Increased platelet reactivity is associated with circulating platelet-monocyte complexes and macrophages in human atherosclerotic plaques

Objective: Platelet reactivity, platelet binding to monocytes and monocyte infiltration play a detrimental role in atherosclerotic plaque progression. We investigated whether platelet reactivity was associated with levels of circulating platelet-monocyte complexes (PMCs) and macrophages in human atherosclerotic carotid plaques. Methods: Platelet reactivity was determined by measuring platelet P-selectin expression after platelet stimulation with increasing concentrations of adenosine diphosphate (ADP), in two independent cohorts: the Circulating Cells cohort (n = 244) and the Athero-Express cohort (n = 91). Levels of PMCs were assessed by flow cytometry in blood samples of patients who were scheduled for percutaneous coronary intervention (Circulating Cells cohort). Monocyte infiltration was semi-quantitatively determined by histological examination of atherosclerotic carotid plaques collected during carotid endarterectomy (Athero-Express cohort). Results: We found increased platelet reactivity in patients with high PMCs as compared to patients with low PMCs (median (interquartile range): 4153 (1585-11267) area under the curve (AUC) vs. 9633 (3580-21565) AUC, P<0.001). Also, we observed increased pl

Fractional flow reserve is not associated with inflammatory markers in patients with stable coronary artery disease.

BACKGROUND: Atherosclerosis is an inflammatory condition and increased blood levels of inflammatory biomarkers have been observed in acute coronary syndromes. In addition, high expression of inflammatory markers is associated with worse prognosis of coronary artery disease. The presence and extent of inducible ischemia in patients with stable angina has previously been shown to have strong prognostic value. We hypothesized that evidence of inducible myocardial ischemia by local lesions, as measured by fractional flow reserve (FFR), is associated with increased levels of blood based inflammatory biomarkers. METHODS: Whole blood samples of 89 patients with stable angina pectoris and 16 healthy controls were analyzed. The patients with stable angina pectoris underwent coronary angiography and FFR of all coronary lesions. We analyzed plasma levels of cytokines IL-6, IL-8 and TNF-α and membrane expression of Toll-like receptor 2 and 4, CD11b, CD62L and CD14 on monocytes and granulocytes as markers of inflammation. Furthermore, we quantified the severity of hemodynamically significant coronary artery disease by calculating Functional Syntax Score (FSS), an extension of the Syntax Score. RESULTS: For the majority of biomarkers, we observed lower levels in the healthy control group compared with patients with stable angina who underwent coronary catheterization. We found no difference for any of the selected biomarkers between patients with a positive FFR (≤ 0.75) and negative FFR (>0.80). We observed no relationship between the investigated biomarkers and FSS. CONCLUSION: The presence of local atherosclerotic lesions that result in inducible myocardial ischemia as measured by FFR in patients with stable coronary artery disease is not associated with increased plasma levels of IL-6, IL-8 and TNF-α or increased expression of TLR2 and TLR4, CD11b, CD62L and CD14 on circulating leukocytes

Expression levels of TLR2 and TLR4 on monocytes (A) and granulocytes (B) FFR-positive and FFR-negative patients and healthy control subjects.

<p>*Comparison by Kruskal-Wallis test for all groups.** Between subgroups with Mann Whitney U test. TLR = toll-like receptor.</p

Baseline Characteristics of patients with stable angina.

<p> <i>Continuous values are presented as means ± standard deviation (SD). Categorical values are presented as number (percentages).</i></p>*<p> <i>Significance level 0.05. FFR = fractional flow reserve BMI = body mass index ;DM = diabetes mellitus; MI = myocardial infarction; PCI = percutaneous coronary intervention; CABG = coronary artery bypass grafting; LVEF = left ventricular ejection fraction.</i></p>**<p> <i>Defined as a serum creatinin >150 µmol/l.</i></p

Concentrations of cytokines and surface markers on monocytes and granulocytes according to FSS tertiles and FFR-negative patients.

<p>Concentrations of cytokines (A) and surface markers on monocytes (B) and granulocytes (C) according to FSS tertiles (of FFR-positive patients) and FFR-negative patients. Comparison of all subgroups was performed with the Jonckheere-Terpstra test. FSS = functional syntax score.</p

Comparison of levels of cytokines for FFR-positive and FFR-negative patients and healthy controls.

<p>*Comparison by Kruskal-Wallis test.** Between subgroup differences compared with Mann Whitney U test.</p

Medication use of patients with stable angina at inclusion.

<p> <i>Proportions were compared using Chi-square testing.</i></p>*<p> <i>Significance level 0.05. FFR = fractional flow reserve; ASA = Acetylsalicylic acid; ACE = angiotensin-converting enzyme.</i></p

Toll-Like Receptor Induced CD11b and L-Selectin Response in Patients with Coronary Artery Disease

<div><p>Toll-Like Receptor (TLR) -2 and -4 expression and TLR-induced cytokine response of inflammatory cells are related to atherogenesis and atherosclerotic plaque progression. We examined whether immediate TLR induced changes in CD11b and L-selectin (CD62L) expression are able to discriminate the presence and severity of atherosclerotic disease by exploring single dose whole blood TLR stimulation and detailed dose-response curves. Blood samples were obtained from 125 coronary artery disease (CAD) patients and 28 controls. CD11b and L-selectin expression on CD14+ monocytes was measured after whole blood stimulation with multiple concentrations of the TLR4 ligand LPS (0.01–10 ng/ml) and the TLR2 ligand P3C (0.5–500 ng/ml). Subsequently, dose-response curves were created and the following parameters were calculated: hillslope, EC50, area under the curve (AUC) and delta. These parameters provide information about the maximum response following activation, as well as the minimum trigger required to induce activation and the intensity of the response. CAD patients showed a significantly higher L-selectin, but not CD11b response to TLR ligation than controls after single dose stimulations as well as significant differences in the hillslope and EC50 of the dose-response curves. Within the CAD patient group, dose-response curves of L-selectin showed significant differences in the presence of hypertension, dyslipidemia, coronary occlusion and degree of stenosis, whereas CD11b expression had the strongest discriminating power after single dose stimulation. In conclusion, single dose stimulations and dose-response curves of CD11b and L-selectin expression after TLR stimulation provide diverse but limited information about atherosclerotic disease severity in stable angina patients. However, both single dose stimulation and dose-response curves of LPS-induced L-selectin expression can discriminate between controls and CAD patients.</p> </div

Single dose TLR response in relation to BMI.

<p>CD11b expression after stimulation with P3C 5 ng/ml (left) and P3C 500 ng/ml (right) was significantly higher in patients with a BMI>25 as compared to normal weight patients (BMI<25). Whiskers are presented as 5–95 percentile. Data were statistically tested with a Mann Whitney U test.</p