Systematic RNA-interference in primary human monocyte-derived macrophages: A high-throughput platform to study foam cell formation

Macrophage-derived foam cells are key regulators of atherogenesis. They accumulate in atherosclerotic plaques and support inflammatory processes by producing cytokines and chemokines. Identifying factors that regulate macrophage lipid uptake may reveal therapeutic targets for coronary artery disease (CAD). Here, we establish a high-throughput screening workflow to systematically identify genes that impact the uptake of DiI-labeled low-density lipoprotein (LDL) into monocyte-derived primary human macrophages. For this, monocytes isolated from peripheral blood were seeded onto 384-well plates, solid-phase transfected with siRNAs, differentiated in vitro into macrophages, and LDL-uptake per cell was measured by automated microscopy and quantitative image analysis. We applied this workflow to study how silencing of 89 genes impacts LDL-uptake into cells from 16 patients with CAD and 16 age-matched controls. Silencing of four novel genes (APOC1, CMTM6, FABP4, WBP5) reduced macrophage LDL-uptake. Additionally, knockdown of the chemokine receptor CXCR4 reduced LDL-uptake, most likely through a G-protein coupled mechanism that involves the CXCR4 ligand macrophage-induced factor (MIF), but is independent of CXCL12. We introduce a high-throughput strategy to systematically study gene function directly in primary CAD-patient cells. Our results propose a function for the MIF/CXCR4 signaling pathway, as well as several novel candidate genes impacting lipid uptake into human macrophages

Separation anxiety and measures of suicide risk among patients with mood and anxiety disorders

Background: Separation anxiety disorder may be important when considering risk of suicide. The aim of this study was to examine the association between both childhood and adult separation anxiety (disorder) and measures of suicide risk in a large cohort of outpatients with anxiety and mood disorders. Methods: The sample included 509 consecutive adult psychiatric outpatients with DSM-IV mood disorders or anxiety disorders as a principal diagnosis recruited at the Department of Psychiatry, University of Pisa, Italy, between 2015 and 2018. Suicide risk was evaluated by the Hamilton Depression Rating Scale (HDRS) item 3. Patients were classified in 2 groups: those with a score ≥ 1 and those with a score of 0 on HDRS item 3. Suicide risk was also evaluated by specific items within the Mood Spectrum, Self-Report (MOODS-SR), a questionnaire evaluating lifetime suicidal symptoms. Separation anxiety (disorder) was assessed based on the Structured Interview for Separation Anxiety Symptoms in Adulthood/Childhood (SCI-SAS-A/C), the Separation Anxiety Symptom Inventory (SASI), and the Adult Separation Anxiety Scale (ASA-27). Results: Of the 509 patients, 97 had an HDRS item 3 score ≥ 1, and 412 had a score of 0. Adult separation anxiety disorder was more frequent among individuals who had suicidal thoughts (53.6%) than those who did not (39.6%) (P = .01). Dimensional separation anxiety symptoms on all scales were elevated in patients with suicidality when compared to patients without (SASI: P = .02; SCI-SAS-C: P < .001; SCI-SAS-A: P < .001; ASA-27: P = .002). Logistic regression found that adult separation anxiety disorder (odds ratio [OR] = 1.86, 95% CI = 1.16–2.97), major depression (OR = 7.13, 95% CI = 3.18–15.97), bipolar I disorder (8.15, 95% CI = 3.34–19.90), and bipolar II disorder (OR = 8.16, 95% CI = 3.50–19.05) predicted suicidal thoughts. Linear regression found that depression (P = .001) and ASA-27 separation anxiety (P = .001) significantly predicted lifetime suicide risk. Mediation analysis found that separation anxiety significantly mediated the association between depression and suicide risk. Conclusions: This study indicates a substantial role of separation anxiety in predicting suicidal thoughts, both as state-related symptoms (evaluated by HDRS item 3) and as longitudinal dimensional symptoms (as evaluated by MOODS-SR). Greater understanding of the influence of separation anxiety in patients with affective disorders may encourage personalized interventions for reducing suicide risk

The relationship of separation anxiety with the age of onset of panic disorder

AIM: This study aimed to investigate whether separation anxiety (SA) constitutes a dimension related to age at onset of panic disorder (PD), in homogeneous subgroups of outpatients with PD, based on their age of onset and symptom severity.METHODS: A sample of 232 outpatients with PD was assessed with the Panic Disorder Severity Scale (PDSS) and the Sheehan Disability Scale (SDS) for functional impairments. Separation anxiety was evaluated using structured interviews and questionnaires. We applied a K-Means Cluster Analysis based on the standardized "PD age of onset" and "the PDSS total score" to identify distinct but homogeneous groups.RESULTS: We identified three groups of patients: group 1 ("PD early onset/severe", N = 97, 42%, onset 23.2 ± 6.7 years), group 2 ("PD early onset/not severe", N = 76, 33%, onset 23.4 ± 6.0 years) and group 3 ("PD adult onset/not severe", N = 59, 25%, onset 42.8 ± 7.0 years). Patients with early onset/severe PD had significantly higher scores on all SA measures than PD late-onset/not severe. Regression analyses showed that SA scores, but not PDSS scores, were predictive of impairment in SDS work/school, social life, and family functioning domains.CONCLUSIONS: Our data indicate a significant relationship between SA and PD with an earlier age of onset and an impact on individual functioning. This may have important implications for implementing preventive interventions targeting early risk factors for the subsequent onset of PD.</p

