Spatial Distribution Analysis of Novel Texture Feature Descriptors for Accurate Breast Density Classification.

Breast density has been recognised as an important biomarker that indicates the risk of developing breast cancer. Accurate classification of breast density plays a crucial role in developing a computer-aided detection (CADe) system for mammogram interpretation. This paper proposes a novel texture descriptor, namely, rotation invariant uniform local quinary patterns (RIU4-LQP), to describe texture patterns in mammograms and to improve the robustness of image features. In conventional processing schemes, image features are obtained by computing histograms from texture patterns. However, such processes ignore very important spatial information related to the texture features. This study designs a new feature vector, namely, K-spectrum, by using Baddeley's K-inhom function to characterise the spatial distribution information of feature point sets. Texture features extracted by RIU4-LQP and K-spectrum are utilised to classify mammograms into BI-RADS density categories. Three feature selection methods are employed to optimise the feature set. In our experiment, two mammogram datasets, INbreast and MIAS, are used to test the proposed methods, and comparative analyses and statistical tests between different schemes are conducted. Experimental results show that our proposed method outperforms other approaches described in the literature, with the best classification accuracy of 92.76% (INbreast) and 86.96% (MIAS).

AQUEOUS HUMOR CYTOKINE LEVELS AS BIOMARKERS OF DISEASE SEVERITY IN DIABETIC MACULAR EDEMA.

PURPOSE: To determine whether aqueous cytokine levels correlate with disease severity in diabetic macular edema. METHODS: A prospective cross-sectional study of 49 adults with diabetes mellitus, centre-involving diabetic macular edema and central subfield macular thickness ≥310 µm on spectral domain optical coherence tomography. Clinical examination and aqueous sampling were carried out before an initial injection of ranibizumab. Multiplex immunoassay of sample was carried out for vascular endothelial growth factor, placental growth factor, transforming growth factor beta, intercellular adhesion molecule-1, interleukin (IL)-2, IL-3, IL-6, IL-8, IL-10, IL-17, vascular cell adhesion molecule-1, monocyte chemoattractant protein-1, and epidermal growth factor. Multivariate robust regression models were constructed, and adjusted for age, lens status, or severity of retinopathy, and size of foveal avascular zone. RESULTS: Spectral domain optical coherence tomography macular volume was an excellent measure of disease severity, correlating strongly with central subfield macular thickness (P < 0.001), best-corrected Snellen visual acuity (P < 0.001), and baseline diabetic retinopathy severity (P = 0.01). Elevated aqueous intercellular adhesion molecule-1 correlated with greater macular volume (P = 0.002). No aqueous cytokine, including VEGF, correlated with central subfield macular thickness. There was an association between IL-10 levels and best-corrected Snellen visual acuity (P = 0.03). CONCLUSION: Aqueous intercellular adhesion molecule-1 correlates with disease severity as measured by macular volume on spectral domain optical coherence tomography, and IL-10 is associated with BCVA. Intercellular adhesion molecule-1 may be a clinically useful biomarker for diabetic macular edema severity.

Prolonged Ocular Retention of Mucoadhesive Nanoparticle Eye Drop Formulation Enables Treatment of Eye Diseases Using Significantly Reduced Dosage.

Eye diseases, such as dry eye syndrome, are commonly treated with eye drop formulations. However, eye drop formulations require frequent dosing with high drug concentrations due to poor ocular surface retention, which leads to poor patient compliance and high risks of side effects. We developed a mucoadhesive nanoparticle eye drop delivery platform to prolong the ocular retention of topical drugs, thus enabling treatment of eye diseases using reduced dosage. Using fluorescent imaging on rabbit eyes, we showed ocular retention of the fluorescent dye delivered through these nanoparticles beyond 24 h while free dyes were mostly cleared from the ocular surface within 3 h after administration. Utilizing the prolonged retention of the nanoparticles, we demonstrated effective treatment of experimentally induced dry eye in mice by delivering cyclosporin A (CsA) bound to this delivery system. The once a week dosing of 0.005 to 0.01% CsA in NP eye drop formulation demonstrated both the elimination of the inflammation signs and the recovery of ocular surface goblet cells after a month. Thrice daily administration of RESTASIS on mice only showed elimination without recovering the ocular surface goblet cells. The mucoadhesive nanoparticle eye drop platform demonstrated prolonged ocular surface retention and effective treatment of dry eye conditions with up to 50- to 100-fold reduction in overall dosage of CsA compared to RESTASIS, which may significantly reduce side effects and, by extending the interdosing interval, improve patient compliance.

