ITF2357 regulates NF-κB signaling pathway to protect barrier integrity in retinal pigment epithelial cells.

The robust integrity of the retinal pigment epithelium (RPE), which contributes to the outer brain retina barrier (oBRB), is compromised in several retinal degenerative and vascular disorders, including diabetic macular edema (DME). This study evaluates the role of a new generation of histone deacetylase inhibitor (HDACi), ITF2357, in regulating outer blood-retinal barrier function and investigates the underlying mechanism of action in inhibiting TNFα-induced damage to RPE integrity. Using the immortalized RPE cell line (ARPE-19), ITF2357 was found to be non-toxic between 50 nM and 5 µM concentrations. When applied as a pre-treatment in conjunction with an inflammatory cytokine, TNFα, the HDACi was safe and effective in preventing epithelial permeability by fortifying tight junction (ZO-1, -2, -3, occludin, claudin-1, -2, -3, -5, -19) and adherens junction (E-cadherin, Nectin-1) protein expression post-TNFα stress. Mechanistically, ITF2357 depicted a late action at 24 h via attenuating IKK, IκBα, and p65 phosphorylation and ameliorated the expression of IL-1ß, IL-6, and MCP-1. Also, ITF2357 delayed IκBα synthesis and turnover. The use of Bay 11-7082 and MG132 further uncovered a possible role for ITF2357 in non-canonical NF-κB activation. Overall, this study revealed the protection effects of ITF2357 by regulating the turnover of tight and adherens junction proteins and modulating NF-κB signaling pathway in the presence of an inflammatory stressor, making it a potential therapeutic application for retinal vascular diseases such as DME with compromised outer blood-retinal barrier.

HTRA1 Regulates Subclinical Inflammation and Activates Proangiogenic Response in the Retina and Choroid.

High-temperature requirement A1 (HtrA1) has been identified as a disease-susceptibility gene for age-related macular degeneration (AMD) including polypoidal choroidal neovasculopathy (PCV). We characterized the underlying phenotypic changes of transgenic (Tg) mice expressing ubiquitous CAG promoter (CAG-HtrA1 Tg). In vivo imaging modalities and histopathology were performed to investigate the possible neovascularization, drusen formation, and infiltration of macrophages. Subretinal white material deposition and scattered white-yellowish retinal foci were detected on CFP [(Tg­33% (20/60) and wild-type (WT)­7% (1/15), p < 0.05]. In 40% (4/10) of the CAG-HtrA1 Tg retina, ICGA showed punctate hyperfluorescent spots. There was no leakage on FFA and OCTA failed to confirm vascular flow signals from the subretinal materials. Increased macrophages and RPE cell migrations were noted from histopathological sections. Monocyte subpopulations were increased in peripheral blood in the CAG-HtrA1 Tg mice (p < 0.05). Laser induced CNV in the CAG-HtrA1 Tg mice and showed increased leakage from CNV compared to WT mice (p < 0.05). Finally, choroidal explants of the old CAG-HtrA1 Tg mice demonstrated an increased area of sprouting (p < 0.05). Signs of subclinical inflammation was observed in CAG-HtrA1 Tg mice. Such subclinical inflammation may have resulted in increased RPE cell activation and angiogenic potential.

Diagnosing Portal Hypertension with Noninvasive Subharmonic Pressure Estimates from a US Contrast Agent.

Background The current standard for assessing the severity of portal hypertension is the invasive acquisition of hepatic venous pressure gradient (HVPG). A noninvasive US-based technique called subharmonic-aided pressure estimation (SHAPE) could reduce risk and enable routine acquisition of these pressure estimates. Purpose To compare quantitative SHAPE to HVPG measurements to diagnose portal hypertension in participants undergoing a transjugular liver biopsy. Materials and Methods This was a prospective cross-sectional trial conducted at two hospitals between April 2015 and March 2019 (ClinicalTrials.gov identifier, NCT02489045). This trial enrolled participants who were scheduled for transjugular liver biopsy. After standard-of-care transjugular liver biopsy and HVPG pressure measurements, participants received an infusion of a US contrast agent and saline. During infusion, SHAPE data were collected from a portal vein and a hepatic vein, and the difference was compared with HVPG measurements. Correlations between data sets were determined by using the Pearson correlation coefficient, and statistical significance between groups was determined by using the Student t test. Receiver operating characteristic analysis was performed to determine the sensitivity and specificity of SHAPE. Results A total of 125 participants (mean age ± standard deviation, 59 years ± 12; 80 men) with complete data were included. Participants at increased risk for variceal hemorrhage (HVPG ≥12 mm Hg) had a higher mean SHAPE gradient compared with participants with lower HVPGs (0.79 dB ± 2.53 vs -4.95 dB ± 3.44; P < .001), which is equivalent to a sensitivity of 90% (13 of 14; 95% CI: 88, 94) and a specificity of 80% (79 of 99; 95% CI: 76, 84). The SHAPE gradient between the portal and hepatic veins was in good overall agreement with the HVPG measurements (r = 0.68). Conclusion Subharmonic-aided pressure estimation is an accurate noninvasive technique for detecting clinically significant portal hypertension. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Kiessling in this issue.

