We underline the importance of the homogenization of the parameters of isolation of EVs and their characterization, which allow considering EVs as excellent biomarkers for diagnosis and prognosis. Antioxid. Redox Signal. 00, 000-000.

BackgroundSeveral translational studies have identified the differential role between saturated and unsaturated fatty acids at cardiovascular level. However, the molecular mechanisms that support the protective role of oleate in cardiovascular cells are poorly known. For these reasons, we studied the protective role of oleate in the insulin resistance and in the atherosclerotic process at cellular level such as in cardiomyocytes (CMs), vascular smooth muscle cells (VSMCs) and endothelial cells (ECs).MethodsThe effect of oleate in the cardiovascular insulin resistance, vascular dysfunction, inflammation, proliferation and apoptosis of VSMCs were analyzed by Western blot, qRT-PCR, BrdU incorporation and cell cycle analysis.ResultsPalmitate induced insulin resistance. However, oleate not only did not induce cardiovascular insulin resistance but also had a protective effect against insulin resistance induced by palmitate or TNFα. One mechanism involved might be the prevention by oleate of JNK-1/2 or NF-κB activation in response to TNF-α or palmitate. Oleate reduced MCP-1 and ICAM-1 and increased eNOS expression induced by proinflammatory cytokines in ECs. Furthermore, oleate impaired the proliferation induced by TNF-α, angiotensin II or palmitate and the apoptosis induced by TNF-α or thapsigargin in VSMCs.ConclusionsOur data suggest a differential role between oleate and palmitate and support the concept of the cardioprotector role of oleate as the main lipid component of virgin olive oil. Thus, oleate protects against cardiovascular insulin resistance, improves endothelial dysfunction in response to proinflammatory signals and finally, reduces proliferation and apoptosis in VSMCs that may contribute to an ameliorated atherosclerotic process and plaque stability.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-015-0237-9) contains supplementary material, which is available to authorized users.

Current pharmacological therapies for treating obesity are of limited efficacy. Genetic ablation or loss of function of AMP-activated protein kinase alpha 1 (AMPKα1) in steroidogenic factor 1 (SF1) neurons of the ventromedial nucleus of the hypothalamus (VMH) induces feeding-independent resistance to obesity due to sympathetic activation of brown adipose tissue (BAT) thermogenesis. Here, we show that body weight of obese mice can be reduced by intravenous injection of small extracellular vesicles (sEVs) delivering a plasmid encoding an AMPKα1 dominant negative mutant (AMPKα1-DN) targeted to VMH-SF1 neurons. The beneficial effect of SF1-AMPKα1-DN loaded sEVs is feeding-independent and involves sympathetic nerve activation and increased BAT UCP1-dependent thermogenesis in BAT. Our results underscore the potential of sEVs to specifically target AMPK in hypothalamic neurons and introduce a broader strategy to manipulate body weight and reduce obesity.

The antigen recognition interface formed by T helper precursors (Thps) and antigen-presenting cells (APCs), called the immunological synapse (IS), includes receptors and signaling molecules necessary for Thp activation and differentiation. We have recently shown that recruitment of the interferon-γ receptor (IFNGR) into the IS correlates with the capacity of Thps to differentiate into Th1 effector cells, an event regulated by signaling through the functionally opposing receptor to interleukin-4 (IL4R). Here, we show that, similar to IFN-γ ligation, TCR stimuli induce the translocation of signal transducer and activator of transcription 1 (STAT1) to IFNGR1-rich regions of the membrane. Unexpectedly, STAT1 is preferentially expressed, is constitutively serine (727) phosphorylated in Thp, and is recruited to the IS and the nucleus upon TCR signaling. IL4R engagement controls this process by interfering with both STAT1 recruitment and nuclear translocation. We also show that in cells with deficient Th1 or constitutive Th2 differentiation, the IL4R is recruited to the IS. This observation suggest that the IL4R is retained outside the IS, similar to the exclusion of IFNGR from the IS during IL4R signaling. This study provides new mechanistic cues for the regulation of lineage commitment by mutual immobilization of functionally antagonistic membrane receptors.

To assess the role of insulin receptor (IR) isoforms (IRA and IRB) in the proliferation of vascular smooth muscle cells (VSMCs) involved in the atherosclerotic process, we generated new VSMC lines bearing IR (wild-type VSMCs; IRLoxP(+/+) VSMCs), lacking IR (IR(-/-) VSMCs) or expressing IRA (IRA VSMCs) or IRB (IRB VSMCs). Insulin and different proatherogenic stimuli induced a significant increase of IRA expression in IRLoxP(+/+) VSMCs. Moreover, insulin, through ERK signaling, and the proatherogenic stimuli, through ERK and p38 signaling, induced a higher proliferation in IRA than IRB VSMCs. The latter effect might be due to IRA cells showing a higher expression of angiotensin II, endothelin 1, and thromboxane 2 receptors and basal association between IRA and these receptors. Furthermore, TNF-α induced in a ligand-dependent manner a higher association between IRA and TNF-α receptor 1 (TNF-R1). On the other hand, IRA overexpression might favor the atherogenic actions of IGF-II. Thereby, IGF-II or TNF-α induced IRA and IGF-I receptor (IGF-IR) overexpression as well as an increase of IRA/IGF-IR hybrid receptors in VSMCs. More importantly, we observed a significant increase of IRA, TNF-R1, and IGF-IR expression as well as higher association of IRA with TNF-R1 or IGF-IR in the aorta from ApoE(-/-) and BATIRKO mice, 2 models showing vascular damage. In addition, anti-TNF-α treatment prevented those effects in BATIRKO mice. Finally, our data suggest that the IRA isoform and its association with TNF-R1 or IGF-IR confers proliferative advantage to VSMCs, mainly in response to TNF-α or IGF-II, which might be of significance in the early atherosclerotic process.

