Higher Endothelial Microparticles Research Articles

Abstract 11165: Stimulated Release of Apoptosis- Induced Endothelial Microparticles in Patients with Diabetes and Coronary Artery Disease

Introduction: High endothelial microparticles (EMPs) levels could be used as a marker of vascular dysfunction. Hypothesis: The release of EMPs with apoptotic characteristics in the blood of patients with diabetes is different according to the presence of CAD, and further distinct between chronic CAD and ACS. Methods: A total of 80 participants were recruited and equally classified as: 1) healthy participants without diabetes, 2) healthy diabetics without micro or macro-vascular complications, 3) participants with T2D and chronic CAD, and 4) participants with T2D and ACS. MPs were isolated using ExoQuick-LP TM and analysed using flow cytometry of CD42 and CD31. CD62E was used to determine the apoptotic/activation state of the EMPs. MP content was extracted and profiled using angiogenesis array (R&D systems). Results: Mean age of participants was 56 ±10. Levels of CD42- CD31+ EMPs were significantly increased in diabetics with acute ACS (257.5±35.58) when compared to healthy subjects (105.7±12.96, p<0.01). There was no significant difference when comparing diabetics, with and without chronic CAD. The ratio of CD42-CD62+/ CD42-CD31+ EMPs was reduced in all diabetic groups, with further reduction in ACS when compared to chronic CAD, reflecting a release by apoptotic endothelial cells. The angiogenic content of the full population of MPs was analysed and revealed a significant differential expression of 5 factors in patients with ACS and diabetes: TGF-β1, PD-ECGF, platelet factor 4, serpin E1 and thrombospondin 1 (Figure 1). Ingenuity Pathway Analysis revealed of those 5 differentially expressed molecules, mainly TGF-β1, causes an inhibition of key pathways involved in normal endothelial function. Conclusions: Our data showed that the release of apoptosis-induced EMPs is increased in diabetes, irrespective of the presence of CAD, ACS patients having the highest levels. The protein contents of MPs interact in networks that are indicative of vascular dysfunction.

Abstract 171: Endothelial Microparticle Levels and Silent Stroke in the Framingham Heart Study

Introduction: Endothelial microparticles (EMP) are membrane vesicles, with a characteristic diameter of 0.1-1.0μ, shed by endothelial cells during cellular activation or apoptosis. EMP are associated with cardiovascular disease and risk factors, but data are lacking on the relations of EMP with subclinical vascular brain injury. Methods: We studied 487 individuals (mean age 66.9±8.8 years, 55% women) from the Framingham Offspring Cohort, who were free from clinical stroke. CD31+/CD41- EMP were identified and quantified using flow cytometry. Silent cerebral infarcts (SCI) were quantified as parenchymal lesions on brain MRI >3mm in size with characteristics of previous infarcts. Logistic regression models were used to investigate cross-sectional associations of EMP levels with prevalent SCI. We tested for interaction between EMP levels and statin use in their relationship with SCI. Results: SCI was observed in 49 (10%) participants. Figure 1 displays results of the logistic regression analysis. Circulating CD31+/CD41- EMP levels were inversely associated with SCI, adjusting for age, sex, and time interval between blood draw and brain MRI (OR 0.90 [0.82-0.98], p=0.016). Similar results were observed when adjusting for additional vascular risk factors. We observed an interaction between statin use and EMP levels in their association with SCI. There was an inverse association (OR 0.76 [0.64-0.89], p=0.001) among statin users and no association among non-statin users. Conclusions: In our analysis of the relationship of CD31+/CD41- EMP with SCI, in a well-defined community cohort, we observed an inverse association of higher EMP levels, primarily in participants on statins. Our data question whether constitutive or induced release of EMP, associated with a pleiotropic effect of statin medication, might mitigate vascular risk factor exposure and reduce risk of SCI. Further studies are needed to determine the associations of EMP with SCI and clinical stroke.

Endothelial dysfunction is associated with activation of the type I interferon system and platelets in patients with systemic lupus erythematosus

ObjectivesEndothelial dysfunction may be connected to cardiovascular disease (CVD) in systemic lupus erythematosus (SLE). Type I interferons (IFNs) are central in SLE pathogenesis and are suggested to induce both endothelial...

High glucose derived endothelial microparticles increase active caspase-3 and reduce microRNA-Let-7a expression in endothelial cells

The experimental aim of this study was to determine the effects of high glucose-induced endothelial microparticles (EMPs) on endothelial cell susceptibility to apoptosis. Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and plated in 6-well plates at a density of 5.0 × 105 cells/condition. Cells were incubated with media containing 25 mM d-glucose (concentration representing a diabetic glycemic state) or 5 mM d-glucose (normoglycemic condition) for 48 h to generate EMPs. EMP identification (CD144+ expression) and concentration was determined by flow cytometry. HUVECs (3 × 106 cells/condition) were treated with EMPs generated from either the normal or high glucose conditions for 24 h. Intracellular concentration of active caspase-3 was determined by enzyme immunoassay. Cellular expression of miR-Let7a, an anti-apoptotic microRNA, was determined by RT-PCR using the ΔΔCT normalized to RNU6. High glucose-derived EMPs significantly increased both basal (1.5 ± 0.1 vs 1.0 ± 0.1 ng/mL) and staurosporine-stimulated (2.2 ± 0.2 vs 1.4 ± 0.1 ng/mL) active caspase-3 compared with normal glucose EMPs. Additionally, the expression of miR-Let-7a was markedly reduced (∼140%) by high glucose EMPs (0.43 ± 0.17 fold vs control). These results demonstrate that hyperglycemic-induced EMPs increase endothelial cell active caspase-3. This apoptotic effect may be mediated, at least in part, by a reduction in miR-Let-7a expression.