Largazole Inhibits Ocular Angiogenesis by Modulating the Expression of VEGFR2 and p21.

Ocular angiogenic diseases, characterized by abnormal blood vessel formation in the eye, are the leading cause of blindness. Although Anti-VEGF therapy is the first-line treatment in the market, a substantial number of patients are refractory to it or may develop resistance over time. As uncontrolled proliferation of vascular endothelial cells is one of the characteristic features of pathological neovascularization, we aimed to investigate the role of the class I histone deacetylase (HDAC) inhibitor Largazole, a cyclodepsipeptide from a marine cyanobacterium, in ocular angiogenesis. Our study showed that Largazole strongly inhibits retinal vascular endothelial cell viability, proliferation, and the ability to form tube-like structures. Largazole strongly inhibits the vessel outgrowth from choroidal explants in choroid sprouting assay while it does not affect the quiescent choroidal vasculature. Largazole also inhibits vessel outgrowth from metatarsal bones in metatarsal sprouting assay without affecting pericytes coverage. We further demonstrated a cooperative effect between Largazole and an approved anti-VEGF drug, Alflibercept. Mechanistically, Largazole strongly inhibits the expression of VEGFR2 and leads to an increased expression of cell cycle inhibitor, p21. Taken together, our study provides compelling evidence on the anti-angiogenic role of Largazole that exerts its function through mediating different signaling pathways.

How to make a synaptic ribbon: RIBEYE deletion abolishes ribbons in retinal synapses and disrupts neurotransmitter release.

Synaptic ribbons are large proteinaceous scaffolds at the active zone of ribbon synapses that are specialized for rapid sustained synaptic vesicles exocytosis. A single ribbon-specific protein is known, RIBEYE, suggesting that ribbons may be constructed from RIBEYE protein. RIBEYE knockdown in zebrafish, however, only reduced but did not eliminate ribbons, indicating a more ancillary role. Here, we show in mice that full deletion of RIBEYE abolishes all presynaptic ribbons in retina synapses. Using paired recordings in acute retina slices, we demonstrate that deletion of RIBEYE severely impaired fast and sustained neurotransmitter release at bipolar neuron/AII amacrine cell synapses and rendered spontaneous miniature release sensitive to the slow Ca(2+)-buffer EGTA, suggesting that synaptic ribbons mediate nano-domain coupling of Ca(2+) channels to synaptic vesicle exocytosis. Our results show that RIBEYE is essential for synaptic ribbons as such, and may organize presynaptic nano-domains that position release-ready synaptic vesicles adjacent to Ca(2+) channels.

Investigating the Role of PPARß/δ in Retinal Vascular Remodeling Using Pparß/δ-Deficient Mice.

Peroxisome proliferator-activated receptor (PPAR)ß/δ is a member of the nuclear receptor superfamily of transcription factors, which plays fundamental roles in cell proliferation and differentiation, inflammation, adipogenesis, and energy homeostasis. Previous studies demonstrated a reduced choroidal neovascularization (CNV) in Pparß/δ-deficient mice. However, PPARß/δ's role in physiological blood vessel formation and vessel remodeling in the retina has yet to be established. Our study showed that PPARß/δ is specifically required for disordered blood vessel formation in the retina. We further demonstrated an increased arteriovenous crossover and wider venous caliber in Pparß/δ-haplodeficient mice. In summary, these results indicated a critical role of PPARß/δ in pathological angiogenesis and blood vessel remodeling in the retina.

Level dominance effect and selective attention in a dichotic sample discrimination task.