Spontaneously occurring fundus findings observed using confocal scanning laser ophthalmoscopy in wild type Sprague Dawley rats.

PURPOSE: Non-invasive in vivo imaging is an increasingly used component of pre-clinical research. However, to reliably interpret data, it may be necessary to identify and document pre-existent findings prior to initiating long-term or intensive protocols, particularly where toxicity or efficacy is under investigation. Here we report here spontaneously occurring findings from the Sprague Dawley (SD) rat eye using multi-modal confocal scanning laser ophthalmoscopy (cSLO). METHODS: As part of ongoing studies, with the goal of excluding animals with abnormalities from further investigation, a total of 165 wild type SD rats (312 eyes) were assessed using cSLO imaging at baseline prior to initiating experiments to detect, describe, and determine the prevalence of spontaneous fundus findings. RESULTS: Using fundus autofluorescence (FAF) as the primary screening modality, over 30% of analyzed eyes possessed some fundus finding that differed from the normal composite reference image. Unexpectedly, 100% of eyes demonstrated a diffuse hyperfluorescent region in the posterior pole that was ultimately considered normal, and formed part of the reference. Evaluated by three independent reviewers, five groups of FAF abnormalities were defined, based primarily on shape and size of the lesion. Of these, the most extensive lesions were further analyzed using infrared reflectance (IR) and red free (RF) imaging. White light and autofluorescent microscopy of excised tissue confirmed that the extensive lesions were derived from abnormalities in both the isolated retina and posterior eyecups. CONCLUSIONS: Given the newly described hyperfluorescent glow that appears in all eyes, and the high basal rate of spontaneous lesions in the outbred SD rat, we suggest that investigators be aware of the variants of normal, and that baseline in vivo screening be considered prior to initiating intensive or expensive investigation.

Hydrophobically-modified poly(vinyl pyrrolidone) as a physically-associative, shear-responsive ophthalmic hydrogel.

The potential of hydrophobically-modified poly(vinyl pyrrolidone) as a shear-responsive, self-associative hydrogel for ophthalmic applications is demonstrated. Hydrophobic modification was achieved via random copolymerization of N-vinylpyrrolidone with N-vinylformamide, the latter of which can be hydrolyzed to expose a desired degree of reactive amine groups permitting grafting of alkyl chlorides of varying alkyl chain lengths. The resulting materials formed highly shear-responsive physical hydrogels, exhibiting tunable shear thinning over 4-5 decades of viscosity from infinite shear to zero shear conditions that facilitates lubrication upon blinking and/or facile injection or drop-based delivery to the anterior or posterior segments of the eye. Viscosity changes due to self-association over time can also be tuned by changing the length of the hydrophobe, with C18-grafted materials exhibiting prolonged thickening over several weeks to form extremely stiff hydrogels and shorter grafts equilibrating significantly faster but forming weaker gels. The hydrogels remained transparent even at very high polymer concentrations (20 wt%) and are demonstrated to facilitate controlled release of a model drug (doxorubicin). The polymers exhibit minimal cytotoxicity in vitro to human corneal epithelial cells and retinal pigment epithelial cells, particularly when lower molecular weight backbone polymers were used. In vivo assessments in rabbits indicated no significant conjunctival edema or redness, secretion, corneal opacity, or iris involvement upon anterior application. Following intravitreal injection in rat eyes, no opacification of the lens, cornea or vitreous, nor any morphological or functional change to the posterior segment was observed. Examination of wholemount tissues and histology demonstrated no adverse effect from the injection or deposition of material. As such, these shear-thinning materials offer potential for drug delivery in both the anterior and posterior segments or as a vitreal replacement that can be easily administered or removed.

