Cardiovascular inflammation in healthy women: multilevel associations with state-level prosperity, productivity and income inequality

<p>Abstract</p> <p>Background</p> <p>Cardiovascular inflammation is a key contributor to the development of atherosclerosis and the prediction of cardiovascular events among healthy women. An emerging literature suggests biomarkers of inflammation vary by geography of residence at the state-level, and are associated with individual-level socioeconomic status. Associations between cardiovascular inflammation and state-level socioeconomic conditions have not been evaluated. The study objective is to estimate whether there are independent associations between state-level socioeconomic conditions and individual-level biomarkers of inflammation, in excess of individual-level income and clinical covariates among healthy women.</p> <p>Methods</p> <p>The authors examined cross-sectional multilevel associations among state-level socioeconomic conditions, individual-level income, and biomarkers of inflammation among women (n = 26,029) in the Women's Health Study, a nation-wide cohort of healthy women free of cardiovascular diseases at enrollment. High sensitivity C-reactive protein (hsCRP), soluble intercellular adhesion molecule-1 (sICAM-1) and fibrinogen were measured between 1993 and 1996. Biomarker levels were examined among women within quartiles of state-level socioeconomic conditions and within categories of individual-level income.</p> <p>Results</p> <p>The authors found that favorable state-level socioeconomic conditions were correlated with lower hsCRP, in excess of individual-level income (e.g. state-level real per capital gross domestic product fixed effect standardized Βeta coefficient [Std B] -0.03, 95% CI -0.05, -0.004). Individual-level income was more closely associated with sICAM-1 (Std B -0.04, 95% CI -0.06, -0.03) and fibrinogen (Std B -0.05, 95% CI -0.06, -0.03) than state-level conditions.</p> <p>Conclusions</p> <p>We found associations between state-level socioeconomic conditions and hsCRP among healthy women. Personal household income was more closely associated with sICAM-1 and fibrinogen than state-level socioeconomic conditions. Additional research should examine these associations in other cohorts, and investigate what more-advantaged states do differently than less-advantaged states that may influence levels of cardiovascular inflammation among healthy women.</p

Clinical and Epidemiologic Research Visual Function in Older Eyes in Normal Macular Health: Association with Incident Early Age-Related Macular Degeneration 3 Years Later

PURPOSE. In older eyes in normal macular health, we examined associations between impaired photopic acuity, mesopic acuity, spatial contrast sensitivity, light sensitivity, and the presence of low luminance deficit (difference between photopic and mesopic acuity) at baseline and incident AMD 3 years later. Associations were compared with an association between delayed rod-mediated dark adaptation and incident AMD, previously reported for this cohort. METHODS. Enrollees were 60 years or older. Eyes at step 1 in the AREDS nine-step classification system based on masked grading of color fundus photographs were included. Photopic and mesopic acuity, contrast sensitivity, and light sensitivity, and the presence of low luminance deficit, were measured at baseline. Demographic, lifestyle, general health, and blood markers were assessed at baseline as potential confounders. Three years later fundus grading was repeated to determine AMD presence. RESULTS. For the analysis, 827 eyes of 467 persons were eligible. Impaired mesopic acuity at baseline was associated with incident AMD, age-adjusted rate ratio (RR) 1.57 (95% confidence interval [CI] 1.04-2.35), whereas impaired photopic acuity, contrast sensitivity and macular light sensitivity, and the presence of a low luminance deficit were not. The mesopic acuity association was slightly weaker than the association between abnormal dark adaptation and incident AMD (RR 1.85, 95% CI 1.07-3.20). CONCLUSIONS. Impaired mesopic acuity in eyes in normal macular health is a risk factor for incident early AMD 3 years later, however, photopic acuity, contrast sensitivity, and light sensitivity, and the presence of a low luminance deficit are not risk factors

ATP6V0C knockdown in neuroblastoma cells alters autophagy-lysosome pathway function and metabolism of proteins that accumulate in neurodegenerative disease.

ATP6V0C is the bafilomycin A1-binding subunit of vacuolar ATPase, an enzyme complex that critically regulates vesicular acidification. We and others have shown previously that bafilomycin A1 regulates cell viability, autophagic flux and metabolism of proteins that accumulate in neurodegenerative disease. To determine the importance of ATP6V0C for autophagy-lysosome pathway function, SH-SY5Y human neuroblastoma cells differentiated to a neuronal phenotype were nucleofected with non-target or ATP6V0C siRNA and following recovery were treated with either vehicle or bafilomycin A1 (0.3-100 nM) for 48 h. ATP6V0C knockdown was validated by quantitative RT-PCR and by a significant decrease in Lysostracker Red staining. ATP6V0C knockdown significantly increased basal levels of microtubule-associated protein light chain 3-II (LC3-II), α-synuclein high molecular weight species and APP C-terminal fragments, and inhibited autophagic flux. Enhanced LC3 and LAMP-1 co-localization following knockdown suggests that autophagic flux was inhibited in part due to lysosomal degradation and not by a block in vesicular fusion. Knockdown of ATP6V0C also sensitized cells to the accumulation of autophagy substrates and a reduction in neurite length following treatment with 1 nM bafilomycin A1, a concentration that did not produce such alterations in non-target control cells. Reduced neurite length and the percentage of propidium iodide-positive dead cells were also significantly greater following treatment with 3 nM bafilomycin A1. Together these results indicate a role for ATP6V0C in maintaining constitutive and stress-induced ALP function, in particular the metabolism of substrates that accumulate in age-related neurodegenerative disease and may contribute to disease pathogenesis