Differences in individual listening patterns are reported for a dichotic sample discrimination task. Seven tones were drawn from normal distributions with means of 1000 or 1100 Hz on each trial. Even-numbered tones (2, 4, and 6) and odd-numbered tones (1, 3, 5, and 7) were drawn, respectively, from distributions with a 50-Hz and 200-Hz standard deviation. Task difficulty was manipulated by presenting odd and even tones at different intensities. In easy conditions, high and low informative tones were presented at 70 dB and 50 dB, respectively. In difficult conditions, high informative and low informative tones were presented at 50 dB and 70 dB, respectively. Participants judged whether the sample was from high- or low-mean distribution. Decision weights, efficiency, and sensitivity showed a range of abilities to attend to high informative tones, with d' from 2.4-0.7. Most listeners showed a left-ear advantage, while no listeners showed a right ear advantage. Some listeners, but not all, showed no loudness dominance effect with the ability to selectively attend to quiet tones in difficult conditions. These findings show that the influence of an attentional strategy in dichotic listening can overcome the loudness dominance effect for some listeners.

Discrimination bandwidths for amplitude modulated and quasi-frequency modulated tones with spectral cues degraded by a roving-level.

Theoretically, discriminating an amplitude modulated tone (AM) from a quasi-frequency modulated tone (QFM) is an ideal task for measuring the bandwidth of phase sensitivity because the stimuli have identical amplitude spectra but different phase spectra. The stimuli are perfectly discriminable at narrow bandwidths, but become indistinguishable at wide bandwidths. Measurements, however, are thought to be compromised by auditory distortion products, particularly a cubic distortion tone which interacts with the lower sideband of the stimulus to create an intensity cue. The results and implications of using a roving level procedure to eliminate distortion product effects are discussed.

System-wide vitreous proteome dissection reveals impaired sheddase activity in diabetic retinopathy.

Rationale: Diabetic retinopathy (DR) is a major complication of diabetes mellitus causing significant vision loss. DR is a multifactorial disease involving changes in retinal microvasculature and neuronal layers, and aberrations in vascular endothelial growth factors (VEGF) and inflammatory pathways. Despite the success of anti-VEGF therapy, many DR patients do not respond well to the treatment, emphasizing the involvement of other molecular players in neuronal and vascular aberrations in DR. Methods: We employed advanced mass spectrometry-based proteome profiling to obtain a global snapshot of altered protein abundances in vitreous humor from patients with proliferative DR (PDR) in comparison to individuals with epiretinal membrane without active DR or other retinal vascular complications. Global proteome correlation map and protein-protein interaction networks were used to probe into the functional inclination of proteins and aberrated molecular networks in PDR vitreous. In addition, peptide-centric analysis of the proteome data was carried out to identify proteolytic processing, primarily ectodomain shedding events in PDR vitreous. Functional validation experiments were performed using preclinical models of ocular angiogenesis. Results: The vitreous proteome landscape revealed distinct dysregulations in several metabolic, signaling, and immune networks in PDR. Systematic analysis of altered proteins uncovered specific impairment in ectodomain shedding of several transmembrane proteins playing critical roles in neurodegeneration and angiogenesis, pointing to defects in their regulating sheddases, particularly ADAM10, which emerged as the predominant sheddase. We confirmed that ADAM10 protease activity was reduced in animal models of ocular angiogenesis and established that activation of ADAM10 can suppress endothelial cell activation and angiogenesis. Furthermore, we identified the impaired ADAM10-AXL axis as a driver of retinal angiogenesis. Conclusion: We demonstrate restoration of aberrant ectodomain shedding as an effective strategy for treating PDR and propose ADAM10 as an attractive therapeutic target. In all, our study uncovered impaired ectodomain shedding as a prominent feature of PDR, opening new possibilities for advancement in the DR therapeutic space.

Adverse childhood experiences and depressive symptoms: Protective effects of dietary flavonoids.

OBJECTIVES: Adverse childhood experiences (ACEs) are associated with increased inflammation, stress, and depression. Diet patterns rich in flavonoids may buffer the effects of ACEs on depression through neuroprotective mechanisms. No studies have examined the protective effects of dietary flavonoids on depressive symptoms after ACEs. We examine the relationships among ACEs, perceived stress, depressive symptoms, and flavonoid intake in older adults. METHODS: In this longitudinal cohort study, flavonoid intake was provided by 6404 Seventh-day Adventist adults in North America who, as part of the Adventist Health Study-2, completed a validated food frequency questionnaire in 2002-6. ACEs, perceived stress, and depressive symptoms were assessed in the Biopsychosocial Religion and Health Study in 2006-7 and 2010-11. Bootstrapping models predicting depression were tested after controls. RESULTS: ACEs were associated with adult depressive symptoms and perceived stress mediated this relationship. A moderated mediation model indicates that flavonoid intake buffers the association between perceived stress and depressive symptoms after ACEs. Flavonoid consumption was negatively associated with depressive symptoms (ß = -0.034, p = .03). As ACEs increased by one standard deviation, depressive symptoms increased through the interaction of perceived stress and flavonoids when flavonoids were consumed a standard deviation below the mean (effect = 0.040 SD, BC 95% CI [0.030, 0.052]). Depressive symptoms were lower for those that consumed flavonoids a standard deviation above the mean (effect =. 035 SD, BC 95% CI [0.025, 0.046]). CONCLUSION: A varied diet rich in flavonoids may reduce depressive symptoms associated with perceived stress following ACEs exposure.