Origin-specific epigenetic program correlates with vascular bed-specific differences in Rgs5 expression.

Cells from multiple origins contribute to vascular smooth muscle cell (VSMC) development. Phenotypic heterogeneity of VSMCs is associated with their point of developmental origin; however, the mechanisms driving such differences are unknown. We here examined the mechanisms controlling vascular bed-specific differences in Rgs5 expression during development. Rgs5 levels were similar across different regions of the vasculature in neonatal animals but were >15-fold higher in descending aortas compared with carotid arteries of adult mice. Thus, vessel bed-specific changes in regulation of Rgs5 expression occurred during vessel maturation. Examination of adult Rgs5-LacZ reporter mice revealed lower Rgs5 expression in VSMCs originating from the third (carotid artery) branchial arch compared with those originating in the fourth and sixth (aortic B segment, right subclavian, and ductus arteriosus) branchial arches. Indeed, a mosaic Rgs5 expression pattern, with discreet LacZ boundaries between VSMCs derived from different developmental origins, was observed. Furthermore, Rgs5-LacZ expression was correlated with the site of VSMC origin (splanchic mesoderm ≈ local mesenchyme > somites > proepicardium > mesothelium). Surprisingly, Rgs5 reporter activity in cultured carotid artery- and descending aorta-derived cells did not recapitulate the differences observed in vivo. Consistent with a developmental origin-specific epigenetic mechanism driving the observed expression differences in vivo, the Rgs5 promoter showed increased methylation on CpG dinucleotides in carotid arteries compared with that in descending aortas in adult but not in neonatal mice. In vitro methylation of the Rgs5 promoter confirmed that its activity is sensitive to transcriptional down-regulation by CpG methylation. These data suggest that an origin-dependent epigenetic program regulates vascular bed- and maturation state-dependent regulation of VSMC-specific gene transcription.

Monkeypox-Associated Disciform Keratitis.

PURPOSE: The purpose of this study was to describe a case of monkeypox (MPX)-associated disciform keratitis. METHODS: This is a case report. RESULTS: A 36-year-old male patient presented to the infectious diseases clinic with a 1-week history of disseminated pustular skin lesions, a 4-day history of constitutional symptoms, and redness in the left eye. Testing of blood, 2 skin lesions, and a conjunctival swab confirmed the presence of MPX virus by polymerase chain reaction. On ophthalmologic examination on the 17th day of illness, there was a corneal epithelial ridge that stained with fluorescein with disciform corneal edema and underlying keratic precipitates. The patient was treated with oral tecovirimat 600 mg twice a day for 14 days and topical prednisolone acetate 1% 4 times daily, starting 2 days later. On completion of oral treatment, his corneal findings had resolved except for a small subepithelial scar at which time topical steroids were tapered. CONCLUSIONS: MPX may cause disciform keratitis and scarring that closely resembles other ocular viral infections. Clinical trials are urgently needed to define the optimal management of human MPX infections and reduce vision loss.

Biphasic effect of danazol on human vascular endothelial cell permeability and f-actin cytoskeleton dynamics.

Breakdown of endothelial barrier function is a hallmark event across a variety of pathologies such as inflammation, cancer, and diabetes. It has also been appreciated that steroid hormones impart direct biological activity on endothelial cells at many levels. The purpose of this investigation was to explore the effect of the androgen-like steroid, danazol, on endothelial cell barrier function in vitro. Primary human endothelial cells exposed to 0.01-50 µM danazol were evaluated for changes in permeability. We found that danazol altered endothelial permeability in a biphasic manner in which nanomolar concentrations enhance barrier function while micromolar concentrations are detrimental. Monitoring of trans-endothelial electrical resistance demonstrated that these barrier enhancing effects were rapid (within 5 min) and lasted for over 24h. Analysis of intracellular f-actin organization showed that barrier enhancement also correlated with the formation of a submembranous cortical actin ring. Conversely, at higher danazol concentrations, contractile cell phenotypes were observed, represented by stress fiber formation. Competitive binding studies performed using steroid hormone receptor antagonists proved that this activity is the result of androgen and estrogen receptor ligation. These findings suggest that low dose danazol may provide a therapeutic window for diseases involving vascular leakage.

