Erythrocyte-derived microvesicles induce arterial spasms in JAK2V617F myeloproliferative neoplasm.

Arterial cardiovascular events are the leading cause of death in patients with JAK2V617F myeloproliferative neoplasms (MPNs). However, their mechanisms are poorly understood. The high prevalence of myocardial infarction without significant coronary stenosis or atherosclerosis in patients with MPNs suggests that vascular function is altered. The consequences of JAK2V617F mutation on vascular reactivity are unknown. We observe here increased responses to vasoconstrictors in arteries from Jak2V617F mice resulting from a disturbed endothelial NO pathway and increased endothelial oxidative stress. This response was reproduced in WT mice by circulating microvesicles isolated from patients carrying JAK2V617F and by erythrocyte-derived microvesicles from transgenic mice. Microvesicles of other cellular origins had no effect. This effect was observed ex vivo on isolated aortas, but also in vivo on femoral arteries. Proteomic analysis of microvesicles derived from JAK2V617F erythrocytes identified increased expression of myeloperoxidase as the likely mechanism accounting for their effect. Myeloperoxidase inhibition in microvesicles derived from JAK2V617F erythrocytes suppressed their effect on oxidative stress. Antioxidants such as simvastatin and N-acetyl cysteine improved arterial dysfunction in Jak2V617F mice. In conclusion, JAK2V617F MPNs are characterized by exacerbated vasoconstrictor responses resulting from increased endothelial oxidative stress caused by circulating erythrocyte-derived microvesicles. Simvastatin appears to be a promising therapeutic strategy in this setting.

Intra-Cardiac Release of Extracellular Vesicles Shapes Inflammation Following Myocardial Infarction.

RATIONALE: A rapid and massive influx of inflammatory cells occurs into ischemic area after myocardial infarction (MI), resulting in local release of cytokines and growth factors. Yet, the mechanisms regulating their production are not fully explored. The release of extracellular vesicles (EVs) in the interstitial space curbs important biological functions, including inflammation, and influences the development of cardiovascular diseases. To date, there is no evidence for in situ release of cardiac EVs after MI. OBJECTIVE: The present study tested the hypothesis that local EV generation in the infarcted heart coordinates cardiac inflammation after MI. METHODS AND RESULTS: Coronary artery ligation in mice transiently increases EV levels in the left ventricle when compared with sham animals. EVs from infarcted hearts were characterized as large vesicles (252±18 nm) expressing cardiomyocyte and endothelial markers and small EVs (118±4 nm) harboring exosomal markers, such as CD (cluster of differentiation) 63 and CD9. Cardiac large EVs generated after MI, but not small EVs or sham EVs, increased the release of IL (interleukin)-6, CCL (chemokine ligand) 2, and CCL7 from fluorescence-activated cell-sorted Ly6C+ cardiac monocytes. EVs of similar diameter were also isolated from fragments of interventricular septum obtained from patients undergoing aortic valve replacement, thus supporting the clinical relevance of our findings in mice. CONCLUSIONS: The present study demonstrates that acute MI transiently increases the generation of cardiac EVs characterized as both exosomes and microvesicles, originating mainly from cardiomyocytes and endothelial cells. EVs accumulating in the ischemic myocardium are rapidly taken up by infiltrating monocytes and regulate local inflammatory responses.

Endothelial autophagic flux hampers atherosclerotic lesion development.

Blood flowing in arteries generates shear forces at the surface of the vascular endothelium that control its anti-atherogenic properties. However, due to the architecture of the vascular tree, these shear forces are heterogeneous and atherosclerotic plaques develop preferentially in areas where shear is low or disturbed. Here we review our recent study showing that elevated shear forces stimulate endothelial autophagic flux and that inactivating the endothelial macroautophagy/autophagy pathway promotes a proinflammatory, prosenescent and proapoptotic cell phenotype despite the presence of atheroprotective shear forces. Specific deficiency in endothelial autophagy in a murine model of atherosclerosis stimulates the development of atherosclerotic lesions exclusively in areas of the vasculature that are normally resistant to atherosclerosis. Our findings demonstrate that adequate endothelial autophagic flux limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence and inflammation.

Autophagy is required for endothelial cell alignment and atheroprotection under physiological blood flow.

