly myopic patients with dome-shaped macula (DSM) and ridge-shaped macula (RSM). METHODS: This was a cross-sectional, noninterventional study. 57 eyes of 38 different patients were included. They were classified as DSM or RSM based on the number of radial scans that showed an inward protrusion ≥50 µm in the swept-source optical coherence tomography (SS-OCT) (12 = DSM; <12 = RSM). All patients underwent a complete ophthalmological examination in addition to SS-OCT. They were graded using the ATN system for myopic maculopathy by 2 masked retina specialists that assessed the atrophic (A), tractional (T), and neovascular (N) components in order to analyze the differences between the groups. As complementary measurements, age, axial length, and best-corrected visual acuity were collected. Height and orientation of the macular bulge and the presence of Bruch's membrane defects, scleral perforating vessels, and staphyloma were recorded. RESULTS: Out of total 57 eyes, 13 eyes (22.8%) were classified as DSM. Regarding the atrophic component (A), there were statistically significant differences between groups, with DSM group showing a greater stage of atrophy (predominantly stage A3 in 69.2% of the sample) compared to the RSM group (predominantly stage A2 in 61.3% of the sample) (p < 0.05). For the T and N components, there were no significant differences between groups. The presence of Bruch's membrane defects was more frequently seen in DSM (p < 0.05). CONCLUSIONS: DSM group showed more Bruch's membrane defects and a greater stage of the atrophy component, based on the ATN grading system, compared with RSM group. As Bruch's membrane may have biomechanical properties in terms of strength, the defects found around the macula, added to the major atrophic component, may be a cause of a local relaxation that induce a central bulge forming the dome.

Topical Nanoemulsion of a Runt-related Transcription Factor 1 Inhibitor for the Treatment of Pathologic Ocular Angiogenesis.

Purpose: To test the efficacy of runt-related transcription factor 1 (RUNX1) inhibition with topical nanoemulsion containing Ro5-3335 (eNano-Ro5) in experimental ocular neovascularization. Design: Preclinical experimental study. Participants: In vitro primary culture human retinal endothelial cell (HREC) culture. C57BL/6J 6- to 10-week-old male and female mice. Methods: We evaluated the effect of eNano-Ro5 in cell proliferation, cell toxicity, and migration of HRECs. We used an alkali burn model of corneal neovascularization and a laser-induced model of choroidal neovascularization to test in vivo efficacy of eNano-Ro5 in pathologic angiogenesis in mice. We used mass spectrometry to measure penetration of Ro5-3335 released from the nanoemulsion in ocular tissues. Main Outcome Measures: Neovascular area. Results: RUNX1 inhibition reduced cell proliferation and migration in vitro. Mass spectrometry analysis revealed detectable levels of the active RUNX1 small-molecule inhibitor Ro5-3335 in the anterior and posterior segment of the mice eyes. Topical treatment with eNano-Ro5 significantly reduced corneal neovascularization and improved corneal wound healing after alkali burn. Choroidal neovascularization lesion size and leakage were significantly reduced after treatment with topical eNano-Ro5. Conclusions: Topical treatment with eNano-Ro5 is an effective and viable platform to deliver a small-molecule RUNX1 inhibitor. This route of administration offers advantages that could improve the management and outcomes of these sight-threatening conditions. Topical noninvasive delivery of RUNX1 inhibitor could be beneficial for many patients with pathologic ocular neovascularization.

Dome-Shaped Macula versus Ridge-Shaped Macula Eyes in High Myopia Based on the 12-line Radial Optical Coherence Tomography Scan Pattern. Differences in Clinical Features.

PURPOSE: To study clinical features in patients with ridge-shaped macula (RSM) compared with those with dome-shaped macula (DSM) having been previously classified by the number of swept-source optical coherence tomography (SS-OCT) radial scans affected. METHODS: Retrospective observational study including 49 highly myopic eyes from 31 patients who underwent SS-OCT. DSM eyes were defined as those that showed a complete round inward convexity in all their axes, presenting an inward convexity ≥50 µm in the 12-line radial OCT scans. Eyes that did not meet this criterion and had at least one flat radial scan were grouped into the RSM group, defined as a macular inward convexity in some meridians across the fovea, whereas the opposite perpendicularly oriented meridians were flat. Age, spherical equivalent, axial length (AL), and best-corrected visual acuity (BCVA) were collected. Height of the bulge, scleral and choroidal thicknesses, Bruch´s membrane defects, and presence of perforating scleral vessels were recorded. RESULTS: Thirty-seven (75.5%) eyes were classified into the RSM group and 12 (24.5%) into the DSM group. Twenty-six (53.0%) eyes showed macular elevation only in the horizontal direction. Mean AL showed statistically significant differences (28.8 ± 2.7 vs. 30.5 ± 1.5 mm in the RMS vs. DSM group, respectively) and the presence of Bruch´s membrane defects was more frequently seen in DSM (p < 0.001). Mean age, spherical equivalent, BCVA, height of the inward convexity, retinal foveal thickness, foveal scleral thickness, subfoveal choroidal thickness, and the presence of perforating scleral vessels did not show significant differences between groups. CONCLUSION: This study shows the reliability of using the 12 equal radial OCT scans as an objective method to define and differentiate DSM versus RSM. Patients with RSM showed differences in AL compared with those with DSM, being longer in DSM, and regarding the presence of Bruch´s membrane defects, being more common in DSM. This may contribute to identifying those patients that, in daily clinical practice, have a higher risk of developing complications due to their myopia.

