Endothelial Microparticles (EMP) for the Assessment of Endothelial Function: An In Vitro and In Vivo Study on Possible Interference of Plasma Lipids

BACKGROUND: Circulating endothelial microparticles (EMP) reflect the condition of the endothelium and are of increasing interest in cardiovascular and inflammatory diseases. Recently, increased numbers of EMP following oral fat intake, possibly due to acute endothelial injury, have been reported. On the other hand, the direct interference of lipids with the detection of EMP has been suggested. This study aimed to investigate the effect of lipid-rich solutions, commonly administered in clinical practice, on the detection, both in vitro and in vivo, of EMP. METHODS: For the in vitro assessment, several lipid-rich solutions were added to whole blood of healthy subjects (n = 8) and patients with coronary heart disease (n = 5). EMP (CD31+/CD42b-) were detected in platelet poor plasma by flow cytometry. For the in vivo study, healthy volunteers were evaluated on 3 different study-days: baseline evaluation, following lipid infusion and after a NaCl infusion. EMP quantification, lipid measurements and peripheral arterial tonometry were performed on each day. RESULTS: Both in vitro addition and in vivo administration of lipids significantly decreased EMP (from 198.6 to 53.0 and from 272.6 to 90.6/µl PPP, respectively, p = 0.001 and p = 0.012). The EMP number correlated inversely with the concentration of triglycerides, both in vitro and in vivo (r = -0.707 and -0.589, p<0.001 and p = 0.021, respectively). The validity of EMP as a marker of endothelial function is supported by their inverse relationship with the reactive hyperemia index (r = -0.758, p = 0.011). This inverse relation was confounded by the intravenous administration of lipids. CONCLUSION: The confounding effect of high circulating levels of lipids, commonly found in patients that receive intravenous lipid-based solutions, should be taken into account when flow cytometry is used to quantify EMP

The Endothelium, A Protagonist in the Pathophysiology of Critical Illness: Focus on Cellular Markers

The endotheliumis key in the pathophysiology of numerous diseases as a result of its precarious function in the regulation of tissue homeostasis. Therefore, its clinical evaluation providing diagnostic and prognostic markers, as well as its role as a therapeutic target, is the focus of intense research in patientswith severe illnesses. In the critically ill with sepsis and acute brain injury, the endothelium has a cardinal function in the development of organ failure and secondary ischemia, respectively. Cellular markers of endothelial function such as endothelial progenitor cells (EPC) and endothelialmicroparticles (EMP) are gaining interest as biomarkers due to their accessibility, although the lack of standardization of EPC and EMP detection remains a drawback for their routine clinical use. In this paper we will review data available on EPC, as a general marker of endothelial repair, and EMP as an equivalent of damage in critical illnesses, in particular sepsis and acute brain injury. Their determination has resulted in new insights into endothelial dysfunction in the critically ill. It remains speculative whether their determination might guide therapy in these devastating acute disorders in the near future

Endothelium dependent vasomotion and in vitro markers of endothelial repair in patients with severe sepsis: an observational study.

BACKGROUND: Outcome in sepsis is mainly defined by the degree of organ failure, for which endothelial dysfunction at the macro- and microvascular level is an important determinant. In this study we evaluated endothelial function in patients with severe sepsis using cellular endothelial markers and in vivo assessment of reactive hyperaemia. MATERIALS AND METHODS: Patients with severe sepsis (n = 30) and 15 age- and gender- matched healthy volunteers were included in this study. Using flow cytometry, CD34+/KDR+ endothelial progenitor cells (EPC), CD31+ T-cells, and CD31+/CD42b- endothelial microparticles (EMP) were enumerated. Migratory capacity of cultured circulating angiogenic cells (CAC) was assessed in vitro. Endothelial function was determined using peripheral arterial tonometry at the fingertip. RESULTS: In patients with severe sepsis, a lower number of EPC, CD31+ T-cells and a decreased migratory capacity of CAC coincided with a blunted reactive hyperaemia response compared to healthy subjects. The number of EMP, on the other hand, did not differ. The presence of organ failure at admission (SOFA score) was inversely related with the number of CD31+ T-cells. Furthermore, the number of EPC at admission was decreased in patients with progressive organ failure within the first week. CONCLUSION: In patients with severe sepsis, in vivo measured endothelial dysfunction coincides with lower numbers and reduced function of circulating cells implicated in endothelial repair. Our results suggest that cellular markers of endothelial repair might be valuable in the assessment and evolution of organ dysfunction

Baseline characteristics of the severe sepsis patients (n = 30).

<p>BMI = body mass index; ICU =  intensive care unit; MAP =  Mean arterial pressure; MII =  Modified Inotropic Index; POD = persistent organ failure; SAPS =  Simplified Acute Physiology; SOFA =  Sequential of Organ Failure Assessment; VDI =  Vasopressor Dependency Index; h =  hour; d =  day.</p><p>Data presented as mean ± SEM or as number (percentage).</p

Outline of the study.

<p>Flow chart representing the timeline used for measurements and assessment of disease severity.CAC =  circulating angiogenic cell, EMP =  endothelial microparticle, EPC =  endothelial progenitor cell, RHI =  reactive hyperaemia, SAPS =  Simplified Acute Physiology; SOFA =  Sequential of Organ Failure Assessment.</p

Comparison of markers of endothelial function between patients with and those without progression of organ failure in the first week.

<p>CAC =  circulating angiogenic cell, EPC =  endothelial progenitor cell; EMP =  endothelial microparticle; PBMC =  peripheral blood mononuclear cells; RHI = reactive hyperaemia index, SAPS =  Simplified Acute Physiology; SOFA =  Sequential of Organ Failure Assessment.</p><p>Data presented as mean ± SEM or as number (percentage).</p>*<p>n =  number of patients analysed with progression and no progression of organ failure respectively.</p

Decrease in EMP after in vitro administration of lipid-rich solutions.

<p>Whole blood was taken from 8 healthy volunteers (black) and 5 patients with coronary heart disease (red). PPP was prepared from different aliquots to which lipid-rich solutions were added in different concentrations. EMP were detected by flow cytometry as particles <1 µm and CD31+/CD42b−. The figure shows EMP numbers/µl PPP from samples without lipid-rich solutions (PPP), and samples with added lipid-rich solutions in the lowest concentration (Lipid). The number of EMP detected by flow cytometry decreased significantly (all data taken together p = 0.001). EMP = endothelial microparticles, PPP = platelet poor plasma.</p

In vivo study: results for the different study-days.

<p>Results are expressed as median (min-max).</p><p>*Friedman test p<0.05 and Wilcoxon signed rank test Day C vs. Day B and Day C vs. Day A p<0.05.</p><p>5 healthy volunteers were evaluated on 3 different study days. <u>Day A</u>: blood was collected after an overnight fast and PAT was performed. EMP were detected by flow cytometry as particles <1 µm and CD31+/CD42b− in PPP. <u>Day B</u>: a NaCl 0.9% infusion was administered in fasting conditions and the same measurements as on day A were performed. <u>Day C</u>: the same protocol was used as on day B, but a pure lipid solution was infused instead of NaCl.</p><p>CH = cholesterol, chyl = chylomicrons, EMP = endothelial microparticles, HDL = High density lipoproteins, LDL = low density lipoproteins, PAT = peripheral arterial tonometry, RHI = reactive hyperemia index, TG = triglycerides, VLDL = very low density lipoproteins.</p

Characteristics of cardiovascular patients (n = 5).

<p>CV disease = cardiovascular disease.</p