It has been known for some time that atherosclerotic lesions preferentially develop in areas exposed to low SS and are characterized by a proinflammatory, apoptotic, and senescent endothelial phenotype. Conversely, areas exposed to high SS are protected from plaque development, but the mechanisms have remained elusive. Autophagy is a protective mechanism that allows recycling of defective organelles and proteins to maintain cellular homeostasis. We aimed to understand the role of endothelial autophagy in the atheroprotective effect of high SS. Atheroprotective high SS stimulated endothelial autophagic flux in human and murine arteries. On the contrary, endothelial cells exposed to atheroprone low SS were characterized by inefficient autophagy as a result of mammalian target of rapamycin (mTOR) activation, AMPKα inhibition, and blockade of the autophagic flux. In hypercholesterolemic mice, deficiency in endothelial autophagy increased plaque burden only in the atheroresistant areas exposed to high SS; plaque size was unchanged in atheroprone areas, in which endothelial autophagy flux is already blocked. In cultured cells and in transgenic mice, deficiency in endothelial autophagy was characterized by defects in endothelial alignment with flow direction, a hallmark of endothelial cell health. This effect was associated with an increase in endothelial apoptosis and senescence in high-SS regions. Deficiency in endothelial autophagy also increased TNF-α-induced inflammation under high-SS conditions and decreased expression of the antiinflammatory factor KLF-2. Altogether, these results show that adequate endothelial autophagic flux under high SS limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence, and inflammation.

A prospective study of the utility of plasma biomarkers to diagnose alcoholic hepatitis.

The diagnosis of alcoholic hepatitis (AH) often requires a transjugular liver biopsy (TJLB), a procedure that is not always readily accessible. We analyzed plasma biomarkers to estimate the presence of histological features of AH among patients with clinical suspicion of AH. Using enzyme-linked immunosorbent assay, we tested M65 and M30 (circulating fragments of cytokeratin-18) and their respective fraction carried by microvesicles (MVs), CCL20 and TREM1. Leukocyte, platelet, and endothelial-derived MVs were quantified by way of flow cytometry. Test and validation cohorts prospectively included patients with clinical features of AH undergoing TJLB. In the test cohort, 46 of 83 (55%) patients showed histological features of AH. Age, bilirubin, INR, and creatinine (ABIC) score was B or C in 83%. Patients with histologically proven AH had higher levels of total and MV-bound M65 and total and MV-bound M30 and CCL20 than those without (P < 0.001 for all tests). Levels of TREM-1 and of subpopulations of MVs were not different between groups. M65 and M30 both had an area under the receiver operating characteristics curve of 0.84 to estimate the presence of AH. For M65, a cutoff of 2000 IU/L had a positive predictive value of 91%, whereas a cutoff of 641 IU/L had a negative predictive value of 88%. In the validation cohort, AH was histologically confirmed in 48 of 68 (71%) patients. ABIC score was B or C in 69% of patients. For M65, the above cutoffs had a diagnostic accuracy of 81%. Even better results were obtained in patients with suspicion of severe AH (ABIC B or C) in both cohorts. CONCLUSION: Plasma levels of cytokeratin-18 fragments are reliable noninvasive markers of AH. Using the proposed cutoffs for M65, two thirds of TJLB can be avoided, which can be useful in centers where this technique is not readily available. (Hepatology 2017;66:555-563).

Liver microRNA-21 is overexpressed in non-alcoholic steatohepatitis and contributes to the disease in experimental models by inhibiting PPARα expression.