Effectiveness at 1 year of monthly versus variable-dosing intravitreal ranibizumab in the treatment of choroidal neovascularization secondary to age-related macular degeneration.

PURPOSE: To evaluate the visual acuity results of monthly ranibizumab injections compared with a variable-dosing schedule for the treatment of neovascular age-related macular degeneration. METHODS: A retrospective study that compared two cohorts of consecutive patients. All patients were treatment naive, with baseline visual acuity of 20/400 or better, and completed 12 months of therapy. In the first group all patients received monthly injections. In the other group, after 3 monthly loading doses, a variable-dosing schedule was used, based on a monthly clinical assessment and optical coherence tomography. RESULTS: Fifty-six consecutive patients (60 eyes) were included. At 12 months the median number of injections were 12 and 8, respectively, and the mean change in Snellen visual acuity was an improvement of 0.27 logarithm of the minimum angle of resolution in the monthly treated group versus 0.21 logarithm of the minimum angle of resolution improvement in the variable-dosing group (P = 0.53). In the monthly treated group 96.8% of eyes lost <0.3 logarithm of the minimum angle of resolution versus 96.6% of eyes in the variable-dosing group (P = 1.0). CONCLUSION: We were able to show that in our clinical setting patients achieved similar visual acuity results with either monthly injections or with a variable-dosing protocol. There was a trend toward better results with monthly treatment.

The PI3K/Akt/mTOR pathway mediates retinal progenitor cell survival under hypoxic and superoxide stress.

Oxygen (O2) tension has emerged as a major regulator of stem cell (SC) biology. Low O2 concentrations that are toxic to mature cells can confer advantage to stem and early progenitors, while superoxide stress remains a constant threat in aerobic biology and may be partially avoided through sequestration of SCs in the relatively hypoxic stem or regenerative niche. Using primary retina-derived retinal progenitor cells (RPCs) and the R28 progenitor cell line in vitro, we show that RPCs are sensitive to hydrogen peroxide (H2O2) induced damage and resistant to moderate levels of low oxygen stress (1% O2). Under hypoxic conditions, multipotent RPCs upregulate Epo receptors, and Epo, along with insulin, protects against both superoxide- and severe hypoxia- (0.25% O2) induced apoptosis through activation of the canonical PI3K/Akt/mTOR pathway. This survival advantage is sensitive to inhibitors of PI3K and mTOR. We further demonstrate phosphorylation of the p70S6 ribosomal kinase, a downstream mediator of PI3K/Akt/mTOR and translational activator. Overall, these data confirm that RPCs are sensitive to superoxide stress and resistant to hypoxia and that this resistance is mediated in part by Epo. They further suggest that manipulation of RPCs ex vivo prior to ocular delivery, or the in vivo delivery of exogenous survival factors at the time of cell implantation, could enhance the success of regenerative therapies aimed to restore sight.

Evolving Patterns of Hyperfluorescent Fundus Autofluorescence Accompany Retinal Atrophy in the Rat and Mimic Atrophic Age-Related Macular Degeneration.

PURPOSE: Complex two-dimensional (2D) patterns of hyperfluorescent short-wave fundus autofluorescence (FAF) at the border of geographic atrophy (GA) can predict its expansion in patients with late non-exudative "dry" AMD. However, preclinical models do not phenocopy this important feature of disease. We sought to describe the spatiotemporal changes in hyperfluorescent FAF patterns that occur following acute oxidative stress, potentially in association with GA expansion. METHODS: Sprague Dawley rats (n = 54) received systemic sodium iodate (25-45 mg/kg, n = 90 eyes) or saline (n = 18 eyes) and underwent serial full fundus imaging by confocal scanning laser ophthalmoscopy, including blue FAF and delayed near-infrared analysis. Composite images of the fundus were assembled, and the 2D patterns were described qualitatively and quantitatively. A subset of eyes underwent tissue analysis, and four underwent optical coherence tomography (OCT) imaging. RESULTS: Reproducibly changing, complex patterns of hyperfluorescent FAF emerge at the borders of toxin-induced damage; however, in the absence of GA expansion, they percolate inward within the region of retinal pigment epithelium loss, evolving, maturing, and senescing in situ over time. Unexpectedly, the late FAF patterns most closely resemble the diffuse tricking form of clinical disease. A five-stage classification system is presented. CONCLUSIONS: Longitudinal, full-fundus imaging of outer retinal atrophy in the rat eye identifies evolving, complex patterns of hyperfluorescent FAF that phenocopy aspects of disease. TRANSLATIONAL RELEVANCE: This work provides a novel tool to assess hyperfluorescent FAF in association with progressive retinal atrophy, a therapeutic target in late AMD.

