Computing of Low Shear Stress-Driven Endothelial Gene Network Involved in Early Stages of Atherosclerotic Process.

BACKGROUND: In the pathogenesis of atherosclerosis, a central role is represented by endothelial inflammation with influx of chemokine-mediated leukocytes in the vascular wall. Aim of this study was to analyze the effect of different shear stresses on endothelial gene expression and compute gene network involved in atherosclerotic disease, in particular to homeostasis, inflammatory cell migration, and apoptotic processes. METHODS: HUVECs were subjected to shear stress of 1, 5, and 10 dyne/cm2 in a Flow Bioreactor for 24 hours to compare gene expression modulation. Total RNA was analyzed by Affymetrix technology and the expression of two specific genes (CXCR4 and ICAM-1) was validated by RT-PCR. To highlight possible regulations between genes and as further validation, a bioinformatics analysis was performed. RESULTS: At low shear stress (1 dyne/cm2) we observed the following: (a) strong upregulation of CXCR4; (b) mild upregulation of Caspase-8; (c) mild downregulation of ICAM-1; (d) marked downexpression of TNFAIP3. Bioinformatics analysis showed the presence of network composed by 59 new interactors (14 transcription factors and 45 microRNAs) appearing strongly related to shear stress. CONCLUSIONS: The significant modulation of these genes at low shear stress and their close relationships through transcription factors and microRNAs suggest that all may promote an initial inflamed endothelial cell phenotype, favoring the atherosclerotic disease.

Medium-term effect of sublingual l-glutathione supplementation on flow-mediated dilation in subjects with cardiovascular risk factors.

OBJECTIVE: Supplementation of glutathione (GSH) may be a positive strategy to improve the endogenous antioxidant defense required to counteract many acute and chronic diseases. However, the efficacy of GSH treatment seems to be closely related to type of administration, degree of absorption, and increase of its concentrations. The aim of this study was to test a new sublingual formulation of L-GSH, which enters directly the systemic circulation, to assess its efficacy on circulating biochemical markers of hepatic metabolism, lipid profile, and oxidative stress and on peripheral vascular function compared with placebo in patients with cardiovascular risk factors (CVRF). METHODS: We enrolled 16 healthy men with CVRF in a double-blinded, randomized placebo-controlled crossover study. At each visit, blood samples were collected for biochemistry analyses and peripheral endothelial function (reactive hyperemia index [RHI]) and stiffness were measured by Endo-PAT2000. RESULTS: In the overall population, a decrease in total and low-density lipoprotein cholesterol was highlighted after L-GSH supplementation compared with placebo (P = 0.023 and P = 0.04, respectively). On the contrary, no difference was observed in RHI and oxidative stress markers between L-GSH and placebo in the study population. However, seven participants with baseline abnormal RHI (≤1.67) compared with those with normal RHI showed a significant reduction of arterial stiffness after L-GSH administration, (P = 0.007 and P = 0.037, respectively). CONCLUSIONS: Supplementation of L-GSH compared with placebo influences the lipid profile of patients with CVRF. Sublingual L-GSH may represent a valid prevention of vascular damage in patients with CVRF and endothelial dysfunction.

Biological and proteomic characterization of a composite mesh for abdominal wall hernia treatment: Reference Study.

AIMS: The industrial development of a product requires performing a deep analysis to highlight its characteristics useful for future design. The clinical use of a product stimulates knowledge improvement about it in a constant effort of progress. This work shows the biological characterization of CMC composite mesh. CMC polypropylene prosthesis was seeded with Human fibroblast BJ. Samples (cells and medium) were collected at different time points in order to perform different analysis: inflammatory markers quantification; collagens immunohistochemistry; matrix metalloproteinases zimography; extracellular matrix proteomic profile. FINDINGS: CMC presented a good cell viability rate and cell growth during the 21 days. The inflammatory profile showed an initial secretion of anti-inflammatory IL-10 and a final increase of pro-inflammatory IL-6. Immunocytochemistry highlighted a similar Collagen type I/type III ratio. The proteomic analysis evidenced the ECM protein content profile composed, mainly, by collagens, fibronectin, laminin. MMPs resulted both expressed when in contact to mesh. CONCLUSIONS: CMC shows a good cell biocompatibility and growth. The increase of pro-inflammatory markers could stimulate proliferation, influencing the integration process in human body. Proteomics highlights the ECM modulation by CMC. An integrated investigation of these biological analyses with mechanical data should improve the design process of a new product. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2045-2052, 2017.

Antioxidant and inflammatory biomarkers for the identification of prodromal Parkinson's disease.