Local photoreceptor cell death differences in the murine model of retinal detachment.

To investigate local cell death differences in the attached and detached retina at different regions in a murine experimental retinal detachment model. Subretinal injection of sodium hyaluronate was performed in eight-week-old C57BL/6J mice. Retinal regions of interest were defined in relation to their distance from the peak of the retinal detachment, as follows: (1) attached central; (2) attached paracentral; (3) detached apex; and (4) detached base. At day 0, the outer nuclear layer cell count for the attached central, attached paracentral, detached apex, and detached base was 1247.60 ± 64.62, 1157.80 ± 163.33, 1264.00 ± 150.7, and 1013.80 ± 67.16 cells, respectively. There were significant differences between the detached base vs. attached central, and between detached base vs. detached apex at day 0. The detached apex region displayed a significant and progressive cell count reduction from day 0 to 14. In contrast, the detached base region did not show progressive retinal degeneration in this model. Moreover, only the detached apex region had a significant and progressive cell death rate compared to baseline. Immediate confounding changes with dramatic differences in cell death rates are present across regions of the detached retina. We speculate that mechanical and regional differences in the bullous detached retina can modify the rate of cell death in this model.

Monochromatic higher order aberrations in highly myopic eyes with Staphyloma.

BACKGROUND: Prevalence of high myopia is continuously increasing, thus, patients affected with staphyloma are abundant worldwide. Assessment of the quality of vision in these patients is mandatory for a proper clinical counselling, specially when undergoing surgical procedures that require intraocular lenses implantation. Thus, the purpose of the study was to assess monochromatic higher order aberrations (HOAs) in highly myopic eyes with staphyloma with or without a dome-shaped macula. METHODS: Participants underwent spectral-domain optical coherence tomography, ocular axial biometry, dual Scheimpflug photography and integrated Placido disk topography, and Hartmann-Shack wavefront analysis. Five groups were evaluated: a low-moderate myopia control group (< 6.00 diopters, n = 31) and four high myopia (≥6.00 diopters) groups: eyes without staphyloma (n = 18), eyes with inferior staphyloma (n = 14), eyes with posterior staphyloma without dome-shaped macula (n = 15) and eyes with posterior staphyloma with dome-shaped macula (n = 17). Subsequently, two new groups (including all participants) were created to assess differences between myopia with and without staphyloma. One-way analysis of covariance was performed using age and lens densitometry as covariates. RESULTS: Statistically significant (p ≤ 0.05) differences in anterior corneal fourth-order HOAs were observed between the low-moderate myopia and no-dome-shaped macula (Mean: 0.16 µm) and dome-shaped macula posterior staphyloma groups (Mean: 0.12 µm) in younger patients (≤45 years old). The same groups also showed (p ≤ 0.05) significant differences for anterior corneal primary spherical aberration (Mean: 0.19 and 0.13 µm, respectively). In addition, anterior corneal tetrafoil was significantly higher (p = 0.04) in dome-shaped macula compared to no-dome-shaped macula (Mean: 0.18 vs 0.06 µm, respectively). When all participants were grouped together, significantly lower mean anterior corneal primary spherical aberration (0.15 µm vs. 0.27 µm, p = 0.004) and higher internal primary spherical aberration (0.04 µm vs. -0.06 µm, p = 0.04) was observed in staphyloma compared to no-staphyloma myopic patients. CONCLUSIONS: Eyes with high myopia and staphyloma have less positive anterior corneal primary spherical aberration and less negative internal primary spherical aberration, suggesting that the anterior corneal surface tends to mimic in a specular fashion the posterior pole profile. This corneal behaviour appears to change in patients older than 45 years.

TNF-α signaling regulates RUNX1 function in endothelial cells.