OBJECTIVE: Previous studies suggested that microRNA-21 may be upregulated in the liver in non-alcoholic steatohepatitis (NASH), but its role in the development of this disease remains unknown. This study aimed to determine the role of microRNA-21 in NASH. DESIGN: We inhibited or suppressed microRNA-21 in different mouse models of NASH: (a) low-density lipoprotein receptor-deficient (Ldlr-/-) mice fed a high-fat diet and treated with antagomir-21 or antagomir control; (b) microRNA-21-deficient and wild-type mice fed a methionine-choline-deficient (MCD) diet; (c) peroxisome proliferation-activator receptor α (PPARα)-deficient mice fed an MCD diet and treated with antagomir-21 or antagomir control. We assessed features of NASH and determined liver microRNA-21 levels and cell localisation. MicroRNA-21 levels were also quantified in the liver of patients with NASH, bland steatosis or normal liver and localisation was determined. RESULTS: Inhibiting or suppressing liver microRNA-21 expression reduced liver cell injury, inflammation and fibrogenesis without affecting liver lipid accumulation in Ldlr-/- fed a high-fat diet and in wild-type mice fed an MCD diet. Liver microRNA-21 was overexpressed, primarily in biliary and inflammatory cells, in mouse models as well as in patients with NASH, but not in patients with bland steatosis. PPARα, a known microRNA-21 target, implicated in NASH, was decreased in the liver of mice with NASH and restored following microRNA-21 inhibition or suppression. The effect of antagomir-21 was lost in PPARα-deficient mice. CONCLUSIONS: MicroRNA-21 inhibition or suppression decreases liver injury, inflammation and fibrosis, by restoring PPARα expression. Antagomir-21 might be a future therapeutic strategy for NASH.

Shear stress regulates endothelial microparticle release.

RATIONALE: Endothelial activation and apoptosis release membrane-shed microparticles (EMP) that emerge as important biological effectors. OBJECTIVE: Because laminar shear stress (SS) is a major physiological regulator of endothelial survival, we tested the hypothesis that SS regulates EMP release. METHODS AND RESULTS: EMP levels were quantified by flow cytometry in medium of endothelial cells subjected to low or high SS (2 and 20 dyne/cm(2)). EMP levels augmented with time in low SS conditions compared with high SS conditions. This effect was sensitive to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and Rho kinases inhibitors but unaffected by caspase inhibitors. Low SS-stimulated EMP release was associated with increased endothelial Rho kinases and ERK1/2 activities and cytoskeletal reorganization. Overexpression of constitutively active RhoA stimulated EMP release under high SS. We also examined the effect of nitric oxide (NO) in mediating SS effects. L-NG-nitroarginine methyl ester (L-NAME), but not D-NG-nitroarginine methyl ester, increased high SS-induced EMP levels by 3-fold, whereas the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) decreased it. L-NAME and SNAP did not affect Rho kinases and ERK1/2 activities. Then, we investigated NO effect on membrane remodeling because microparticle release is abolished in ABCA1-deficient cells. ABCA1 expression, which was greater under low SS than under high SS, was augmented by L-NAME under high SS and decreased by SNAP under low SS conditions. CONCLUSIONS: Altogether, these results demonstrate that sustained atheroprone low SS stimulates EMP release through activation of Rho kinases and ERK1/2 pathways, whereas atheroprotective high SS limits EMP release in a NO-dependent regulation of ABCA1 expression and of cytoskeletal reorganization. These findings, therefore, identify endothelial SS as a physiological regulator of microparticle release.

Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis.

BACKGROUND & AIMS: Circulating membrane-shed microparticles (MPs) participate in regulation of vascular tone. We investigated the cellular origins of MPs in plasma from patients with cirrhosis and assessed the contribution of MPs to arterial vasodilation, a mechanism that contributes to portal hypertension. METHODS: We analyzed MPs from blood samples of 91 patients with cirrhosis and 30 healthy individuals (controls) using flow cytometry; their effects on the vascular response to vasoconstrictors were examined in vitro and in vivo. RESULTS: Circulating levels of leuko-endothelial (CD31(+)/41(-)), pan-leukocyte (CD11a(+)), lymphocyte (CD4(+)), and erythrocyte (CD235a(+)) MPs were higher in patients with cirrhosis than in controls. Plasma of patients with cirrhosis contained hepatocyte-derived MPs (cytokeratin-18(+)), whereas plasma from controls did not. The severity of cirrhosis and systemic inflammation were major determinants of the levels of leuko-endothelial and hepatocyte MPs. MPs from patients with advanced cirrhosis significantly impaired contraction of vessels in response to phenylephrine, whereas MPs from healthy controls or from patients of Child-Pugh class A did not. This effect depended on cyclooxygenase type 1 and required phosphatidylserine on the surface of MPs. Intravenous injection of MPs from patients with cirrhosis into BALB/C mice decreased mean arterial blood pressure. CONCLUSIONS: Cirrhosis is associated with increases in circulating subpopulations of MPs, likely resulting from systemic inflammation and liver cell damage. The overall pool of circulating MPs from patients with advanced cirrhosis impairs vasoconstrictor responses and decreases blood pressure, contributing to the arterial vasodilation associated with portal hypertension.