OBJECTIVES: We explored the role of oxidative stress and inflammatory molecules as potential Parkinson (PD) biomarkers and correlated biological with non-motor abnormalities (olfactory impairment and dysautonomia), in patients with idiopathic REM behavior disorder (iRBD) (prodromal PD) and established PD. METHODS: We recruited 11 iRBD and 15 patients with idiopathic PD (Hohen&Yahr 1-3, on L-DOPA and dopamine agonists combination therapy) and 12 age- and sex-matched controls (CTRL). We measured total olfactory score (TOS), autonomic function [deep breathing (DB), lying to standing (LS) and Valsalva manoeuvre (VM) ratios], blood reduced glutathione (Br-GSH), oxidative stress and inflammatory markers (neopterin). RESULTS: Anosmia was similarly prevalent in iRBD (36%) and PD (33%) patients, but absent in CTRL. Orthostatic hypotension was more common among iRBD (73%) and PD (60%) than in CTRL (25%). By univariable ordinal logistic regression, TOS, Br-GSH, LS and VM ratio worsened from CTRL to iRBD and PD groups. Only reduced Br-GSH levels (p=0.037, OR=0.994; 95%CI 0.988-1.000) were independently associated to PD. TOS correlated with Br-GSH (R=0.34, p=0.037), VM ratio (R=0.43, p=0.015), and neopterin (rho=0.39, p=0.016). CONCLUSIONS: Reduced systemic antioxidant capacity is found in prodromal and overt PD and may represent, in association with olfactory loss and cardiovascular dysautonomia, a useful biomarker for an integrative, early diagnosis of PD.

Inflammatory and antioxidant pattern unbalance in "clopidogrel-resistant" patients during acute coronary syndrome.

BACKGROUND: In acute coronary syndrome (ACS), inflammation and redox response are associated with increased residual platelet reactivity (RPR) on clopidogrel therapy. We investigated whether clopidogrel interaction affects platelet function and modulates factors related to inflammation and oxidation in ACS patients differently responding to clopidogrel. MATERIAL AND METHODS: Platelet aggregation was measured in 29 ACS patients on dual (aspirin/clopidogrel) antiplatelet therapy. Nonresponders (NR) were defined as RPR ≥70% by ADP. Several inflammatory and redox parameters were assayed and platelet proteome was determined. RESULTS: Eight (28%) out of 29 ACS patients resulted NR to clopidogrel. At 24 hours, the levels of Th2-type cytokines IL-4, IFNγ, and MCP-1 were higher in NR, while blood GSH (r-GSHbl) levels were lower in NR than responders (R). Proteomic analysis evidenced an upregulated level of platelet adhesion molecule, CD226, and a downregulation of the antioxidant peroxiredoxin-4. In R patients the proinflammatory cytokine IL-6 decreased, while the anti-inflammatory cytokine IL-1Ra increased. CONCLUSIONS: In patients with high RPR on clopidogrel therapy, an unbalance of inflammatory factors, platelet adhesion molecules, and circulatory and platelet antioxidant molecules was observed during the acute phase. Proinflammatory milieu persists in nonresponders for a long time after the acute event while antioxidant blood factors tend to conform to normal responsiveness.

Differential regulation of microRNAs in end-stage failing hearts is associated with left ventricular assist device unloading.

Mechanical unloading by left ventricular assist devices (LVADs) in advanced heart failure (HF), in addition to improving symptoms and end-organ perfusion, is supposed to stimulate cellular and molecular responses which can reverse maladaptive cardiac remodeling. As microRNAs (miRNAs) are key regulators in remodeling processes, a comparative miRNA profiling in transplanted hearts of HF patients with/without LVAD assistance could aid to comprehend underlying molecular mechanisms. Next generation sequencing (NGS) was used to analyze miRNA differential expression in left ventricles of HF patients who underwent heart transplantation directly (n = 9) or following a period of LVAD support (n = 8). After data validation by quantitative real-time PCR, association with functional clinical parameters was investigated. Bioinformatics' tools were then used for prediction of putative targets of modulated miRNAs and relative pathway enrichment. The analysis revealed 13 upregulated and 10 downregulated miRNAs in failing hearts subjected to LVAD assistance. In particular, the expression level of some of them (miR-338-3p, miR-142-5p and -3p, miR-216a-5p, miR-223-3p, miR-27a-5p, and miR-378g) showed correlation with off-pump cardiac index values. Predicted targets of these miRNAs were involved in focal adhesion/integrin pathway and in actin cytoskeleton regulation. The identified miRNAs might contribute to molecular regulation of reverse remodeling and heart recovery mechanisms.

Myocardial interleukin-6 in the setting of left ventricular mechanical assistance: relation with outcome and C-reactive protein.