Endothelial microparticles delivering microRNA-155 into T lymphocytes are involved in the initiation of acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation.

Endothelial microparticles (EMPs) upregulation has been observed in acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the role of EMPs remains unclear. We found that EMPs derived from TNF-α-stimulated human umbilical vein endothelial cells (EA.hy926) concentrated more microRNA-155 (miR-155) compared with maternal cells. The miR-155 levels in MPs from peripheral blood of aGVHD patients and mice were remarkably elevated and significantly higher than the levels in plasma. Moreover, the rising peak of miR-155 in MPs occurred significantly prior to the peak in T lymphocytes. Additionally, we observed fluorescently-labeled miR-155 in EMPs actively transported into recipient T lymphocytes. Inhibition of miR-155 in EMPs by antagomir-155 did not influence the proliferation and apoptosis of T lymphocytes, but induced defective differentiation toward Th1, Th9 and Th17 cells and skewed differentiation toward Th2 and Treg cells. Furthermore, intravenous injection of miR-155-deficient-EMPs into aGVHD mice significantly attenuated the exacerbation of aGVHD manifestations and abnormal T lymphocytes differentiation induced by high concentration EMPs. Taken together, these data provide a mechanistic framework in which miR-155 delivered by EMPs is involved in aGVHD pathogenesis by activating specific T lymphocytes functions. The results may provide new therapeutic approaches for aGVHD while preserving graft-versus-leukemia (GVL) effect.

Endothelial microparticles mediate inflammation-induced vascular calcification.

Stimulation of endothelial cells (ECs) with TNF-α causes an increase in the expression of bone morphogenetic protein-2 (BMP-2) and the production of endothelial microparticles (EMPs). BMP-2 is known to produce osteogenic differentiation of vascular smooth muscle cells (VSMCs). It was found that EMPs from TNF-α-stimulated endothelial cells (HUVECs) contained a significant amount of BMP-2 and were able to enhance VSMC osteogenesis and calcification. Calcium content was greater in VSMCs exposed to EMPs from TNF-α-treated HUVECs than EMPs from nontreated HUVECs (3.56 ± 0.57 vs. 1.48 ± 0.56 µg/mg protein; P < 0.05). The increase in calcification was accompanied by up-regulation of Cbfa1 (osteogenic transcription factor) and down-regulation of SM22α (VSMC lineage marker). Inhibition of BMP-2 by small interfering RNA reduced the VSMC calcification induced by EMPs from TNF-α-treated HUVECs. Similar osteogenic capability was observed in EMPs from both patients with chronic kidney disease and senescent cells, which also presented a high level of BMP-2 expression. Labeling of EMPs with CellTracker shows that EMPs are phagocytized by VSMCs under all conditions (with or without high phosphate, control, and EMPs from TNF-α-treated HUVECs). Our data suggest that EC damage results in the release of EMPs with a high content of calcium and BMP-2 that are able to induce calcification and osteogenic differentiation of VSMCs.

Annual FEV1 changes and numbers of circulating endothelial microparticles in patients with COPD: a prospective study

ObjectiveGrowing evidence suggests that endothelial injury is involved in the pathophysiology of chronic obstructive pulmonary disease (COPD). Circulating endothelial microparticles (EMPs) increase in patients with COPD because of the presence...

E003 Shear stress modulates endothelial microparticles shedding

Plasma levels of endothelial microparticles (EMPs) are markers of cardiovascular diseases and contribute to the pathogenesis of atherosclerosis. Laminar shear stress (SS) protects against plaque formation contrarily to oscillatory and low SS. We thus investigated whether different flow patterns (laminar, low or static) could affect EMP release. HUVECs were subjected to high and low SS (15, 1.5 dynes /cm 2 ) or no SS (static) for 24 hours. EMPs were isolated from the culture medium and characterized by annexin V labeling using flow cytometry analysis. HUVECs exposed to high SS for 24 hours emitted 4 fold less AnnexinV+ EMPs compared to static (p < 0.001) and 3 fold less compared to low SS (p < 0.01) conditions. This flowdependent EMP shedding was associated with the presence of 1.7 % ± 0.05, 3.75 % ± 0.1, 0.4 % ± 0.06 of TUNEL positive HUVECs in static, low and high conditions, respectively. Treatment of HUVECs with L-NAME (10-4M) significantly increased EMPs in all conditions when compared to untreated cells. Similar EMPs increase was obtained when cells were treated with KT5720, a PKA inhibitor (10-6M) under low and high flow. We further characterized EMPs by determining their surface expression of ICAM-1. ICAM-1 expression on EMPs was significantly increased in low SS and inhibited by high SS when compared to static conditions. Adhesion assays with EMPs-stimulated HUVECs (24hours) increased U937 cells capacity to attach in low SS-EMPs exposed to HUVECs compared to static and high SS EMPs stimulations. This effect was abolished with LFA-pretreated U937 before adhesion suggesting the pro-adhesive properties of low SS-EMPs bearing ICAM-1 at their surface. Altogether, these findings indicate that high SS decreased the thrombotic and adhesive properties of EMPs, which might in part explain their anti-atherogenic effects, whereas low SS induced the shedding of prothrombotic, ICAM-1 positive EMPs, suggesting a novel way by which low SS might affect atherosclerosis.