Runt-related transcription factor 1 (RUNX1) acts as a mediator of aberrant retinal angiogenesis and has been implicated in the progression of proliferative diabetic retinopathy (PDR). Patients with PDR, retinopathy of prematurity (ROP), and wet age-related macular degeneration (wet AMD) have been found to have elevated levels of Tumor Necrosis Factor-alpha (TNF-α) in the eye. In fibrovascular membranes (FVMs) taken from patients with PDR RUNX1 expression was increased in the vasculature, while in human retinal microvascular endothelial cells (HRMECs), TNF-α stimulation causes increased RUNX1 expression, which can be modulated by RUNX1 inhibitors. Using TNF-α pathway inhibitors, we determined that in HRMECs, TNF-α-induced RUNX1 expression occurs via JNK activation, while NF-κB and p38/MAPK inhibition did not affect RUNX1 expression. JNK inhibitors were also effective at stopping high D-glucose-stimulated RUNX1 expression. We further linked JNK to RUNX1 through Activator Protein 1 (AP-1) and investigated the JNK-AP-1-RUNX1 regulatory feedback loop, which can be modulated by VEGF. Additionally, stimulation with TNF-α and D-glucose had an additive effect on RUNX1 expression, which was downregulated by VEGF modulation. These data suggest that the downregulation of RUNX1 in conjunction with anti-VEGF agents may be important in future treatments for the management of diseases of pathologic ocular angiogenesis.

Treatment of Experimental Choroidal Neovascularization via RUNX1 Inhibition.

Choroidal neovascularization (CNV) is a prevalent cause of vision loss in patients with age-related macular degeneration. Runt-related transcription factor 1 (RUNX1) has been identified as an important mediator of aberrant retinal angiogenesis in proliferative diabetic retinopathy and its modulation has proven to be effective in curbing pathologic angiogenesis in experimental oxygen-induced retinopathy. However, its role in CNV remains to be elucidated. This study demonstrates RUNX1 expression in critical cell types involved in a laser-induced model of CNV in mice. Furthermore, the preclinical efficacy of Ro5-3335, a small molecule inhibitor of RUNX1, in experimental CNV is reported. RUNX1 inhibitor Ro5-3335, aflibercept-an FDA-approved vascular endothelial growth factor (VEGF) inhibitor, or a combination of both, were administered by intravitreal injection immediately after laser injury. The CNV area of choroidal flatmounts was evaluated by immunostaining with isolectin B4, and vascular permeability was analyzed by fluorescein angiography. A single intravitreal injection of Ro5-3335 significantly decreased the CNV area 7 days after laser injury, and when combined with aflibercept, reduced vascular leakage more effectively than aflibercept alone. These data suggest that RUNX1 inhibition alone or in combination with anti-VEGF drugs may be a new therapy upon further clinical validation for patients with neovascular age-related macular degeneration.

Topical delivery of a small molecule RUNX1 transcription factor inhibitor for the treatment of proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is the leading cause of retinal detachment surgery failure. Despite significant advances in vitreoretinal surgery, it still remains without an effective prophylactic or therapeutic medical treatment. After ocular injury or retinal detachment, misplaced retinal cells undergo epithelial to mesenchymal transition (EMT) to form contractile membranes within the eye. We identified Runt-related transcription factor 1 (RUNX1) as a gene highly expressed in surgically-removed human PVR specimens. RUNX1 upregulation was a hallmark of EMT in primary cultures derived from human PVR membranes (C-PVR). The inhibition of RUNX1 reduced proliferation of human C-PVR cells in vitro, and curbed growth of freshly isolated human PVR membranes in an explant assay. We formulated Ro5-3335, a lipophilic small molecule RUNX1 inhibitor, into a nanoemulsion that when administered topically curbed the progression of disease in a novel rabbit model of mild PVR developed using C-PVR cells. Mass spectrometry analysis detected 2.67 ng/mL of Ro5-3335 within the vitreous cavity after treatment. This work shows a critical role for RUNX1 in PVR and supports the feasibility of targeting RUNX1 within the eye for the treatment of an EMT-mediated condition using a topical ophthalmic agent.

Predictive models of long-term anatomic outcome in age-related macular degeneration treated with as-needed Ranibizumab.

BACKGROUND: To analyze predictors and develop predictive models of anatomic outcome in neovascular age-related macular degeneration (AMD) treated with as-needed ranibizumab after 4 years of follow-up. METHODS: A multicenter consecutive case series non-interventional study was performed. Clinical, funduscopic and OCT characteristics of 194 treatment-naïve patients with AMD treated with as-needed ranibizumab for at least 2 years and up to 4 years were analyzed at baseline, 3 months and each year until the end of the follow-up. Baseline demographic and angiographic characteristics were also evaluated. R Statistical Software was used for statistical analysis. Main outcome measure was final anatomic status. RESULTS: Factors associated with less probability of preserved macula were diagnosis in 2009, older age, worse vision, presence of atrophy/fibrosis, pigment epithelium detachment, and geographic atrophy/fibrotic scar/neovascular AMD in the fellow eye. Factors associated with higher probability of GA were presence of atrophy and greater number of injections, whereas male sex, worse vision, lesser change in central macular thickness and presence of fibrosis were associated with less probability of GA as final macular status. Predictive model of preserved macula vs. GA/fibrotic scar showed sensibility of 77.78% and specificity of 69.09%. Predictive model of GA vs. fibrotic scar showed sensibility of 68.89% and specificity of 72.22%. CONCLUSIONS: We identified predictors of final macular status, and developed two predictive models. Predictive models that we propose are based on easily harvested variables, and, if validated, could be a useful tool for individual patient management and clinical research studies.