Novel Texture Feature Descriptors Based on Multi-Fractal Analysis and LBP for Classifying Breast Density in Mammograms.

This paper investigates the usefulness of multi-fractal analysis and local binary patterns (LBP) as texture descriptors for classifying mammogram images into different breast density categories. Multi-fractal analysis is also used in the pre-processing step to segment the region of interest (ROI). We use four multi-fractal measures and the LBP method to extract texture features, and to compare their classification performance in experiments. In addition, a feature descriptor combining multi-fractal features and multi-resolution LBP (MLBP) features is proposed and evaluated in this study to improve classification accuracy. An autoencoder network and principal component analysis (PCA) are used for reducing feature redundancy in the classification model. A full field digital mammogram (FFDM) dataset, INBreast, which contains 409 mammogram images, is used in our experiment. BI-RADS density labels given by radiologists are used as the ground truth to evaluate the classification results using the proposed methods. Experimental results show that the proposed feature descriptor based on multi-fractal features and LBP result in higher classification accuracy than using individual texture feature sets.

Limiting factors to the OCT axial resolution for in-vivo imaging of human and rodent retina in the 1060 nm wavelength range.

A computational model was developed to evaluate the limitations to the highest axial resolution, achievable with ultrahigh resolution optical coherence tomography (UHROCT) in the 1060 nm wavelength region for in-vivo imaging of the human and rodent retina. The model considers parameters such as the wavelength dependent water absorption, the average length of the human and rodent eyes, and the power limitations for the imaging beam as defined in the ANSI standard. A custom-built light source with re-shaped spectrum was used to verify experimentally the results from the computational model. Axial OCT resolution of 4.2 microm and 7.7 microm was measured from a mirror reflection with the custom light source by propagating the imaging beam through water cells with 5 mm and 25 mm thickness, corresponding to the average axial length of the rodent and human eye respectively. Assuming an average refractive index of 1.38 for retinal tissue, the expected axial OCT resolution in the rodent and human retina is 3 microm and 5.7 microm respectively. Retinal tomograms acquired in-vivo from the rat eye with the modified light source show clear visualization of all intraretinal layers, as well as a network of capillaries (approximately 10 microm in diameter) in the inner- and outer plexiform layers of the retina.

Aqueous Humor Cytokines and Long-Term Response to Anti-Vascular Endothelial Growth Factor Therapy in Diabetic Macular Edema.

PURPOSE: To determine the association of aqueous humor cytokine concentrations with long-term treatment response to anti-vascular endothelial growth factor (VEGF) agents in diabetic macular edema (DME). DESIGN: Retrospective case series. METHODS: Pooled data of aqueous humor cytokine concentrations collected at baseline and 2-month follow-up (2 injections) for treatment-naïve eyes with center-involving DME previously enrolled in a prospective study were reviewed. Subjects receiving intravitreal anti-VEGF injections outside of study protocol as per standard of care were classified into Responders versus Nonresponders based on qualitative assessment of optical coherence tomography for persistence of DME at longitudinal follow-up visits. RESULTS: Of the 41 eyes, 85% were classified as Responders with a significant decline in baseline central subfield thickness and macular volume (P values < .001), and 15% were identified as Nonresponders to anti-VEGF therapy over 51.4 ± 18.7 months of follow-up. No significant difference in baseline aqueous humor VEGF concentration was noted, while at the 2-month follow-up the Nonresponder group had a significantly higher VEGF concentration compared with the Responder group (451.5 ± 690.9 pg/mL vs 113.7 ± 211.4 pg/mL; P = .02). The Responder group also demonstrated a significant decline from baseline to 2-month follow-up in concentration of intercellular adhesion molecule-1 (P < .001), interleukin-10 (P = .041), monocyte chemotactic protein-1 (P = .046), placental growth factor (P = .027), and transforming growth factor-ß2 (P = .017). CONCLUSIONS: Aqueous humor cytokine concentrations serve as an early biomarker for long-term response to anti-VEGF therapy and may enable more effective treatment regimens that improve anatomical outcomes in eyes with DME.