ATP6V0C regulates basal and stress-induced metabolism of alpha synuclein.

<p>Representative western blot for α-syn (A) from lysates following nucleofection and subsequent treatment for 48 h with 0–100 nM bafilomycin A1 (BafA1), indicating α-syn high molecular weight (HMW) species (>50 kDa, suggesting multimeric species) and α-syn monomer (∼17 kDa). Blots were stripped and re-probed for actin (42 kDa) to normalize for gel loading. Data from at least six independent experiments are presented graphically in panels B–E. Within groups comparisons of BafA1 concentration responsiveness (Non-target siRNA, open columns, left; ATP6V0C siRNA, filled columns, right) were determined for α-syn HMW species (C) or monomer (D) by expressing mean ± SEM band intensities relative to actin loading control (results of one-way ANOVA were not significant, p>0.05). Comparisons between groups (Non-target vs. ATP6V0C siRNA) for α-syn HMW species (D) or monomer (E) were determined for each concentration of BafA1 by expressing mean ± SEM fold changes for each ATP6V0C siRNA condition relative to its companion Non-target control. ^#p<0.05 vs. corresponding Non-target siRNA control using one-sample t-test.</p

Multidisciplinary Ophthalmic Imaging RefMoB, a Reflectivity Feature Model-Based Automated Method for Measuring Four Outer Retinal Hyperreflective Bands in Optical Coherence Tomography

Citation: Ross DH, Clark ME, Godara P, et al. RefMoB, a reflectivity feature model-based automated method for measuring four outer retinal hyperreflective bands in optical coherence tomography. Invest Ophthalmol Vis Sci. 2015;56:4166-4176. DOI:10.1167/iovs.14-15256 PURPOSE. To validate a model-driven method (RefMoB) of automatically describing the four outer retinal hyperreflective bands revealed by spectral-domain optical coherence tomography (SDOCT), for comparison with histology of normal macula; to report thickness and position of bands, particularly band 2 (ellipsoid zone [EZ], commonly called IS/OS). METHODS. Foveal and superior perifoveal scans of seven SDOCT volumes of five individuals aged 28 to 69 years with healthy maculas were used (seven eyes for validation, five eyes for measurement). RefMoB determines band thickness and position by a multistage procedure that models reflectivities as a summation of Gaussians. Band thickness and positions were compared with those obtained by manual evaluators for the same scans, and compared with an independent published histological dataset. RESULTS. Agreement among manual evaluators was moderate. Relative to manual evaluation, RefMoB reported reduced thickness and vertical shifts in band positions in a band-specific manner for both simulated and empirical data. In foveal and perifoveal scans, band 1 was thick relative to the anatomical external limiting membrane, band 2 aligned with the outer one-third of the anatomical IS ellipsoid, and band 3 (IZ, interdigitation of retinal pigment epithelium and photoreceptors) was cleanly delineated. CONCLUSIONS. RefMoB is suitable for automatic description of the location and thickness of the four outer retinal hyperreflective bands. Initial results suggest that band 2 aligns with the outer ellipsoid, thus supporting its recent designation as EZ. Automated and objective delineation of band 3 will help investigations of structural biomarkers of dark-adaptation changes in aging. Keywords: optical coherence tomography, reflectivity, segmentation, retina, ellipsoid, interdigitation, photoreceptors, retinal pigment epithelium, age-related macular degeneration S pectral-domain optical coherence tomography (SDOCT) is an interferometry technique that uses low coherence light to achieve depth-resolved, comprehensive, and noninvasive cross-sectional views of chorioretinal structure in vivo. 5 By assuming that the ELM and RPE-BrM bands were correctly designated, we found that band 2 closely aligned with the IS ellipsoid (ISel), a conclusion supported by direct imaginghistology correlations in laboratory animals. 5 These fine processes, up to 15 lm long, form a candelabra-like arrangement with cone OS, which are shorter than rod OS. Othe

ATP6V0C regulates autophagic flux.

