Microvesicles (MVs) which include microparticles (MPs) and exosomes are found in blood circulation in normal physiologic conditions and are increased in a variety of diseases. This study evaluated the effects of MVs on human umbilical vein endothelial cells (HUVEC) by morphologic changes, apoptosis, and thrombogenicty, in vitro. Stimulation of monocyte cell line (THP-1) by starvation or by endotoxin and calcium ionophore A23187 resulted in the release of MVs which express exosome marker Tsg 101, negative phospholipids in their leaflets, monocyte markers (CD18, CD14) and active tissue factor (TF). MVs were found to disrupt EC integrity and rapidly induce membrane blebbing. Brief exposure (2–4 hours) to MVs resulted in EC membrane phospholipids “flip-flop” while longer stimulation (20 hours) led to two contradicting outcomes – tube formation as well as apoptosis, as assessed by nuclear fragmentation. Additionally, MVs exposure resulted in increased cell surface thrombogenicity and perturbation of the endothelial haemostatic balance, which were enhanced during longer exposure time. Activity, antigen level and mRNA expression of the coagulation initiator TF were elevated due to (i) adherence of MVs derived TF to the EC membrane, and (ii) an increase in endothelial TF expression. Furthermore, levels of the anticoagulant tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) were decreased. These findings demonstrate that monocyte MVs increase endothelial thrombogenicity and apoptosis. In addition, they induce tube formation which may indicate their angiogenic effect. These findings may clarify, in part, the role of MVs in EC dysfunction associated with inflammatory diseases and hypercoagulable states.

Summary: Type 2 diabetes mellitus (T2DM) is associated with increased coagulability and vascular complications. Circulating microparticles (MPs) are involved in thrombosis, inflammation, and angiogenesis. However, the role of MPs in T2DM vascular complications is unclear. We characterised the cell origin and pro-coagulant profiles of MPs obtained from 41 healthy controls and 123 T2DM patients with coronary artery disease, retinopathy and foot ulcers. The effects of MPs on endothelial cell coagulability and tube formation were evaluated. Patients with severe diabetic foot ulcers expressed the highest levels of MPs originated from platelet and endothelial cells and negatively-charged phospholipidbearing MPs. MP coagulability, calculated from MP tissue factor (TF) and TF pathway inhibitor (TFPI) ratio, was low in healthy controls and in diabetic retinopathy patients (1.8, p≥0.002). MPs of all T2DM patients induced a more than two-fold increase in endothelial cell TF (antigen and gene expression) but did not affect TFPI levels. Tube networks were longest and most stable in endothelial cells that were incubated with MPs of healthy controls, whereas no tube formation occurred in MPs of diabetic patients with coronary artery disease. MPs of diabetic retinopathy and diabetic foot ulcer patients induced branched tube networks that were unstable and collapsed over time. This study demonstrates that MP characteristics are related to the specific type of vascular complications and may serve as a bio-marker for the procoagulant state and vascular pathology in patients with T2DM.

The function of endothelial progenitor cells (EPCs), which are key cells in vascular repair, is impaired in diabetes mellitus. Nitric oxide (NO) and reactive oxygen species can regulate EPC functions. EPCs tolerate oxidative stress by upregulating superoxide dismutase (SOD), the enzyme that neutralizes superoxide anion (O2-). Therefore, we investigated the roles of NO and SOD in glucose-stressed EPCs.

Microvesicles shedding from cell membrane affect inflammation, apoptosis, and angiogenesis. We hypothesize that microvesicles of women with gestational vascular complications reflect pathophysiological state of the patients and affect their endothelial and trophoblast cell function. Microvesicles of healthy pregnant women, women with gestational hypertension, mild, or severe preeclampsia/toxemia, were characterized, and their effects on early-stage or term trophoblasts and endothelial cells were evaluated using apoptosis, migration, and tube formation assays. Patient subgroups differed significantly only in proteinuria levels, therefore their microvesicles were assessed as 1 group, demonstrating higher levels of inflammatory and angiogenic proteins compared with those of healthy pregnant women. In endothelial cells, microvesicles of healthy pregnant women reduced caspase 3/7 activity, increased migration, and induced tube formation. These processes were suppressed by microvesicles of women with gestational vascular complications. In early-stage trophoblasts, microvesicles of healthy pregnant women decreased apoptosis compared with untreated cells (6±5% versus 13.8±5.8%; P <0.001) and caspase 3/7 activity and induced higher migration (39.7±10.1 versus 20.3±8.3 mm ² ; P <0.001). This effect was mediated through extracellular signal-regulated kinase pathway. Conversely, microvesicles of women with gestational vascular complications increased term trophoblast apoptosis compared with cells exposed to microvesicles of healthy pregnant women (15.1±3.3% versus 6.5±2.1%; P <0.001) and inhibited early-stage trophoblasts migration (21.4±18.5 versus 39.7±10.1 mm ² ; P <0.001). In conclusion, microvesicle content and effects on endothelial and trophoblast cells vary according to the physiological/pathological state of a pregnant woman. Microvesicles seem to play a pivotal role in the course of pregnancy, which could potentially result in gestational vascular complications.