Critical limb ischaemia is characterised by an increased production of whole blood reactive oxygen species and expression of TREM-1 on neutrophils.

BACKGROUND: Atherosclerosis is a chronic inflammatory process involving polymorphonuclear neutrophils (PMN) and formation of reactive oxygen species (ROS). The aim of the present study was to investigate the phenotype of inflammatory cells in regard to the expression of triggering receptor expressed on myeloid cells (TREM)-1 and its soluble form (sTREM-1) as well as its relationship with oxidative stress in peripheral artery disease (PAD) patients. METHODS: In total 90 patients with PAD (N = 30 intermittent claudication (IC) > 300 m absolute walking distance, N = 30 IC < 300 m absolute walking distance, N = 30 critical limb ischaemia (CLI)) and 30 control persons were included. ROS formation was measured at basal or stimulated conditions using the luminol analogue L-012 chemiluminescence. Peripheral blood leucocytes were analysed from whole blood by flow cytometry using different gating strategies to identify PMN and monocytes and analyse TREM-1 expression. RESULTS: CLI patients showed a significant higher ROS production at basal levels (p < 0.05) and upon stimulation with PDBu (p < 0.0001), LPS (p < 0.05) and zymosan A (p < 0.0001). TREM-1 was expressed significantly more on PMN of CLI patients (p < 0.01) in comparison to all other groups, whereas monocytic expression of TREM-1 was similar between all 4 groups. The serum concentration of its soluble form sTREM-1 however was increased in CLI and IC < 300 m patients (p < 0.0001). sTREM-1 concentrations correlated with basal ROS levels as wells with ROS production upon stimulation. Furthermore, we found the walking distance of IC patients to inversely correlate with sTREM-1 (rs = - 0.29; p = 0.03). CONCLUSIONS: We found an increased oxidative stress as well as an increased expression of TREM-1 and serum levels of sTREM-1 in patients with CLI. IC < 300 m patients showed a similar patter in regard to oxidative stress, TREM-1 expression and sTREM-1 concentration. Thus, sTREM-1 might represent a potential inflammatory biomarker to evaluate the severity of PAD. Whether this implies the potential for therapeutic recommendations, i.e. conservative vs. interventional/operative treatment, or a possibility to monitor the efficacy of interventions, requires further studies.

Phenotypic characterisation of pro-inflammatory monocytes and dendritic cells in peripheral arterial disease.

Atherosclerosis is a chronic inflammatory process involving antigen-presenting cells like monocytes and dendritic cells (DC). The aim of this study was to perform a phenotypic characterisation of these cell types in patients with different degrees of peripheral arterial disease (PAD). Sixty patients with PAD [N= 30 intermittent claudication (IC), N= 30 critical limb ischemia (CLI)] and 30 controls were included. Peripheral blood leucocytes were analysed from peripheral blood by flow cytometry using different gating strategies to directly identify and analyse monocytes, myeloid DC, (mDC) and plasmacytoid DC (pDC). PAD patients showed a significantly higher proportion of proinflammatory CD14++CD16+ monocytes (p<0.0001) compared with healthy individuals. We found an increased number of mDC/ml and a reduced number of pDC/ml (both p<0.01) in PAD patients, leading to a shift in the mDC/pDC ratio (p<0.01). As compared to patients with intermittent claudication, CLI patients presented a reduced expression of HLA-DR (p<0.01), CD86 and CD40 on both mDCs and pDCs (p<0.01). Peripheral blood monocytes show a proinflammatory phenotype in PAD patients compared to controls. In contrast, CLI patients show a reduced expression of proinflammatory markers. We hypothesise that severe ischaemia and/or prolonged inflammation in CLI might lead to a paradoxical attenuation in the proinflammatory membrane pattern of circulating mononuclear cells, possibly hindering an adequate regulatory function of mDCs and pDCs and favouring the progression of disease.

Endothelial function and hemorheological parameters modulate coronary blood flow in patients without significant coronary artery disease.