Endothelial cell-derived microparticles loaded with iron oxide nanoparticles: feasibility of MR imaging monitoring in mice.

PURPOSE: To assess the feasibility of loading iron oxide nanoparticles in endothelial microparticles (EMPs), thereby enabling their noninvasive monitoring with magnetic resonance (MR) imaging in mice. MATERIALS AND METHODS: Experiments were approved by the French Ministry of Agriculture. Endothelial cells, first labeled with anionic superparamagnetic nanoparticles, were stimulated to generate EMPs, carrying the nanoparticles in their inner compartment. C57BL/6 mice received an intravenous injection of nanoparticle-loaded EMPs, free nanoparticles, or the supernatant of nanoparticle-loaded EMPs. A 1-week follow-up was performed with a 4.7-T MR imaging device by using a gradient-echo sequence for imaging spleen, liver, and kidney and a radial very-short-echo time sequence for lung imaging. Comparisons were performed by using the Student t test. RESULTS: The signal intensity loss induced by nanoparticle-loaded EMPs or free nanoparticles was readily detected within 5 minutes after injection in the liver and spleen, with a more pronounced effect in the spleen for the magnetic EMPs. The kinetics of signal intensity attenuation differed for nanoparticle-loaded EMPs and free nanoparticles. No signal intensity changes were observed in mice injected with the supernatant of nanoparticle-loaded EMPs, confirming that cells had not released free nanoparticles, but only in association with EMPs. The results were confirmed by using Perls staining and immunofluorescence analysis. CONCLUSION: The strategy to generate EMPs with magnetic properties allowed noninvasive MR imaging assessment and follow-up of EMPs and opens perspectives for imaging the implications of these cellular vectors in diseases.

Microparticles from human atherosclerotic plaques promote endothelial ICAM-1-dependent monocyte adhesion and transendothelial migration.

RATIONALE AND OBJECTIVE: Membrane-shed submicron microparticles (MPs) released following cell activation or apoptosis accumulate in atherosclerotic plaques, where they stimulate endothelial proliferation and neovessel formation. The aim of the study was to assess whether or not MPs isolated from human atherosclerotic plaques contribute to increased endothelial adhesion molecules expression and monocyte recruitment. METHOD AND RESULTS: Human umbilical vein and coronary artery endothelial cells were exposed to MPs isolated from endarterectomy specimens (n=62) and characterized by externalized phosphatidylserine. Endothelial exposure to plaque, but not circulating, MPs increased ICAM-1 levels in a concentration-dependant manner (3.4-fold increase) without affecting ICAM-1 mRNA levels. Plaque MPs harbored ICAM-1 and transferred this adhesion molecule to endothelial cell membrane in a phosphatidylserine-dependent manner. MP-borne ICAM-1 was functionally integrated into cell membrane as demonstrated by the increased ERK1/2 phosphorylation following ICAM-1 ligation. Plaque MPs stimulated endothelial monocyte adhesion both in culture and in isolated perfused mouse carotid. This effect was also observed under flow condition and was prevented by anti-LFA-1 and anti-ICAM-1 neutralizing antibodies. MPs isolated from symptomatic plaques were more potent in stimulating monocyte adhesion than MPs from asymptomatic patients. Plaque MPs did not affect the release of interleukin-6, interleukin-8, or MCP-1, nor the expression of VCAM-1 and E-selectin. CONCLUSION: These results demonstrate that MPs isolated from human atherosclerotic plaques transfer ICAM-1 to endothelial cells to recruit inflammatory cells and suggest that plaque MPs promote atherosclerotic plaque progression.

Magnetic tagging of cell-derived microparticles: new prospects for imaging and manipulation of these mediators of biological information.