Functional characterization of rs2229094 (T>C) polymorphism in the tumor necrosis factor locus and lymphotoxin alpha expression in human retina: the Retina 4 project.

PURPOSE: The objective of this study is to determine the expression and localization of lymphotoxin alpha (LTA) in human retinas and the functionality of one of its polymorphisms rs2229094 (C13R) (T>C), previously associated with proliferative vitreoretinopathy (PVR) development. MATERIALS AND METHODS: Total RNA from three healthy human retinas were extracted and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis, using flanking primers of LTA cDNA. In addition, three human eyes with retinal detachment (RD) and three healthy control eyes were subjected to immunohistochemistry (IHC) with a specific antibody against LTA. The functionality of T and C alleles was assessed by using pCEFL-Flag expression vector and transient transfection assays in COS-1 cell line. In addition, expression analysis by RT-PCR, Western blot and subcellular localization of both alleles and by immunofluorescence assay was performed. RESULTS: RT-PCR analysis revealed no significant levels of messenger RNA (mRNA) LTA in healthy human retinas. Sequential IHC staining showed differences between healthy human and RD retinas. No differences in mRNA and protein expression levels and in subcellular localization between both alleles were found. Both alleles were located in the cytoplasm of COS-1 cells. CONCLUSION: Although results suggest lack of functionality, the differences found in IHC study and its strong association with PVR and its relationship with tumor necrosis factor locus, warrant further studies and could justify the use of this polymorphism as a valid biomarker to identify high-risk patients to develop PVR after RD.

ACUTE RETINAL DAMAGE AFTER USING A TOXIC PERFLUORO-OCTANE FOR VITREO-RETINAL SURGERY.

PURPOSE: To describe a series of retinal acute toxicity cases with severe visual loss after intraocular use of a toxic perfluoro-octane (PFO). The clinical presentation is described, and the likely causes are analyzed. New biological methods for testing safety of intraocular medical devices are proposed. METHODS: Information regarding a series of eyes suffering acute severe events after intraocular use of a toxic PFO was analyzed. Four types of spectroscopy, nuclear magnetic resonance, and chromatography were used to identify the potential PFO contaminants. Cultures of human retinal pigment epithelial cells (ARPE-19) and porcine neuroretina were used to quantify the toxicity of the suspect PFO lots. RESULTS: Of 117 cases of intraocular toxicity, 96 were considered clearly related to the use of PFO. Fifty-three cases had no light perception, and 97 had no measurable visual acuity. Retinal necrosis (n = 38) and vascular occlusion (n = 33) were the most characteristic findings. Two hydroxyl compounds, perfluorooctanoic acid and dodecafluoro-1-heptanol, and benzene derivatives were identified as the suspected toxic agents. While existing toxicity testing failed, we proposed new tests that demonstrated clear toxicity. CONCLUSION: Protocols to determine cytotoxicity of intraocular medical devices should be revised to assure safety. Acute toxic events should be reported to health authorities and scientific media.

Proliferative vitreoretinopathy: A new concept of disease pathogenesis and practical consequences.

During the last four decades, proliferative vitreoretinopathy (PVR) has defied the efforts of many researchers to prevent its occurrence or development. Thus, PVR is still the major complication following retinal detachment (RD) surgery and a bottle-neck for advances in cell therapy that require intraocular surgery. In this review we tried to combine basic and clinical knowledge, as an example of translational research, providing new and practical information for clinicians. PVR was defined as the proliferation of cells after RD. This idea was used for classifying PVR and also for designing experimental models used for testing many drugs, none of which were successful in humans. We summarize current information regarding the pathogenic events that follow any RD because this information may be the key for understanding and treating the earliest stages of PVR. A major focus is made on the intraretinal changes derived mainly from retinal glial cell reactivity. These responses can lead to intraretinal PVR, an entity that has not been clearly recognized. Inflammation is one of the major components of PVR, and we describe new genetic biomarkers that have the potential to predict its development. New treatment approaches are analyzed, especially those directed towards neuroprotection, which can also be useful for preventing visual loss after any RD. We also summarize the results of different surgical techniques and clinical information that is oriented toward the identification of high risk patients. Finally, we provide some recommendations for future classification of PVR and for designing comparable protocols for testing new drugs or techniques.