BackgroundAbnormal proliferation and migration of vascular smooth muscle cells (VSMCs) is a major contributor to the development of atherosclerotic process. In a previous work, we demonstrated that the insulin receptor isoform A (IRA) and its association with the insulin-like growth factor-I receptor (IGF-IR) confer a proliferative advantage to VSMCs. However, the role of IR and IGF-IR in VSMC migration remains poorly understood.MethodsWound healing assays were performed in VSMCs bearing IR (IRLoxP+/+ VSMCs), or not (IR−/− VSMCs), expressing IRA (IRA VSMCs) or expressing IRB (IRB VSMCs). To study the role of IR isoforms and IGF-IR in experimental atherosclerosis, we used ApoE−/− mice at 8, 12, 18 and 24 weeks of age. Finally, we analyzed the mRNA expression of total IR, IRB isoform, IGF-IR and IGFs by qRT-PCR in the medial layer of human aortas.ResultsIGF-I strongly induced migration of the four cell lines through IGF-IR. In contrast, insulin and IGF-II only caused a significant increase of IRA VSMC migration which might be favored by the formation of IRA/IGF-IR receptors. Additionally, a specific IGF-IR inhibitor, picropodophyllin, completely abolished insulin- and IGF-II-induced migration in IRB, but not in IRA VSMCs. A significant increase of IRA and IGF-IR, and VSMC migration were observed in fibrous plaques from 24-week-old ApoE−/− mice. Finally, we observed a marked increase of IGF-IR, IGF-I and IGF-II in media from fatty streaks as compared with both healthy aortas and fibrolipidic lesions, favoring the ability of medial VSMCs to migrate into the intima.ConclusionsOur data suggest that overexpression of IGF-IR or IRA isoform, as homodimers or as part of IRA/IGF-IR hybrid receptors, confers a stronger migratory capability to VSMCs as might occur in early stages of atherosclerotic process.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-016-0477-3) contains supplementary material, which is available to authorized users.

Type 2 diabetes mellitus is a complex metabolic disease and its pathogenesis involves abnormalities in both peripheral insulin action and insulin secretion. Previous in vitro data showed that insulin receptor isoform A, but not B, favours basal glucose uptake through its specific association with endogenous GLUT1/2 in murine hepatocytes and beta cells. With this background, we hypothesized that hepatic expression of insulin receptor isoform A in a mouse model of type 2 diabetes could potentially increase the glucose uptake of these cells, decreasing the hyperglycaemia and therefore ameliorating the diabetic phenotype. To assure this hypothesis, we have developed recombinant adeno-associated viral vectors expressing insulin receptor isoform A (IRA) or isoform B (IRB) under the control of a hepatocyte­-specific promoter. Our results demonstrate that in the long term, hepatic expression of IRA in diabetic mice is more efficient than IRB in ameliorating glucose intolerance. Consequently, it impairs the induction of compensatory mechanisms through beta cell hyperplasia and/or hypertrophy that finally lead to beta cell failure, reverting the diabetic phenotype in about 8 weeks. Our data suggest that long-term hepatic expression of IRA could be a promising therapeutic approach for the treatment of type 2 diabetes mellitus.

Rationale: Metabolic syndrome (MetS) is a cluster of interrelated risk factors for cardiovascular diseases and atherosclerosis. Circulating levels of large extracellular vesicles (lEVs), submicrometer-sized vesicles released from plasma membrane, from MetS patients were shown to induce endothelial dysfunction but their role in early stage of atherosclerosis and on vascular smooth muscle cells (SMC) remain to be fully elucidated. Objective: To determine the mechanisms by which lEVs lead to the progression of atherosclerosis in the setting of MetS. Methods and Results: Proteomic analysis revealed that the small GTPase, Rap1 was overexpressed in lEVs from MetS patients compared to those from non-MetS (nMetS) subjects. Rap1 was in GTP-associated active state in both types of lEVs and Rap1-lEVs levels correlated with increased cardiovascular risks including stenosis. MetS-lEVs, but not nMetS-lEVs, increased Rap1-dependent endothelial cell permeability. MetS-lEVs significantly promoted migration and proliferation of human aortic SMC and increased expression of pro-inflammatory molecules and activation of ERK5/p38 pathways. Neutralization of Rap1 by specific antibody or pharmacological inhibition of Rap1 with GGTi-298, completely prevented the effects of lEVs from MetS patients. HFD-fed ApoE -/- mice displayed an increased expression of Rap1 both in aortas and circulating lEVs. lEVs accumulated in plaque atherosclerotic lesions depending on the progression of atherosclerosis. lEVs from HFD-fed ApoE -/- mice, but not those from mice fed with a standard diet, enhanced SMC proliferation. Human atherosclerotic lesions were enriched in lEVs expressing Rap1. Conclusions: These data demonstrate that Rap1 carried by MetS-lEVs participates in the enhanced SMC proliferation, migration, pro-inflammatory profile and activation of ERK5/p38 pathways leading to vascular inflammation and remodeling, and atherosclerosis. These results highlight that Rap1 carried by MetS-lEVs may be a novel determinant of diagnostic value for cardiometabolic risk factors and suggest Rap1 as a promising therapeutic target against the development of atherosclerosis.