BACKGROUND: In left ventricular assist device (LVAD) recipients, plasma levels of interleukin (IL)-6 are associated with Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) profiles, reflecting post-operative risk. However, it is not clear how the cardiac level of IL-6, detectable on the tissue samples at the time of implantation, can contribute to predict the post-operative outcome. METHODS: In 40 LVAD recipients, blood and myocardial samples from LV-apex were collected at the time of implantation to assess plasma and cardiac IL-6 levels. Serum C-reactive protein (CRP) levels were considered as inflammatory variable routinely used in LVAD-based therapy. RESULTS: Cardiac IL-6 levels did not correlate with either plasma IL-6 levels (R=0.296, p=0.063) and tissue IL-6 mRNA expression (R=-0.013, p=0.954). Contrary to what happened for the plasma IL-6 and CRP, no differences were observed in cardiac IL-6 levels with respect to INTERMACS profiles (p=0.090). Furthermore, cardiac IL-6 concentrations, unlike IL-6 and CRP circulating levels, were not correlated with the length of intensive care unit stay and hospitalization. CONCLUSIONS: Cardiac IL-6 levels do not contribute to improve risk profile of LVAD recipients in relation to clinical inpatient post-implantation. Instead, plasma IL-6 and serum CRP concentrations are more effective in predicting the severity of the clinical course in the early phase of LVAD therapy.

Relationship between early inflammatory response and clinical evolution of the severe multiorgan failure in mechanical circulatory support-treated patients.

BACKGROUND: The mechanical circulatory support (MCS) is an effective treatment in critically ill patients with end-stage heart failure (ESHF) that, however, may cause a severe multiorgan failure syndrome (MOFS) in these subjects. The impact of altered inflammatory response, associated to MOFS, on clinical evolution of MCS postimplantation patients has not been yet clarified. METHODS: Circulating cytokines, adhesion molecules, and a marker of monocyte activation (neopterin) were determined in 53 MCS-treated patients, at preimplant and until 2 weeks. MOFS was evaluated by total sequential organ failure assessment score (tSOFA). RESULTS: During MCS treatment, 32 patients experienced moderate MOFS (tSOFA < 11; A group), while 21 patients experienced severe MOFS (tSOFA ≥ 11) with favorable (B group) or adverse (n = 13, C group) outcomes. At preimplant, higher values of left ventricular ejection fraction (LVEF) and estimated glomerular filtration rate (eGFR) were the only parameter independently associated with A group. In C group, during the first postoperative week, high levels of interleukin-8 (IL-8) and tumor necrosis factor (TNF)-α, and an increase of neopterin and adhesion molecules, precede tSOFA worsening and exitus. CONCLUSIONS: The MCS patients of C group show an excessive release to IL-8 and TNF-α, and monocyte-endothelial activation after surgery, that might contribute to the unfavourable evolution of severe MOFS.

Vascular injury post stent implantation: different gene expression modulation in human umbilical vein endothelial cells (HUVECs) model.

To explore whether stent procedure may influence transcriptional response of endothelium, we applied different physical (flow changes) and/or mechanical (stent application) stimuli to human endothelial cells in a laminar flow bioreactor (LFB) system. Gene expression analysis was then evaluated in each experimental condition. Human umbilical vein endothelial cells (HUVECs) were submitted to low and physiological (1 and 10 dyne/cm(2)) shear stress in absence (AS) or presence (PS) of stent positioning in a LFB system for 24 h. Different expressed genes, coming from Affymetrix results, were identified based on one-way ANOVA analysis with p values <0.01 and a fold changed >3 in modulus. Low shear stress was compared with physiological one in AS and PS conditions. Two major groups include 32 probes commonly expressed in both 1AS versus 10AS and 1PS versus 10PS comparison, and 115 probes consisting of 83 in addition to the previous 32, expressed only in 1PS versus 10PS comparison. Genes related to cytoskeleton, extracellular matrix, and cholesterol transport/metabolism are differently regulated in 1PS versus 10PS condition. Inflammatory and apoptotic mediators seems to be, instead, closely modulated by changes in flow (1 versus 10), independently of stent application. Low shear stress together with stent procedure are the experimental conditions that mainly modulate the highest number of genes in our human endothelial model. Those genes belong to pathways specifically involved in the endothelial dysfunction.

Determination of different forms of aminothiols in red blood cells without washing erythrocytes.

Detection and quantification of different aminothiols forms (reduced and total) in biological fluids are important for the investigation of oxidative stress-related diseases and cell homeostasis study. The aim of this study was to optimize a HPLC method in order to determine both reduced and total thiol forms in red blood cells (RBC) at low temperature without washing erythrocytes. Analytical recoveries for total and reduced thiols were 91.6-98.5 and 94.9-98.2% respectively. The relative standard deviations intra-assay for total and reduced thiols were 1.14-3.64 and 0.83-2.3% respectively and the relative standard deviations inter-assay for total and reduced thiols were 1.12-3.54 and 0.84-2.03%, respectively. This method allows specific analysis of the aminothiol state inside the RBC, as a model of intracellular metabolism functioning.