Latency to selective serotonin reuptake inhibitor vs benzodiazepine treatment in patients with panic disorder: a naturalistic study

BACKGROUND: Panic disorder (PD) is a prevalent and impairing anxiety disorder with previous reports suggesting that the longer the condition remains untreated, the greater the likelihood of nonresponse. However, patients with PD may wait for years before receiving a guideline-recommended pharmacological treatment. The widespread prescription of benzodiazepines (BDZ) for managing anxiety symptoms and disorders might delay the administration of pharmacotherapy according to guidelines (eg, selective serotonin reuptake inhibitors, SSRIs). The present study aimed to determine the mean duration of untreated illness (DUI) in a sample of PD patients, to quantify and compare DUI-SSRI to DUI-BDZ, and to compare findings with those from previous investigations.METHODS: Three hundred and fourteen patients with a Diagnostic and Statistical Manual of Mental Disorders, fifth edition diagnosis of PD were recruited from an Italian outpatient psychotherapy unit, and epidemiological and clinical variables were retrieved from medical records. Descriptive statistical analyses were undertaken for sociodemographic and clinical variables, Wilcoxon matched-pair signed rank test was applied to compare the distribution of DUI-SSRI vs DUI-BDZ, and Welch's t test was performed to compare findings with those from previous studies.RESULTS: The mean DUI-SSRI of the total sample was 64.25 ± 112.74 months, while the mean DUI-BDZ was significantly shorter (35.09 ± 78.62 months; P &lt; 0.0001). A significantly longer DUI-SSRI, compared to findings from previous studies, was also observed.CONCLUSIONS: The present results confirm a substantial delay in implementing adequate pharmacological treatments in patients with PD, and highlight the discrepancy between recommendations from international treatment guidelines and common clinical practice in relation to BDZ prescription.</p

CXCL4 Plasma Levels Are Not Associated with the Extent of Coronary Artery Disease or with Coronary Plaque Morphology

<div><p>Background</p><p>CXCL4 is a platelet chemokine released at micromolar concentrations upon platelet activation. CXCL4 has been shown to promote atherogenesis by various mechanisms. However, data on CXCL4 plasma levels in patients with coronary artery disease are largely inconclusive. Computed coronary artery angiography (CCTA) represents an excellent tool to quantify and characterize coronary atherosclerotic plaques. We hypothesized that increased CXCL4 plasma levels may be associated with features of plaque instability resulting in adverse cardiovascular events. Specifically, we sought to determine whether CXCL4 levels are correlated with specific features of coronary artery disease including (1) plaque volume, (2) calcium score, (3) degree of stenosis, or (4) vascular remodeling.</p><p>Methods and Results</p><p>CXCL4 plasma levels were measured by ELISA in 217 patients undergoing CCTA for suspected CAD (mean age 64.2 ± 9.4 years, 107 (49.3%) male). Mean CXCL4 plasma levels were 12.5 ± 4.6 ng/mL. There was no significant correlation between CXCL4 levels and any clinical or demographic parameters including cardiovascular risk factors. CXCL4 plasma levels did not differ between patient with or without coronary artery disease (CAD: 12.5 ± 4.5 ng/ml, no CAD: 12.5 ± 4.8 ng/ml). Neither univariate nor multivariate analysis showed an association between CXCL4 levels and plaque volume, total calcium score, degree of stenosis, or vascular remodeling. Subgroup analysis of patients with CAD as confirmed by CCTA did not show any association of CXCL4 levels with the extent of CAD.</p><p>Conclusions</p><p>While CXCL4 may be present and active within the arterial wall, local increase of CXCL4 may not translate into systemically elevated CXCL4 levels. Further studies will have to test whether CXCL4 may still represent a suitable therapeutic target in human atherosclerosis.</p></div

Plaque characteristics.

<p>Plaque characteristics of all patients and divided by CXCL4 quartiles. For each parameter the coefficient and P value with CXCL4 plasma levels is indicated as calculated by Pearson’s correlation. Continuous variables are indicated as mean ± standard deviation, categorical variables are indicated as absolute number (percentage).</p

Patient characteristics.

<p>Demographic and clinical data of all patients and divided by CXCL4 quartiles. For each parameter the coefficient and P value with CXCL4 plasma levels is indicated as calculated by Pearson’s correlation. Continuous variables are indicated as mean ± standard deviation, categorical variables are indicated as absolute number (percentage).</p

CXCL4 plasma levels in patients with or without CAD.

<p>CXCL4 plasma levels as measured by ELISA in 217 individuals undergoing coronary computed-tomography angiography (CCTA) divided by „no CAD”(n = 107) and „CAD”(n = 110). For each group, box whisker plots and dot plots are shown.</p