Aqueous Humor Cytokine Levels and Anatomic Response to Intravitreal Ranibizumab in Diabetic Macular Edema.

Importance: Variability in response to anti-vascular endothelial growth factor (VEGF) treatment in diabetic macular edema (DME) remains a significant clinical challenge. Biomarkers could help anticipate responses to anti-VEGF therapy. Objectives: To investigate aqueous humor cytokine level changes in response to intravitreal ranibizumab therapy for the management of DME, and to determine the association between baseline aqueous levels and anatomic response. Design, Setting, and Participants: In this prospective multicenter cohort study, 49 participants with diabetes mellitus complicated by center-involving DME, with a central subfield thickness of 310 µm or greater on spectral-domain optical coherence tomography (SD-OCT), were recruited from December 22, 2011, to June 13, 2013 and statistical analysis were performed from March 1, 2017, to June 1, 2017. A total of 48 participants proceeded to follow-up. Interventions: Participants received monthly injections of ranibizumab, 0.5 mg, for 3 months. Aqueous fluid for cytokine analysis was obtained at baseline and repeated at the 2-month visit. Multiplex immunoassay was carried out in duplicate for VEGF, placental growth factor, transforming growth factor beta 2, intercellular adhesion molecule 1 (ICAM-1), interleukin 6 (IL-6), IL-8, IL-10, vascular intercellular adhesion molecule, and monocyte chemoattractant protein 1. Main Outcomes and Measures: Baseline and 2-month change in aqueous cytokine levels, 3-month change in SD-OCT central subfield thickness and macular volume (MV), and the statistical association between baseline aqueous cytokine levels and these measures of anatomic response to ranibizumab in center-involving DME. Results: Among the 48 participants, the mean (SD) age was 61.9 (7.1) years and 36 participants (75.0%) were men. The following cytokines were lower at month 2 vs baseline: ICAM-1 (median change, -190.88; interquartile range [IQR], -634.20 to -26.54; P < .001), VEGF (median change, -639.45; IQR, -1040.61 to -502.61; P < .001), placental growth factor (median change, -1.31; IQR, -5.99 to -0.01; P < .001), IL-6 (median change, -38.61; IQR, -166.72 to -2.80; P < .001), and monocyte chemoattractant protein 1 (median change, -90.13; IQR, -382.74 to 109.47; P = .01). When controlling for age, foveal avascular zone size, and severity of retinopathy, multiple linear regression determined that increasing baseline aqueous ICAM-1 was associated with a favorable anatomic response, in terms of reduced SD-OCT MV at 3 months (every additional 100 pg/mL of baseline ICAM-1 was associated with a reduction of 0.0379 mm3; P = .01). Conversely, increasing baseline aqueous VEGF was associated with a less favorable SD-OCT MV response at 3 months (every additional 100 pg/mL of baseline VEGF was associated with an increase of 0.0731 mm3; P = .02) and was associated with lower odds of being a central subfield thickness responder (odds ratio, 0.868; 95% CI, 0.755-0.998). Conclusions and Relevance: Elevated aqueous ICAM-1 and reduced VEGF levels at baseline are associated with a favorable anatomic response to ranibizumab in DME, although there is not always direct correlation between anatomic and visual acuity response.

Angiopoietin modulation of vascular endothelial growth factor: Effects on retinal endothelial cell permeability.