How do type 2 diabetes mellitus-related chronic complications impact direct medical cost in four major cities of urban China?

OBJECTIVE: The purpose of this study was to evaluate the direct medical costs of type 2 diabetes mellitus with or without complications, and to determine the economic impact of complications on type 2 diabetic patients. METHODS: We performed a cross-sectional study of prevalent type 2 diabetes carried out in four major cities of China. The study populations were 1530 outpatients and 524 inpatients from clinics or wards of a total of 20 hospitals, using a two-phase subject enrollment process, by face-to-face interview with a unique questionnaire. RESULTS: The annual direct medical cost per patient was estimated to be 4800 Chinese Yuan (CNY) in median or 10,164 CNY in mean. There is a difference between annual direct medical costs for patients with or without complications (6056 vs. 3583 CNY; P < 0.001). It is also significantly different for the pay-out-of-pocket proportions (P = 0.015) between the patients with (44.6%) and without complications (40.4%). The direct medical cost varied significantly among the four cities (P < 0.001). Patients who simultaneously suffered microvascular and macrovascular diseases had higher direct medical cost (7600) than those with macrovascular (6000) (P = 0.012) and microvascular disease (5364) (P < 0.001), and those without both (3600) (P < 0.001). The correlation was statistically significant between the number of complications and direct medical costs (P < 0.001). CONCLUSIONS: The high economic burden raised by diabetes and its complications challenges the Chinese health-care system. It implicates an urgent need of intervention to prevent the development of long-term complications among the diabetic population, especially on the development of complications in high-cost body system.

Apratoxin S4 Inspired by a Marine Natural Product, a New Treatment Option for Ocular Angiogenic Diseases.

Purpose: Abnormal blood vessel formation is a defining feature of many blinding eye diseases. Targeting abnormal angiogenesis by inhibiting VEGF has revolutionized the treatment of many ocular angiogenic diseases over the last decade. However, a substantial number of patients are refractory to anti-VEGF treatment or may develop resistance over time. The objective of this study was to determine the efficacy and the mechanism of action of Apratoxin S4 in ocular angiogenesis. Methods: Retinal vascular cell proliferation, migration, and the ability to form tube-like structure were studied in vitro. Ex vivo aortic ring, choroid, and metatarsal assays were used to study Apratoxin S4's impact on vessel outgrowth in a multicellular environment. Apratoxin S4 was also tested in mouse models of oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV), and in a rabbit model of persistent retinal neovascularization (PRNV). Western blot and ELISA were used to determine the expression of key angiogenic regulators after Apratoxin S4 treatment. Results: Apratoxin S4 strongly inhibits retinal vascular cell activation by suppressing multiple angiogenic pathways. VEGF-activated vascular cells and angiogenic vessels are more susceptible to Apratoxin S4 treatment than quiescent vascular cells and vessels. Both intraperitoneal and intravitreal delivery of Apratoxin S4 are able to impede ocular neovascularization in vivo. Apratoxin S4 specifically attenuates pathological ocular angiogenesis and exhibits a combinatorial inhibitory effect with standard-of-care VEGF inhibitor drug (aflibercept). Conclusions: Apratoxin S4 is a potent antiangiogenic drug that inhibits the activation of retinal endothelial cells and pericytes through mediating multiple angiogenic pathways.

ITF2357 transactivates Id3 and regulate TGFß/BMP7 signaling pathways to attenuate corneal fibrosis.

