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.

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.

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.

Hypopyon and White Cataract Due to an Intraocular Helminth.

We describe a case of a 29-year-old man with a history of intravenous drug use and vague history of eye trauma who presented with a hypopyon and white cataract in the right eye. He underwent pars plana vitrectomy and lensectomy; his anterior chamber aspirate revealed a single helminth on calcofluor stain. We suspect that his helminth infection may be secondary to unsanitary eating and drinking practices. As overall hygiene and dietary habits have improved during the years, parasitic helminth infections are relatively rare in nonendemic areas, especially in the nonpediatric population. [Ophthalmic Surg Lasers Imaging Retina. 2022;53:168-171.].

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.

Targeting Runt-Related Transcription Factor 1 Prevents Pulmonary Fibrosis and Reduces Expression of Severe Acute Respiratory Syndrome Coronavirus 2 Host Mediators.

Pulmonary fibrosis (PF) can arise from unknown causes, as in idiopathic PF, or as a consequence of infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current treatments for PF slow, but do not stop, disease progression. We report that treatment with a runt-related transcription factor 1 (RUNX1) inhibitor (Ro24-7429), previously found to be safe, although ineffective, as a Tat inhibitor in patients with HIV, robustly ameliorates lung fibrosis and inflammation in the bleomycin-induced PF mouse model. RUNX1 inhibition blunted fundamental mechanisms downstream pathologic mediators of fibrosis and inflammation, including transforming growth factor-ß1 and tumor necrosis factor-α, in cultured lung epithelial cells, fibroblasts, and vascular endothelial cells, indicating pleiotropic effects. RUNX1 inhibition also reduced the expression of angiotensin-converting enzyme 2 and FES Upstream Region (FURIN), host proteins critical for SARS-CoV-2 infection, in mice and in vitro. A subset of human lungs with SARS-CoV-2 infection overexpress RUNX1. These data suggest that RUNX1 inhibition via repurposing of Ro24-7429 may be beneficial for PF and to battle SARS-CoV-2, by reducing expression of viral mediators and by preventing respiratory complications.

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.

Resistance to autosomal dominant Alzheimer's disease in an APOE3 Christchurch homozygote: a case report.

We identified a PSEN1 (presenilin 1) mutation carrier from the world's largest autosomal dominant Alzheimer's disease kindred, who did not develop mild cognitive impairment until her seventies, three decades after the expected age of clinical onset. The individual had two copies of the APOE3 Christchurch (R136S) mutation, unusually high brain amyloid levels and limited tau and neurodegenerative measurements. Our findings have implications for the role of APOE in the pathogenesis, treatment and prevention of Alzheimer's disease.

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.