PURPOSE: Vascular permeability is important at many sites, but particularly so in diabetic retinopathy where macular oedema is the major cause of blindness. Angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) are important factors involved in neovascularization and vascular leakage, but there is little data on their interaction to promote increased vascular permeability. METHODS: Porcine retinal endothelial cells (PREC) were seeded into permeable inserts and cultured in 24-well plates that permit measurement of permeability using fluorescent dextrans. Cell purity was assessed immunohistochemically. At confluency, PREC were treated with increasing concentrations of VEGF (20-100ng/ml) and Ang-2 (15-75ng/ml). The effect on tight junctions was assessed by visualization with an anti-ZO-1 antibody. RESULTS: Immunohistochemistry showed high purity of isolated PREC. Permeability of untreated PREC monolayers was low. The increase in permeability in Ang-2 treated cells (25-30% compared with non-treated cells) was less than that for cells treated with VEGF only (20-100% compared with untreated cells). Highest permeability was seen with a combination of Ang-2 and VEGF (100-400% compared with untreated cells). Permeability increased with time after growth factor application. Preliminary ZO-1 immunohistochemistry appeared to demonstrate the presence of tight junctions between untreated PREC, and loss of tight junctions after treatment with VEGF and Ang-2. CONCLUSIONS: VEGF alone is twice as potent in interrupting tight junctions in an endothelial cell monolayer as Ang-2. However, both growth factors acting together increase permeability three times as much as VEGF alone. Treatments designed to reduce vascular permeability in diabetic macular oedema should consider that crosstalk between growth factors including VEGF and the Ang-2/Tie-2 system can multiply their effects.

Intact endothelial autophagy is required to maintain vascular lipid homeostasis.

The physiological role of autophagic flux within the vascular endothelial layer remains poorly understood. Here, we show that in primary endothelial cells, oxidized and native LDL stimulates autophagosome formation. Moreover, by both confocal and electron microscopy, excess native or modified LDL appears to be engulfed within autophagic structures. Transient knockdown of the essential autophagy gene ATG7 resulted in higher levels of intracellular (125) I-LDL and oxidized LDL (OxLDL) accumulation, suggesting that in endothelial cells, autophagy may represent an important mechanism to regulate excess, exogenous lipids. The physiological importance of these observations was assessed using mice containing a conditional deletion of ATG7 within the endothelium. Following acute intravenous infusion of fluorescently labeled OxLDL, mice lacking endothelial expression of ATG7 demonstrated prolonged retention of OxLDL within the retinal pigment epithelium (RPE) and choroidal endothelium of the eye. In a chronic model of lipid excess, we analyzed atherosclerotic burden in ApoE(-/-) mice with or without endothelial autophagic flux. The absence of endothelial autophagy markedly increased atherosclerotic burden. Thus, in both an acute and chronic in vivo model, endothelial autophagy appears critically important in limiting lipid accumulation within the vessel wall. As such, strategies that stimulate autophagy, or prevent the age-dependent decline in autophagic flux, might be particularly beneficial in treating atherosclerotic vascular disease.

Fundus autofluorescence (FAF) non-invasively identifies chorioretinal toxicity in a rat model of retinal pigment epithelium (RPE) damage.

INTRODUCTION: Traditional methods of pre-clinical ocular toxicology require that multiple cohorts of animals be sacrificed over time for terminal histological analysis. By contrast, in vivo techniques capable of following the same cohort prospectively have the potential to be efficient and cost-saving. We therefore asked if fundus autofluorescence (FAF), a non-invasive imaging technique, could detect damage to the posterior pole. Results were compared against electroretinography (ERG), another in vivo technique. The systemic toxin sodium iodate (NaIO3) was used to induce retinal pigment epithelium (RPE) damage. METHODS: FAF images (488/510nm excitation/emission) were obtained using a commercially available confocal scanning laser ophthalmoscope (cSLO; Heidelberg, HRAII) and were described qualitatively and quantitatively. NaIO3, over a dose range of 5 to 45mg/kg, or saline, was injected via tail vein in 6-10week old Sprague Dawley rats, and FAF images obtained at baseline and days 3, 7 and 14 thereafter and compared against the ERG response amplitude. RESULTS: Compared against baseline, there was no change in the FAF or ERG responses in the control, 5 or 15mg/kg NaIO3 groups. At 30mg/kg, responses fell into two groups. Half the animals developed small patches of abnormal FAF with modest reductions in the ERG amplitude; the other half developed large areas of damage and had severely reduced ERG responses. At 45mg/kg, all eyes developed extensive areas of abnormal FAF and the ERG was non- or minimally recordable. The en face size of the FAF patches was inversely correlated with the b-wave amplitude. DISCUSSION: This study demonstrates that FAF can detect chorioretinal toxicity in vivo in the rat eye, and that the findings correlate with the ERG. Such in vivo testing can enhance the detection of ocular toxicity.