BACKGROUND: Coronary (micro)vascular resistance is regulated by the complex interplay of several factors. Two potentially important determinants include endothelial function and the rheological properties of blood. However, their impact on the control of the coronary resistance vasculature is poorly understood. METHODS: The corrected Thrombolysis In Myocardial Infarction frame count (TIMIfc, an index of coronary flow velocity), conduit artery endothelial function, intima-media thickness of the common carotid artery and complete blood counts were measured in 145 patients undergoing elective coronary angiography. Patients with obstructive coronary artery disease or systemic conditions thought to be associated with microvascular disease were excluded from the analysis. RESULTS: There was a strong correlation between the TIMIfc measured in the three main coronary artery distributions (R values between 0.71 and 0.85, P < 0.00001). The TIMIfc was higher in males (P < 0.05), but there was no association with traditional risk factors for coronary artery disease (all P > 0.1). There was a correlation between TIMIfc and L-FMC, a parameter of resting endothelial function (R = 0.33, P < 0.0005). TIMIfc also correlated with mean platelet volume (a marker of platelet activation, R = 0.33, P < 0.001), and hematocrit (R = 0.33, P = 0.0002). There was no correlation between TIMIfc and carotid intima-media thickness and the degree of coronary atherosclerosis. Logistic regression analysis showed that L-FMC and hemorheological variables may explain as much as 19% of the variability in TIMIfc. CONCLUSIONS: Resting peripheral endothelial function, as well as parameters of platelet function, correlate with coronary TIMIfc. These data emphasize the existence of an association between endothelial function, hemorheological variables and coronary blood flow velocity.

Endothelial function assessment: flow-mediated dilation and constriction provide different and complementary information on the presence of coronary artery disease.

AIMS: A number of risk factors for atherosclerosis have been identified, but it remains difficult, on an individual patient basis, to predict how these factors interact in determining the development of coronary artery disease (CAD). It also remains unclear whether the study of endothelial function provides information that is additive to that of traditional risk factors. METHODS AND RESULTS: Flow-mediated dilation (FMD) and low-flow-mediated constriction (L-FMC) were measured in 451 consecutive patients before coronary angiography. Low-flow-mediated constriction (P< 0.0001) and FMD (P=0.0005) progressively decreased with the number of diseased vessels, and L-FMC showed a significant linear correlation with the SYNTAX score (R=0.38; P< 0.0001). Logistic regression analysis confirmed the association between endothelial function parameters and CAD (P=0.001 for L-FMC, P=0.02 for FMD). Receiver operating characteristic analysis demonstrated that the addition of L-FMC alone and of the combination of FMD and L-FMC improved the predictive power of a model based on traditional risk factors for CAD (area under the curve of the risk factor model=0.716; risk factor model + FMD=0.734, P=0.1 compared with risk factor model; risk factor model + L-FMC=0.771, P=0.004; risk factor model + L-FMC + FMD=0.779, P=0.002). Reclassification statistics showed that the introduction of FMD to the model based on the traditional risk factors correctly reclassified an additional 5% of patients, and that the introduction of L-FMC net correctly reclassified 19% of the patients. There was no correlation between different parameters of endothelial function. CONCLUSION: Endothelial function assessment provides modest but statistically significant additional information in predicting the presence of CAD.

Peripheral hemorheological and vascular correlates of coronary blood flow.

The slow coronary flow phenomenon (SCF), a condition described by the presence of inappropriate delay in the progression of intracoronary contrast during angiography in the absence of stenoses, has been shown in some patients presenting with chest pain. While several conditions leading to "secondary" slow flow are known, there are no definitive conclusions regarding the exact pathogenesis of "primary" SCF. The present paper outlines the mechanisms that may lead to SCF, emphasizing the role of hemorheological and vascular factors in the pathogenesis of this phenomenon. Small vessel dysfunction has been proposed in the pathogenesis of SCF since the first description of this syndrome in 1972. Abnormalities in coronary microvascular function result from increased microvascular resistances and impaired endothelial release of vasoactive substances, especially in production and bioavailability of endothelium derived NO. Inflammatory conditions (increased levels of C-reactive protein, interleukin-6 and adhesion molecules) and metabolic abnormalities such as impaired glycemic control, hyperuricemia and elevated serum gamma-glutamyltransferase were also found to contribute to microvascular dysfunction in patients with SCF. New studies have also indicated that increased blood viscosity and one of its major determinants, erythrocyte aggregation, is associated with the SCF. Rheological variables play a role in the control of shear stress and contribute to blood flow velocity changes. Although platelets do not have a significant influence on blood viscosity, it has been demonstrated that they are involved in the development of SCF. Increased mean platelet volume (MPV), an indicator of platelet activation and platelet aggregability is also significantly higher in patients with SCF compared with patients with normal coronary flow.

Endothelial functions: Translating theory into clinical application.

The vascular endothelium plays a pivotal role in modulating endothelial homeostasis. A number of methods have been developed to assess the function of this important tissue in humans in vivo, in the hope that such data may contribute to the early diagnosis and risk stratification of patients at risk for, or with, cardiovascular disease. Despite these efforts, a number of issues, both practical and theoretical, arise from the attempt of quantifying the elusive "endothelial function", and from the attempt of defining what is "endothelial dysfunction". The present paper, based on a lecture held at the conference of the European Society of Hemorheology and Microcirculation, will try to deal with these issues.