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Aims Atherosclerosis is an inflammatory disease wherein cholesterol-loaded macrophages play a major role. MicroRNAs and microparticles propagate inflammatory pathways and are involved in cardiovascular disease. We aimed to screen and validate circulating microRNAs correlated with atherosclerosis development in humans, and to dissect the molecular mechanisms associated with atherogenesis using in vitro and in vivo approaches. Methods and results A panel of 179 secreted microRNAs was screened in plasma samples of patients with and without atherosclerosis, and validated cross-sectionally and prospectively in patients followed for up to 11 years. miR-30c-5p was inversely correlated with total and LDL cholesterol, carotid intimal media thickness (CIMT), presence and future development of plaques. Using a human macrophage line and in vitro gene silencing strategies, we found that miR-30c-5p was downregulated by oxidized LDL (oxLDL) via the scavenger receptor CD36 and inhibition miR processing by Dicer. In turn, miR-30c-5p downregulation was responsible for the effects of oxLDL on macrophage IL-1b release, caspase-3 expression, and apoptosis. miR-30c-5p loaded into microparticles was uptaken by macrophages and regulated target genes, like caspase-3, at transcriptional level. To establish the relevance of this pathway on endothelial damage as the earliest step of atherogenesis, we show that systemic miR-30c-5p knockdown induced caspase-3 and impaired endothelial healing after carotid injury in C57Bl/6 J mice. Conclusions With an unbiased screening of secreted microRNAs, we identify reduction of miR-30c-5p in microparticles as a promoter of early atherosclerosis, by conveying pro-inflammatory pro-apoptotic signals and impairing endothelial healing. Therefore, stimulation of miR-30c-5p is a candidate direct anti-atherosclerotic therapy. [ABSTRACT FROM AUTHOR]

Upon activation, neutrophils undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, NETosis can damage tissue. Therefore, we examined the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in nonhealing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components (elastase, histones, neutrophil gelatinase-associated lipocalin, and proteinase-3) were elevated in the blood of patients with DFUs. Circulating elastase and proteinase-3 were associated with infection, and serum elastase predicted delayed healing. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis but an impaired inducible NETosis. In mice, skin PAD4 activity was increased by diabetes, and FACS detection of histone citrullination, together with intravital microscopy, showed that NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing. [ABSTRACT FROM AUTHOR]

Objective: The presence of carotid plaques is associated with future cardiovascular events, with local plaque composition being an independent outcome predictor. We examined the association between ultrasonographically determined carotid plaque calcification and incident major adverse cardiovascular events (MACE) and death in type 2 diabetes (T2D).Research Design and Methods: We enrolled 581 patients with T2D who underwent routine carotid ultrasonography. Plaques were classified as echolucent (lipid rich), heterogenous, and echogenic (calcific). We collected demographic, anthropometric, and clinical data at baseline and followed the patients for up to 9 years.Results: Plaques were detected in 81.8% of the patients (echolucent in 16.4%, heterogenous in 43.2%, and echogenic in 22.2%). During follow-up (4.3 ± 0.1 years), 58 deaths (27 cardiovascular) and 236 fatal and nonfatal MACE occurred. In univariate analyses, presence versus absence of any carotid plaque was associated with incident MACE, and the hazard ratio (95% CI) progressively increased from echolucent (1.97 [0.93-3.44]), to heterogeneous (3.10 [2.09-4.23]), to echogenic (3.71 [2.09-5.59]) plaques. Compared with echolucent plaques, echogenic plaques were associated with incident MACE independently from confounders. This association was attenuated after adjusting for the degree of stenosis, but in patients with stenosis ≤30%, echogenic plaque type still predicted total and atherosclerotic MACE, even after further adjusting for mean intima-media thickness.Conclusions: In T2D, carotid plaque calcification predicts MACE, especially in patients with a low degree of stenosis. The biology of atherosclerotic calcification in diabetes needs to be further elucidated to understand the basis of this association. [ABSTRACT FROM AUTHOR]

There is a concern on the risk of thyroid cancer associated with glucagon-like peptide-1 (GLP-1) analogs including liraglutide and exenatide. In this article, we review related experimental studies, clinical trials and observational human studies currently available. In rodents, liraglutide activated the GLP-1 receptors on C-cells, causing an increased incidence of C-cell neoplasia. Animal experiments with monkeys demonstrated no increase in calcitonin release and no C-cell proliferation after long-term liraglutide administration. Longitudinal 2-year data from clinical trials do not support any significant risk for the activation or growth of C-cell cancer in humans in response to liraglutide. However, an analysis of the FDA adverse event reporting system database suggested an increased risk for thyroid cancer associated with exenatide after its marketing. Noticeably, a recent study discovered that GLP-1 receptor could also be expressed in human papillary thyroid carcinomas (PTC), but the impact of GLP-1 analogs on PTC is not known. Therefore, GLP-1 analogs might increase the risk of thyroid C-cell pathology in rodents, but its risk in humans awaits confirmation. Since GLP-1 receptor is also expressed in PTC besides C-cells, it is important to investigate the actions of GLP-1 on different subtypes of thyroid cancer in the future. [ABSTRACT FROM AUTHOR]