Prostaglandin agonist effect on matrix metalloproteinase aqueous levels in glaucoma patients.

OBJECTIVE: To determine whether the aqueous levels of matrix metalloproteinases (MMPs) differ between patients with glaucoma treated with topical prostaglandin analogues and normal, nonglaucomatous control patients. Also, to note any difference in MMP levels between latanoprost, travoprost, and bimatoprost that might suggest a difference in efficacy or mechanism of action between these drugs. DESIGN: Prospective, observational study. PARTICIPANTS: Patients who were scheduled to undergo routine intraocular surgery (phacoemulsification or combined phacotrabeculectomy) as part of their standard clinical care were included. Eighteen eyes of 18 patients with glaucoma using any 1 prostaglandin analogue (latanoprost, travoprost, or bimatoprost) were compared with 8 normal control patients. METHODS: This was a multicentre study. Aqueous humour (0.2 mL) was aspirated at the beginning of the intraocular surgery through a clear corneal paracentesis. MMP-2 and -9 were quantified in the aqueous of all participants using enzyme-linked immunosorbent assay. RESULTS: There was no significant difference in the levels of either MMP-2 (p = 0.216) or MMP-9 (p = 0.552) between the control patients and the patients with glaucoma on prostaglandins. There was no difference in the levels of MMP-2 or -9 between the latanoprost, travoprost, or bimatoprost groups. CONCLUSIONS: The levels of MMP-2 and -9 in aqueous of glaucomatous eyes on topical prostaglandin analogues were the same as those of normal age-matched control patients. This could reflect either a return to normal levels with efficacious treatment or a lack of difference between disease and nondisease states.

Retinal toxicity of high-dose hydroxychloroquine in patients with chronic graft-versus-host disease.

OBJECTIVE: To evaluate retinal toxicity in patients treated with high-dose hydroxychloroquine (HCQ) (Plaquenil, Sanofi Pharmaceuticals) for chronic graft-versus-host disease (GVHD). DESIGN: Cohort study. PARTICIPANTS: Twelve patients with chronic GVHD treated with 800 mg/day HCQ between June 2005 and December 2010. METHODS: Patients in this study underwent ophthalmologic examination yearly and ancillary studies including colour vision, Amsler grid, fundus photographs, Humphrey 10-2 automated perimetry, spectral-domain optical coherence tomography (SD-OCT), and multifocal electroretinography (mfERG). Evidence of HCQ toxicity was determined by the presence of scotomas in the Amsler grid and Humphrey 10-2 automated perimetry, and confirmed by at least 1 objective test including SD-OCT or mfERG. RESULTS: Of the 12 patients, 7 were male and 5 were female. Mean age was 49 years. Mean best corrected visual acuity at baseline was 20/25 and remained 20/25 at final follow-up. Median duration of HCQ treatment was 22.8 months. Median adjusted daily dosage was 11.5 mg/kg/day. Seven patients developed vortex keratopathy. No signs of pigmentary retinopathy or bull's-eye maculopathy were found in any of the patients. Three patients developed retinal toxicity with scotomas in the Amsler grid and Humphrey 10-2 automated perimetry, as well as abnormal mfERG. Retinal structure measured by SD-OCT was abnormal in 2 of the 3 patients with retinal toxicity. Colour vision measured by Ishihara plates, as well as by 100 Hue colour test, was abnormal in 2 of the 3 patients with retinal toxicity. CONCLUSIONS: High-dose HCQ in patients with GVHD was associated with higher incidence and earlier development of retinal toxicity.