Atheroprotective Krüppel-like factor 4 is downregulated in monocyte subsets of patients with coronary artery disease.

doi:10.1160/TH13-05-0367 Thromb Haemost 2013; 110: 1080–1082 Dear Sirs, The zinc finger transcription factor Krüppel-like factor 4 (KLF4) plays an important role in the regulation of cellular functions, including proliferation, differentiation or activation (1). Circulating monocytes, which give rise to tissue-resident macrophages, are essential cellular mediators of atherosclerosis (2). Recently, it could be demonstrated that KLF4-deficient macrophages adopt a proinflammatory phenotype (3, 4). Moreover, myeloid KLF4 deficiency in mice was associated with increased vascular inflammation and atherosclerotic lesion formation (4). Human monocytes can be divided into so-called classical CD14++CD16and CD16+ monocytes, the latter consisting of intermediate CD14++CD16+ and non-classical CD14(+)CD16+ monocytes (2). Pro-atherogenic properties are mostly attributed to CD16+ monocytes (5), however, CD14++CD16monocytes also were found to be increased in individuals with cardiovascular risk factors in some studies (2). So far, the expression of KLF4 in human monocyte subsets and its relation to atherosclerosis has not been investigated. Therefore, we recruited 52 patients with stable, angiographically confirmed coronary artery disease (CAD; 77% male; median age, 69.5 years; median body mass index 27.1 kg/m2; median blood leukocyte count, 6.7x103/μl; median high-sensitivity C-reactive protein blood level, 2.60 mg/l) at the Department of Cardiology and Pulmonary Medicine and analysed KLF4 expression in their monocyte subsets. Their results were compared to healthy controls without any cardiovascular risk factors (CONTR; n=18; 78% male, p=not significant; 57.5 years, p<0.001; body-mass-index 25.2 kg/m2, p=not significant; leukocyte count, 5.6x103/μl, p<0.05; high-sensitivity C-reactive protein, 0.77 mg/l, p<0.01) which were recruited at the Department of Transfusion Medicine. As these individuals did not undergo cardiac catherisation, we cannot exclude the presence of clinically silent cardiovascular disease in CONTR subjects. Persons with an active infection, malignancy, nephropathy or acute (within the last 2 months) coronary syndrome were excluded. Informed consent was obtained according to the requirements of the local ethics committee. Mononuclear cells were isolated from heparinised peripheral venous blood by Histopaque® (Sigma-Aldrich, St. Louis, MO; USA) density gradient centrifugation. Subsequently, cells were stained with fluorescein isothiocyanate (FITC)-conjugated human CD14 (clone MφP9; BD) and phycoerythrin (PE)-conjugated CD16 (clone 3G8; BD) antibodies, or their respective isotype controls. Next, cells were permeabilised by Flow Cytometry Permeabilization Buffer/Wash Buffer I (R&D Systems, Indianapolis, IN; USA) and then stained with an allophycocyanin (APC)-conjugated human KLF4 antibody (R&D Systems), or its respective isotype control. Cells were investigated on a BD FACS CantoTM II flow cytometer at three wavelengths and analysed using BD FACS DivaTM software. After gating monocytes according to their forward and sideward scatter profiles, cells were subdivided into CD14++CD16-, CD14++CD16+ and CD14(+)CD16+ monocytes and further examined with regard to their KLF4 expression. Moreover, tumour necrosis factor (TNF)-α plasma levels were measured in duplicate using a commercial high sensitivity enzyme-linked immunosorbent assay (eBioscience, San Diego, CA, USA), according to the manufacturer’s instructions. Comparisons were performed using the non-parametric Mann-Whitney U or Kruskal-Wallis tests, the latter followed by Dunn’s multiple comparison test. All statistical tests were two-sided and a probability (p) value of <0.05 was considered significant. The median percentage of circulating CD14++CD16monocytes was 84%, of CD14++CD16+ monocytes 11% and of CD14(+)CD16+ monocytes 5.4% in the CONTR subjects, and 84%, 11% and 4.7%, Letters to the Editor