Diabetes mellitus (DM) alters circulating progenitor cells relevant for the pathophysiology of coronary artery disease (CAD). While endothelial progenitor cells (EPCs) are reduced, there is no data on procalcific polarization of circulating progenitors, which may contribute to vascular calcification in these patients. In a cohort of 107 subjects with and without DM and CAD, we analyzed the pro-calcific versus endothelial differentiation status of circulating CD34+ progenitor cells. Endothelial commitment was determined by expression of VEGFR-2 (KDR) and pro-calcific polarization by expression of osteocalcin (OC) and bone alkaline phosphatase (BAP). We found that DM patients had significantly higher expression of OC and BAP on circulating CD34+ cells than control subjects, especially in the presence of CAD. In patients with DM and CAD, the ratio of OC/KDR, BAP/KDR, and OC+BAP/KDR was about 3-fold increased than in other groups. EPCs cultured from DM patients with CAD occasionally formed structures highly suggestive of calcified nodules, and the expression of osteogenic markers by EPCs from control subjects was significantly increased in response to the toll-like receptor agonist LPS. In conclusion, circulating progenitor cells of diabetic patients show a phenotypic drift toward a pro-calcific phenotype that may be driven by inflammatory signals.

The metabolic syndrome (MS), a predisposing condition for cardiovascular disease, presents disturbances in hemodynamics; impedance cardiography (ICG) can assess these alterations. In subjects with MS, the morphology of the pulses present in the ICG time series is more irregular/complex than in normal subjects. Therefore, the aim of the present study was to quantitatively assess the complexity of ICG times series in 53 patients, with or without MS, through a nonlinear analysis algorithm, the approximate entropy, a method employed in recent years for the study of several biological signals, which provides a scalar index, ApEn. We correlated ApEn computed from ICG times series data during fasting and postprandial phase with the presence of alterations in the parameters defining MS [Adult Treatment Panel (ATP) III (Grundy SM, Brewer HB Jr, Cleeman JI, Smith SC Jr, Lenfant C; National Heart, Lung, and Blood Institute; American Heart Association. Circulation 109: 433-438, 2004) and the International Diabetes Federation (IDF) definition]. Results show that ApEn was significantly higher in subjects with MS compared with those without (1.81 ± 0.09 vs. 1.65 ± 0.13; means ± SD; P = 0.0013, with ATP III definition; 1.82 ± 0.09 vs. 1.67 ± 0.12; P = 0.00006, with the IDF definition). We also demonstrated that ApEn increase parallels the number of components of MS. ApEn was then correlated to each MS component: mean ApEn values of subjects belonging to the first and fourth quartiles of the distribution of MS parameters were statistically different for all parameters but HDL cholesterol. No difference was observed between ApEn values evaluated in fasting and postprandial states. In conclusion, we identified that MS is characterized by an increased complexity of ICG signals: this may have a prognostic relevance in subjects with this condition.

Patients with chronic severe lung disease are prone to develop pulmonary vascular remodeling, possibly through pulmonary endothelial dysfunction. Circulating endothelial progenitor cells (EPCs) are involved in maintenance of endothelial homeostasis. The aim of this study was to assess whether obstructive and restrictive lung diseases are associated with modification of EPC number in peripheral blood. The study was cross-sectional and involved patients with obstructive (n = 15) and restrictive (n = 15) lung disease on oxygen therapy and 15 control subjects. Circulating EPCs were defined by the surface expression of CD34, CD133, and kinase-insert domain receptor. Results from spirometric tests, blood gas analyses, and blood cell counts have been related to EPC numbers. Patients with chronic hypoxia and severe lung disease showed lower levels of all progenitors than do control subjects. A consensual further reduction of EPC was found in restrictive patients in comparison with obstructive patients. Among restrictive patients, EPC reduction was related to reduced lung volumes and impaired alveolo-arterial diffusion, whereas progenitor cell levels were directly related to erythrocyte number. Considering obstructive patients, significant correlations were found between progenitor cell levels and bronchial obstruction and between progenitor cell levels and arterial oxygen tension. These findings demonstrate a reduction of EPCs in patients with chronic lung disease and long-lasting hypoxia. This alteration was more evident in restrictive patients and correlated to disease severity. Depletion of circulating EPCs may be involved in altered endothelial homeostasis of pulmonary circulation in these disorders.