Impact of normalization strategy on cardiac expression of pro-inflammatory cytokines: evaluation of reference genes in different human myocardial regions after Left Ventricular Assist Device support.

OBJECTIVE: New device therapies have expanded the strategies for treating heart failure (HF) patients. Unloading of the heart with a left ventricular assist device (LVAD) can lead to the reversal of many remodeling changes whose underlying mechanisms are not yet completely known. Molecular analysis might play a role in obtaining further insight into the regulatory mechanisms of this process. A critical step in an RT-PCR study is the selection of reference genes for data normalization. This study aimed to determine an optimal combination of stably expressed reference genes in different regions of the human heart in order to study the effects of LVAD implants on cardiac remodeling, and in particular to check their reliability on the evaluation of pro-inflammatory cytokine expression. DESIGN AND METHODS: We validated nine of the most commonly used reference genes in human myocardium samples obtained at heart transplantation from patients with LVAD implant (n=30 from a total of six patients) and from heart transplant (HT from a total of seven patients) recipients as controls (n=35). Samples from both left (LV) and right (RV) ventricles were analyzed. The normalization strategy was tested by analyzing mRNA expression of IL-6, IL-8 and TNF-α, whose protein levels were measured by immunometric assay. RESULTS: The most stable gene combinations changed according to the experimental groups (the LVAD and HT groups and the different myocardial regions). Considering all the cardiac samples as a whole, the three most stably expressed genes were PPIA, RPL13A, and YWHAZ (M=0.70). Using the best normalization strategy, a significant increase in IL-6, IL-8 mRNA expression was observed in LVAD samples compared to HT (p<0.0001). Similar results were obtained by protein analysis. CONCLUSIONS: Our results underline the importance of always selecting reference genes for the specific system studied. The most appropriate normalization strategy is of pivotal importance for understanding the molecular mechanisms associated with the pathophysiology of HF, such as inflammation.

No association of DPP6 with amyotrophic lateral sclerosis in an Italian population.

We have attempted to replicate a recently reported association of polymorphism rs10260404, in the Dipeptidyl-peptidase 6 gene (DPP6), with susceptibility to amyotrophic lateral sclerosis (ALS) in a large independent Italian cohort of 904 cases and 1036 controls. Minor allele frequency was 0.38 in cases and 0.39 in controls and no evidence of association with ALS was observed (P=0.638). Our negative results agree with those recently reported in additional Polish and Italian cohorts.

Lack of association of PON polymorphisms with sporadic ALS in an Italian population.

Paraoxonase (PON) gene polymorphisms have been associated with susceptibility to sporadic amyotrophic lateral sclerosis (ALS). We have investigated the role of the previously associated single nucleotide polymorphisms rs854560, rs662, and rs6954345 in 350 ALS patients and 376 matched controls from Italy. No significant association was observed at genotype and haplotype level. Our data suggest that PON polymorphisms are not involved in ALS pathogenesis in an Italian population.

Mutations of FUS gene in sporadic amyotrophic lateral sclerosis.

BACKGROUND: Mutations in the FUS gene have recently been discovered to be a major cause of familial amyotrophic lateral sclerosis (FALS). OBJECTIVE: To determine the identity and frequency of FUS gene mutations in a large cohort of Italian patients enriched in sporadic cases (SALS). METHODS: Exons 5, 6, 14 and 15 of the FUS gene were screened for mutations in 1009 patients (45 FALS and 964 SALS). The genetic analysis was extended to the entire coding sequence of FUS in all the FALS and 293 of the SALS patients. RESULTS: Seven missense mutations (p.G191S, p.R216C, p.G225V, p.G230C, p.R234C, p.G507D and p.R521C) were identified in nine patients (seven SALS and two FALS), and none in 500 healthy Italian controls. All mutations are novel except for the p.R521C mutation identified in one SALS and one FALS case. Both patients showed a similar unusual presentation, with proximal, mostly symmetrical, upper limb weakness, with neck and axial involvement. With the exception of p.G507D and p.R521C, the mutations identified in SALS patients are all localised in the glycine-rich region encoded by exon 6. In addition, eight different in-frame deletions in two polyglycine motifs were detected, the frequency of which was not significantly different in patients and controls. CONCLUSIONS: The results show that FUS missense mutations are present in 0.7% of Italian SALS cases, and confirm the previous mutational frequency reported in FALS (4.4%). An unusual proximal and axial clinical presentation seems to be associated with the presence of the p.R521C mutation.