Association of Endothelial Microparticles (EMP) and Endothelial Progenitors with Serum Cholesterol Levels.

INTRODUCTION: High cholesterol (HC) is known to adversely affect endothelial cells (EC) and has been shown to correlate with decreased levels of circulating endothelial progenitor cells (CEPC). We assayed endothelial microparticles (EMP), a sensitive indicator of EC perturbation, to investigate relations among HC, CEPC, and injury of coronary artery endothelial cells (CAEC), both in vivo and in vitro.METHODS: Twelve subjects with normal cholesterol (150 ±30 mg/dL, control) and 12 with HC (250 ±25) were studied. EMP were assayed by flow cytometry using fluorescent antibodies and CAEC were cultured as previously described [Jimenez et al, Thromb Res 109:175, 2003]. CEPC were isolated, cultured, and assayed for endothelial colony formation (CFU) as described [Hill et al, NEJM 348:593, 2003].RESULTS: Comparing the two groups, EMP measured by CD31+/CD42b− were nearly 2.5-fold elevated in HC as compared to controls (1.7 ±0.5 ×106/mL vs.0.35 ±0.02 ×106/mL; p<0.01). Cholesterol levels correlated well with this measure of EMP (R=0.60, p=0.002). However, no significant correlation was found between CD62E+ EMP and cholesterol levels. Assay of CEPC revealed a nearly 2.5-fold decrease in CFU in HC vs. controls (10 ±2 vs. 25 ±4; p<0.01). In studies in vitro, CEPC from controls were cultured in presence of 20% 0.1μm filtered plasma from members of both groups. The HC group plasma inhibited CEPC colony formation by almost 50% (23 ±3.5 CFU for control plasma vs. 13 ±4 colonies for HC plasma). We next assessed the longer-term effect of HC plasma on CAEC cultures. Six-day culture of CAEC in the presence of 20% plasma resulted in a significant increase of CD31+/CD42b− EMP from CAEC treated with HC plasma vs. normal plasma (6.5 ±0.7 ×106/mL vs. 0.23 ±0.03 ×106/mL; p=0.02).CONCLUSION: These results suggest that EMP are useful markers to monitor cholesterol mediated-EC changes. High EMP levels inversely reflect the vascular endothelial cell regeneration potential due to decreased circulating endothelial progenitor cells.

Endothelial microparticles correlate with high-risk angiographic lesions in acute coronary syndromes

Background: Endothelial Microparticles (EMP) are small fragments of endothelial cell membrane shed during apoptosis or activation. Our group has previously reported elevations of EMP in patients with coronary artery disease (CAD), thrombotic thrombocytopenic purpura (TTP), pre-eclampsia, multiple sclerosis (MS), and severe hypertension (HTN). In the present study, we evaluate the possible relationship between EMP levels and the angiographic severity and characteristics of coronary obstructive lesions. Methods: We studied a total of 43 patients undergoing coronary angiography. Fifteen had presented with acute myocardial infarction (MI), 20 with unstable anginas (UA), 5 with stable angina (SA) and 3 with congestive heart failure. Coronary angiography was reviewed and coronary lesions were classified using the Ambrose classification. Coronary stenoses were classified as high and low risk. High-risk included lesions with eccentric appearance (type II), presence of thrombi, or multiple irregularities. Low-risk lesions were defined as concentric or type I. Lesions were also analyzed by degree of stenosis and history of acute coronary syndrome (ACS). EMP in plasma was assayed by flow cytometry. Results: EMP in eccentric type II or multiple irregular lesions (high-risk) were 2.5-fold higher than in type I or concentric (low-risk) lesions, p<0.05. Lesions with thrombi had three-fold higher EMP than those without ( p=0.05). Mild stenosis (>20%–<45%) had three-fold higher EMP than more severe (>45%), and five-fold higher than those without stenosis ( p<0.01). Among patients with type II lesions, those with first ACS episode had four-fold higher EMP levels than those with recurrent ACS ( p<0.01). Conclusion: High EMP was associated with high-risk angiographic lesions including eccentric type II, multiple irregular, and lesions with thrombi. Mild to moderate stenosis was associated with higher EMP levels than severe stenosis. EMP may be a useful marker in detecting endothelial injury and risk of ACS as defined by angiography.