Microparticle formation after co‐culture of human whole blood and umbilical artery in a novel in vitro model of flow

Abstract

Cardiovascular disease (CVD) is now the largest killer in western society, and the importance of interactions between vascular endothelium and circulating blood components in disease pathogenesis is well established. Microparticles are a heterogeneous population of \1 lm blood borne particles that arise from blebbing or shedding of cell membranes. The microparticle population includes several classes of apoptotic bodies; however, increased numbers of procoagulant microparticles have been described in plasma from people with CVD. We have previously demonstrated that interactions of monocytes and platelets with isolated inflamed endothelial cells lead to production of pro-coagulant tissue factor bearing microparticles under laminar flow conditions. Here we have investigated microparticle production after perfusion of human whole blood through intact inflamed human umbilical artery. When blood was perfused through umbilical arteries which had been pre-stimulated with tumour necrosis factor (TNFa) for 18 h under flow conditions, there was significantly increased production of microparticles from both platelet and non-platelet sources, in particular from erythrocytes. To determine whether microparticles generated during interactions with inflamed endothelium could induce a pro-inflammatory response in trans, we isolated microparticles by centrifugation after co-culture and incubated with isolated quiescent endothelial cells followed by measurement of reactive oxygen species formation. Microparticles derived from co-culture with inflamed endothelium induced significantly enhanced levels of reactive oxygen species (ROS). These data suggest that presence of an inflamed endothelium causes release of pro-inflammatory microparticles from circulating blood cells, which could contribute to prolonged endothelial activation and subsequent atherosclerotic changes in blood vessels subjected to inflammatory insult. ' 2011 International Society for Advancement of Cytometry

Liraglutide mitigates TNF‐α induced pro‐atherogenic changes and microvesicle release in HUVEC from diabetic women

“…At the basal state and after stimulations, the culture media of C‐HUVEC and GD‐HUVEC was collected. For each sample, 100 μL of supernatant was added to 200 μL of PBS and processed by a common flow cytometry no‐lyse and no‐wash method . Briefly, samples were stained using a cocktail of reagents (only direct immunolabelling was performed, in order to avoid immune complex formation), as described in Table .…”

“…For each sample, 100 μL of supernatant was added to 200 μL of PBS and processed by a common flow cytometry no-lyse and no-wash method. 19 Briefly, samples were stained using a cocktail of reagents (only direct immunolabelling was performed, in order to avoid immune complex formation), as described in Table 1. After 30 minutes of staining (4°C in the dark), 500 μL of PBS was added to each tube, and 1 × 10 6 events/sample were acquired by flow cytometry (FACSVerse, BD-3 laser, 8 colour dilutions were established based on achieving the highest signal (mean fluorescence intensity, MFI) for the positive population and the lowest signal for the negative population, representing the optimal signal to noise ratio, 21 and stain indexes were calculated.…”

Evolving Role of Microparticles in the Pathophysiology of Endothelial Dysfunction

“…Also, inflamed endothelium cells alone can cause the release of proinflammatory MPs from circulating blood cells. This release of MPs could then contribute to prolonged endothelial activation and atherosclerotic changes in blood vessels subjected to inflammatory insult (43 ).…”

Nitric oxide, hemolysis, and the red blood cell storage lesion: interactions between transfusion, donor, and recipient

“…Elevated levels of reactive oxygen species in the patient's circulation can compromise RBC membrane integrity via oxidation leading to changes in rheologic properties and possibly hemolysis. Likewise, increased expression of endothelial adhesion molecules, related to systemic inflammatory or oxidative stress, can increase RBC‐endothelial shear and hemolysis 18 . In addition, drug treatments could affect the survival of transfused RBCs in the circulation, as some xenobiotics, such as phenylhydrazine or aniline, are redox‐active agents that are capable of inducing hemolysis through oxidative denaturation of the Hb 19,20 …”

“…Interestingly, Holtom and colleagues 18 have demonstrated enhanced formation of RBC‐derived membrane microparticles in response to incubation of fresh whole blood with inflamed endothelium under flow. These studies revealed the capacity of inflamed endothelial cells to induce membrane damage in fresh RBCs.…”