Corneal fibrosis is often seen in patients with ocular trauma and infection that compromises corneal transparency resulting in vision loss. Treatment strategies including NSAIDs, steroids, MMC and corneal transplants have shown tremendous success but with several side effects and cellular toxicity. Histone deacetylase inhibitors (HDACi) have been shown to inhibit corneal fibrosis via TGFß signaling pathway. In this study, we investigated safety, efficacy and mechanism of action of a HDACi, ITF2357 in TGFß-stimulated in vitro primary human cornea stromal fibroblasts (pHCSFs) and in vivo in a photorefractive keratectomy-treated rabbit model of corneal fibrosis. We found that in vivo ITF2357 decreased collagen I, collagen IV, fibronectin, integrin αVß3 expression with a reduction in corneal haze. In addition, ITF2357 reduced myofibroblast formation, suppressed phosphorylation of Smad proteins in TGFß pathway and inhibited key responsive protein, P4HA1 involved in pro-collagen synthesis. Treatment of pHCSFs with ITF2357 activated BMP7 levels and expressed all the members of inhibitor of differentiation proteins (Id1-Id4), however, it failed to rescue TGFß-driven transdifferentiation of fibroblasts to myofibroblasts in the presence of siRNA specific to Id3. We conclude that ITF2357 is a potential anti-fibrotic drug that exerts its action via activation of Id3, a downstream target of TGFß/BMP7 signaling pathways.

Characterization of Fatty Acid Binding Protein 7 (FABP7) in the Murine Retina.

PURPOSE: To characterize the mouse retina lacking fatty acid binding protein (FABP7-/-). METHODS: Immunohistochemistry (IHC) was performed in 8-week-old mice to localize FABP7 in the retina. Retinal thickness was measured using image-guided spectral-domain optical coherence topography images. Electroretinography was carried out to assess retinal function. Fundus photography and fundus fluorescein angiography were performed on FABP7-/- and littermate wild-type (WT) mice, and retinal vascular changes were calculated using Singapore I Vessel Assessment (SIVA) analysis. Blood glucose levels were measured in the 8-week-old WT and FABP7-/- mice. In addition, retina was processed for trypsin digestion and retinal flat mounts for isolectin staining. Transcript levels of FABP7, VEGF, GFAP, and Na+K+ATPase were quantified using real-time PCR, and protein expression was analyzed by IHC and Western blot. RESULTS: Fatty acid binding protein 7 is expressed in the inner nuclear layer, outer plexiform layer, and photoreceptor inner segments. No significant difference in retinal thickness and ERG responses was observed between FABP7-deficient and WT retinas. FABP7-/- mice have significantly decreased retinal venular caliber retinal arteriolar fractal dimension compared with WT littermates. FABP7-/- mice showed significant increased areas of fluorescein leakage in the retina. FABP7-/- mice exhibited elevated high blood glucose levels compared with WT mice. Trypsin digested FABP7-/- mice retina showed increased acellular strands and endothelial cell drop outs, and reduced microvasculature branching compared with WT retina. FABP7-/- mice retina also have increased GFAP and VEGF expression. CONCLUSIONS: Fatty acid binding protein 7 is expressed in the retina and might play an important role in maintaining retinal vasculature.

The prevalence of anxiety symptoms and depressive symptoms in patients with somatic disorders in urban China: a multi-center cross-sectional study.

OBJECTIVE: To assess the prevalence of anxiety and depressive symptoms among patients with somatic diseases in urban China. METHOD: A hospital-based cross-sectional study was carried out in four major cities of China from June to August in 2004. There were 2111 eligible subjects with Stroke, Parkinson's Disease, Epilepsy, Irritable Bowel Syndrome, Functional Dyspepsia, and Menopausal Syndrome, and 317 Post-natal women were recruited from general hospitals. Self-completed hospital anxiety and depression scale (HAD) questionnaire was used for screening anxiety and/or depressive symptoms. Subjects with a HAD score of > = 9 were further assessed with Hamilton anxiety scale (HAMA) and Hamilton depression scale (HAMD) by certified psychologists or psychiatrists. RESULTS: The prevalence of "screened" depressive and anxiety symptoms using HAD were 11-19% and 11-22% respectively in patients with above somatic diseases and post-natal women. Assessed by HAMA/HAMD scale, the prevalence of "definite" depressive symptoms was 30%-59% in subjects with "screened" depressive symptoms, and 44%-84% in subjects with "screened" anxiety symptoms. About half of the subjects had co-morbidity depressive and anxiety symptoms. Less than one-fourth of these subjects had ever been diagnosed as depressive/anxiety disorders and been treated prior to the investigation. CONCLUSION: There is a high prevalence and low diagnosis and treatment rate of depressive and anxiety symptoms in patients with these somatic diseases in China.