<p>Representative western blot for autophagosome marker LC3-II (A) to assess autophagic flux by treating in the presence or absence of the lysosome inhibitors (“LI” in panel A and “LYSO INH” in panel B) chloroquine (100 μM) plus leupeptin (200 μM) for the last 2–4 h of a 48 h time course. Blots were stripped and re-probed for actin (42 kDa) to normalize for gel loading. Data from seven independent experiments are presented graphically in panel B. Comparisons between groups (Non-target vs. ATP6V0C siRNA) were determined by normalizing bands to actin loading control and expressing as mean ± SEM fold change for each ATP6V0C siRNA condition (filled columns) relative to its companion Non-target control (open columns). ^#p<0.05 vs. corresponding Non-target siRNA control using one-sample t-test. (C–H) Representative confocal microscopy images of fixed cells following nucleofection with either Non-target siRNA control (C–E) or ATP6V0C siRNA (F–H) and subsequent treatment with DMSO vehicle for 48 h. LC3 immunoreactivity (red, C, F) and LAMP-1 (green, D, G) are shown separately and as merged (E, H). Nuclei are stained in panels E and H with bis-benzimide (blue). Co-localization of LC3 and LAMP-1 immunoreactivity (arrows, panels E, H) suggests inhibition of autophagic flux resulting from accumulation of dysfunctional autolysosomes. Scale bar  = 50 μm.</p

ATP6V0C knockdown exhibit enhanced markers of cytotoxicity.

<p>Representative confocal microscopy images obtained from differentiated SH-SY5Y cells following nucleofection with Non-target (A–D) or ATP6V0C (E–H) siRNA and subsequent treatment for 48 h with 0 (A, E), 1 (B, F), 3 (C, G) or 10 (D, H) nM bafilomycin A1 (BafA1). Arrows indicate neuritic processes. Scale bar  = 50 μm. Neurite length (μm) is expressed graphically (I) and represents results (mean ± SEM) from three independent experiments (10 cells per condition in each experiment). Percent cell death (percentage of propidium iodide (PI)-positive cells quantified using flow cytometry) is expressed graphically (J) as mean ± SEM with data obtained from a total of nine independent experiments. All lines above columns indicate significant within-group differences with respect to concentration (*p<0.05 using one-way ANOVA and Bonferroni's post-hoc test). Comparisons between groups (Non-target vs. ATP6V0C siRNA) were determined for each concentration of BafA1 using two-sample t-test (^#p<0.05).</p

ATP6V0C regulates basal and stress-induced metabolism of alpha synuclein.

<p>Representative western blot for α-syn (A) from lysates following nucleofection and subsequent treatment for 48 h with 0–100 nM bafilomycin A1 (BafA1), indicating α-syn high molecular weight (HMW) species (>50 kDa, suggesting multimeric species) and α-syn monomer (∼17 kDa). Blots were stripped and re-probed for actin (42 kDa) to normalize for gel loading. Data from at least six independent experiments are presented graphically in panels B–E. Within groups comparisons of BafA1 concentration responsiveness (Non-target siRNA, open columns, left; ATP6V0C siRNA, filled columns, right) were determined for α-syn HMW species (C) or monomer (D) by expressing mean ± SEM band intensities relative to actin loading control (results of one-way ANOVA were not significant, p>0.05). Comparisons between groups (Non-target vs. ATP6V0C siRNA) for α-syn HMW species (D) or monomer (E) were determined for each concentration of BafA1 by expressing mean ± SEM fold changes for each ATP6V0C siRNA condition relative to its companion Non-target control. ^#p<0.05 vs. corresponding Non-target siRNA control using one-sample t-test.</p

ATP6V0C regulation of autophagy-lysosome pathway markers.

<p>Representative western blots for lysosome marker LAMP-1 (A) or autophagosome marker LC3-II (B) from lysates collected following nucleofection with Non-target or ATP6V0C siRNA and subsequent treatment for 48 h with 0–100 nM bafilomycin A1 (BafA1). Blots were stripped and re-probed for actin (42 kDa) to normalize for gel loading. Data from at least six independent experiments are presented graphically in panels C–F. Within group comparisons of BafA1 concentration responsiveness (Non-target siRNA, open columns, left; ATP6V0C siRNA, filled columns, right) were determined for LC3-II (C) or LAMP-1 (D) by expressing mean ± SEM band intensities relative to actin loading control. All lines above columns indicate significant within-group differences with respect to concentration (*p<0.05 using one-way ANOVA and Bonferroni's post-hoc test). Comparisons between groups (Non-target vs. ATP6V0C siRNA) for LC3-II (E) and LAMP-1 (F) were determined for each concentration of BafA1 by expressing mean ± SEM fold changes for each ATP6V0C siRNA condition relative to its companion Non-target control. ^#p<0.05 vs. corresponding Non-target siRNA control using one-sample t-test.</p