AIMS: Submicron membrane fragments termed microparticles (MPs), which are released by apoptotic or activated cells, are newly considered as vectors of biological information and actors of pathology development. We propose the tagging of MPs with magnetic nanoparticles as a new approach allowing imaging, manipulation and targeting of cell-derived MPs. MATERIALS & METHODS: MPs generated in vitro from human endothelial cells or isolated from atherosclerotic plaques were labeled using citrate-coated 8 nm iron-oxide nanoparticles. MPs were tagged with magnetic nanoparticles on their surface and detected as Annexin-V positive by flow cytometry. RESULTS: Labeled MPs could be mobilized, isolated and manipulated at a distance in a magnetic field gradient. Magnetic mobility of labeled MPs was quantified by micromagnetophoresis. Interactions of labeled MPs with endothelial cells could be triggered and modulated by magnetic guidance. Nanoparticles served as tracers at different scales: at the subcellular level by electron microscopy, at the cellular level by histology and at the macroscopic level by MRI. CONCLUSION: Magnetic labeling of biogenic MPs opens new prospects for noninvasive monitoring and distal manipulations of these biological effectors.

Proteomic analysis reveals presence of platelet microparticles in endothelial progenitor cell cultures.

The concept of endothelial progenitor cells (EPCs) has attracted considerable interest in cardiovascular research, but despite a decade of research there are still no specific markers for EPCs and results from clinical trials remain controversial. Using liquid chromatography-tandem mass spectrometry, we analyzed the protein composition of microparticles (MPs) originating from the cell surface of EPC cultures. Our data revealed that the conventional methods for isolating mononuclear cells lead to a contamination with platelet proteins. Notably, platelets readily disintegrate into platelet MPs. These platelet MPs are taken up by the mononuclear cell population, which acquires "endothelial" characteristics (CD31, von Willebrand factor [VWF], lectin-binding), and angiogenic properties. In a large population-based study (n = 526), platelets emerged as a positive predictor for the number of colony-forming units and early outgrowth EPCs. Our study provides the first evidence that the cell type consistent with current definitions of an EPC phenotype may arise from an uptake of platelet MPs by mononuclear cells resulting in a gross misinterpretation of their cellular progeny. These findings demonstrate the advantage of using an unbiased proteomic approach to assess cellular phenotypes and advise caution in attributing the benefits in clinical trials using unselected bone marrow mononuclear cells (BMCs) to stem cell-mediated repair.

CD40 ligand+ microparticles from human atherosclerotic plaques stimulate endothelial proliferation and angiogenesis a potential mechanism for intraplaque neovascularization.

OBJECTIVES: Our goal was to demonstrate that microparticles (MPs) are the endogenous signal leading to neovessel formation through CD40 ligation in human atherosclerotic plaques. BACKGROUND: Vulnerable atherosclerotic plaques prone to rupture are characterized by an increased number of vasa vasorum and frequent intraplaque hemorrhage. Although inflammatory cytokines, growth factors, or CD40/CD40 ligand (CD40L) are possible candidates, the mechanism of atherosclerotic plaque neovascularization remains unknown. Atherosclerotic plaques contain large amounts of membrane-shed submicron MPs released after cell activation or apoptosis. METHODS: Microparticles were isolated from endarterectomy specimens surgically obtained from 26 patients and characterized by phosphatidylserine exposure and specific markers of cellular origin. RESULTS: Plaque MPs increased both endothelial proliferation assessed by (3)H-thymidine incorporation and cell number and stimulated in vivo angiogenesis in Matrigel (BD Biosciences, San Diego, California) assays performed in wild-type and BalbC/Nude mice, whereas circulating MPs had no effect. Microparticles from symptomatic patients expressed more CD40L and were more potent in inducing endothelial proliferation, when compared with asymptomatic plaque MPs. Most of CD40L+ MPs (93%) isolated from human plaques were of macrophage origin. Microparticle-induced endothelial proliferation was impaired by CD40L or CD40-neutralizing antibodies and abolished after endothelial CD40-ribonucleic acid silencing. In addition, the proangiogenic effect of plaque MPs was abolished in Matrigel assays performed in the presence of CD40L-neutralizing antibodies or in CD40-deficient mice. CONCLUSIONS: These results demonstrate that MPs isolated from human atherosclerotic lesions express CD40L, stimulate endothelial cell proliferation after CD40 ligation, and promote in vivo angiogenesis. Therefore, MPs could represent a major determinant of intraplaque neovascularization and plaque vulnerability.