Search

Endothelial progenitor cells (EPCs) and monocytic cells from bone marrow (BM) can be recruited to the injured endothelium and contribute to its regeneration. During metabolic diseases such as obesity and diabetes, progenitor cell function is impaired. Several studies have shown that moderate alcohol consumption prevents the development and progression of atherosclerosis in a variety of animal/mouse models and increases mobilization of progenitor cells. Along with these studies, we identify ethanol at low concentration as therapeutic tool to in vitro expand progenitor cells in order to obtain an adequate number of cells for their use in the treatment of cardiovascular diseases. We evaluated the effects of ethanol on the phenotype of BM-derived cells from mice fed with high-fat diet (HFD). HFD did not induce changes in weight of mice but induced metabolic alterations. HFD feeding increased the differentiation of monocytic progenitors but not EPCs. Whereas ethanol at 0.6% is able to increase monocytic progenitor differentiation, 1% ethanol diminished it. Furthermore, ethanol at 0.6% increased the ability of progenitor cells to promote in vivo angiogenesis as well as secretome of BM-derived cells from mice fed with HFD, but not in mice fed normal diet. In conclusion, ethanol at low concentration is able to increase angiogenic abilities of progenitor cells from animals with early metabolic alterations.

Microparticles (MPs) are small vesicles released from the plasma membrane of activated and/or apoptotic cells. We have previously shown that MPs generated from apoptotic T cells induce endothelial dysfunction through a decrease of nitric oxide production (NO). In this study we hypothesize that the mechanism by which MPs induce endothelial dysfunction implicates endoplasmic reticulum (ER) stress activation. For this, human aortic endothelial cells (HAoECs) are treated with 10μg/ml MPs in the absence or in the presence of the ER stress inhibitor TUDCA for 24 hours. Expressions of molecular components of ER stress such as XBP- 1, p-eIF2alpha and CHOP are increased by MP treatment and are prevented in the presence of TUDCA. In addition, in the presence of inhibitors of reactive oxygen species (ROS) sources (allopurinol, apocynin or rotenone), MPs are not able to induce ER stress indicating the implication of ROS in the effects of MPs. Since during ER stress, it has been shown that ER and mitochondria interactions are increased, we analyzed the expression of mitofusin-2 and VDAC-1 channel, two components of mitochondria-associated membranes. MPs are able to increase expression of mitofusin-2 and to decrease VDAC-1 channel suggesting that mitochondria-associated membranes are implicated in the effects of MPs. Finally, the decrease on NO production induced by MPs is prevented in the presence of TUDCA. Altogether, these results show the involvement of ER stress and the mitochondria in endothelial dysfunction induced by MPs via the increase of ROS production.

After ischaemic injury and in patients with atherosclerosis, the pool of inflammatory macrophages is enlarged in the heart and in atherosclerotic plaques. Monocyte/macrophage-derived microparticles (MPs) are part of the pathological process of unstable atherosclerotic plaques. In the present study, we have focused on MPs released by apoptotic murine RAW 264.7 macrophage cell line. The hypothesis that these MPs could induce deleterious effects in adult myocardial cells was evaluated. Flow cytometry and western blot analysis showed that these MPs contained the soluble form of tumour necrosis factor alpha (TNF-α). Furthermore, cardiomyocyte sarcomere shortening amplitudes and kinetics were reduced within 5 min of exposure to MPs. Conversely, Ca2+ transient amplitudes and kinetics were not modified. The contractile effects of MPs were completely prevented after pre-treatment with nitric oxide synthase, guanylate cyclase or TNF-α inhibitors as well as blocking TNF-α receptor 1 with neutralizing antibody. Moreover, confocal microscopy showed that, after 1 h, MPs were clearly surrounding rod-shaped cardiomyocytes, and after 2 h they were internalized into cardiomyocytes undergoing apoptosis. Indeed, after 4 h of treatment with MPs, cardiomyocytes expressed increased apoptotic markers expression (caspase-3, caspase-8, Bax and cytochrome C) as demonstrated by Western Blot. The present study showed that these MPs carry functional TNF-α and activate TNFR1 in isolated adult murine ventricular cardiomyocytes, leading to cell shortening without affecting calcium signalling via a mechanism sensitive to NO synthase inhibitor and that the mitochondrial intrinsic pathway was implicated in their proapoptotic effects. Thus, the present study allows supporting the hypothesis that MPs from human carotid atherosclerotic plaques may contain active TNF-α, which could contribute to MP-induced inflammatory signals in human atherosclerotic lesions leading to myocardial infarction. The author hereby declares no conflict of interest

Recent studies have demonstrated that microparticles (MPs) generated from T lymphocytes undergoing both activation and apoptosis correct endothelial injury, and regulate angiogenesis. These effects are mediated by an increase of NO release and a decrease of reactive oxygen species (ROS) production. Apoptosis is an important mechanism that controls endothelial cell number and regeneration. We studied the effects of these MPs on human umbilical vein endothelial cell (HUVEC) apoptosis induced by actinomycin D (actD). Engineered MPs were obtained by activation of human lymphocyte CEM-T cell line with phytohemagglutinin and then, by stimulation with phorbol-12-myristate-13-acetate and actD. HUVECs were grown for 24 h in absence or presence of the proapoptotic agent actD (1 μg/ml), and/or 10 μg protein/ml of MPs. In another set of experiments, endothelial cells were pre-incubated either with nonselective caspases inhibitor, z-vad.fmk (50 μm), PI3-kinase inhibitor, LY294002 (10 μm), ERK inhibitor, U0126 (10 μm), NOS inhibitor, L-NA (100 μm), or the superoxide dismutase (SOD) mimetic, manganese(III)tetrakis-(1-methyl-4-pyridyl)-porphyrin pentachloride (MnTMPyP, 100 μm). The proportion of nuclei with hypodiploid DNA (sub-G1 peak) corresponding to apoptotic cells was determined by flow cytometry after staining with propidium iodide, and by microscopy using TUNEL labeling. Our results clearly indicate that activated/apoptotic T lymphocytes MP treatment significantly reduces HUVEC apoptosis evoked by actD. Moreover, pancaspases inhibitor reduces the degree of cell death either in presence or in absence of MPs, indicating the implication of caspases in actD-induced apoptosis. Although, the PI3-kinase inhibitor induces apoptosis by itself, it does not blunt the capacity of MPs in reducing apoptosis induced by actD. In contrast, the inhibitory effect of MPs is prevented in the presence either of the ERK inhibitor or the NOS inhibitor. Interestingly, MnTMPyP reduces actD-evoked apoptosis and the protective effects of MPs. Thus, these MPs may act as an SOD mimetic under these experimental conditions. Altogether, T lymphocyte MPs evoked cell protection against apoptosis at the level of caspases inhibition, MAP kinases, NO and ROS formations. They underscore the potential therapeutic of such MPs against vascular pathologies in close association with apoptosis. Supported by ANR-07-PHYSIO-010-01.