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Cardiovascular status after Kawasaki disease in the UK

Objective Kawasaki disease (KD) is an acute vasculitis that causes coronary artery aneurysms (CAA) in young children. Previous studies have emphasised poor long-term outcomes for those with severe CAA. Little is known about the fate of those without CAA or patients with regressed CAA. We aimed to study long-term cardiovascular status after KD by examining the relationship between coronary artery (CA) status, endothelial injury, systemic inflammatory markers, cardiovascular risk factors (CRF), pulse-wave velocity (PWV) and carotid intima media thickness (cIMT) after KD. Methods Circulating endothelial cells (CECs), endothelial microparticles (EMPs), soluble cell-adhesion molecules cytokines, CRF, PWV and cIMT were compared between patients with KD and healthy controls (HC). CA status of the patients with KD was classified as CAA present (CAA+) or absent (CAA−) according to their worst-ever CA status. Data are median (range). Results Ninety-two KD subjects were studied, aged 11.9 years (4.3–32.2), 8.3 years (1.0–30.7) from KD diagnosis. 54 (59%) were CAA−, and 38 (41%) were CAA+. There were 51 demographically similar HC. Patients with KD had higher CECs than HC (p=0.00003), most evident in the CAA+ group (p=0.00009), but also higher in the CAA− group than HC (p=0.0010). Patients with persistent CAA had the highest CECs, but even those with regressed CAA had higher CECs than HC (p=0.011). CD105 EMPs were also higher in the KD group versus HC (p=0.04), particularly in the CAA+ group (p=0.02), with similar findings for soluble vascular cell adhesion molecule 1 and soluble intercellular adhesion molecule 1. There was no difference in PWV, cIMT, CRF or in markers of systemic inflammation in the patients with KD (CAA+ or CAA−) compared with HC. Conclusions Markers of endothelial injury persist for years after KD, including in a subset of patients without CAA.

factor (VEGF), angiopoietin 1 and 2, soluble E-selectin (sE-sel), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble P selectin (sP-Sel), and thrombomodulin (TM) were assessed using a multi-array detection system based on electro-chemiluminescence technology (SECTOR Imager 2400, MesoScale Discovery; see supplementary methods). In brief, this system uses multi-array plates fitted with multi-electrodes per well with each electrode being coated with a different catching antibody. The assay procedure then follows that of a classic sandwich ELISA with the analytes of interest captured on the relevant electrode. These captured analytes were then in turn detected by a secondary analyte-specific ruthenium-conjugated antibody, which is capable of emitting light after electrochemical stimulation.
A particular advantage of this system is the ability to simultaneously measure different biomarkers in small (25ul or 50uL) serum or plasma samples. Tissue Factor (TF) was measured by sandwich enzyme immunoassay using a commercially available kit from R & D Systems, Europe Ltd, (Abingdon, UK).

Circulating endothelial cells
Venous blood (1 ml) collected into tubes containing EDTA was mixed with buffer (1 ml of phosphate buffered saline containing 0.1% bovine serum albumin and 0.6% sodium citrate) and 20 μl of Fc receptor-blocking reagent (Miltenyi Biotec) and incubated for 5 minutes at room temperature. Fifty microliters of a preparation of anti-CD146-coated immunomagnetic beads (clone S-endo-1; BioCytex and Dynal Biotech) was added, and the sample was incubated at 4°C for 30 minutes, with rotation.
Bead-bound cells were separated using a magnet (MPC-L; Dynal Biotech) and washed 3 times with buffer. Cells were then resuspended in 100 μl of buffer containing 10 μl of a 2-mg/ml preparation of FITC-labeled Ulex europaeus lectin (Sigma-Aldrich) and incubated for 1 hour at room temperature in the dark. CECs in the sample were counted using a Nageotte chamber on a fluorescence microscope by an experienced scientist (VS) blinded to the study subject status. CECs were defined as Ulex bright cells that were >10 μm in size, with >5 magnetic beads attached.

Endothelial microparticles (EMPs)
Blood was collected in 3.2% buffered citrate and centrifuged at 5000 g for 5 min twice to obtain

Vascular stiffness and carotid intima media thickness
Carotid-femoral and carotid-radial pulse wave velocity (PWV) was assessed by a trained investigator (VS) using the Vicorder device (Skidmore Medical Limited) as per manufacturer instructions, and in accordance with American heart Association recommendations (23).
The technique most widely used to measure arterial stiffness is the determination of arterial pulse wave velocity (PWV). PWV is the speed of travel of the pulse along an arterial segment. Carotidfemoral PWV is a direct measurement, and it corresponds to the widely accepted propagative model of the arterial system. However, carotid-radial PWV may also provide other relevant information of arterial stiffness. For this study, PWV measurements were obtained using the Vicorder device (Skidmore Medical Devices) by placing a 100 mm wide blood pressure cuff around the upper thigh to measure the femoral pulse, and a 30mm partial cuff around the neck at the level of the carotid artery. High quality waveforms were recorded simultaneously for 3 seconds with the subject in the supine position, and the foot-to-foot transit time was determined using an in-built cross-correlation algorithm centered around the peak of the second derivative of pressure. For carotid to femoral PWV, path length was defined as the distance from the suprasternal notch to the middle of the thigh cuff as indicated by the manufacturer (mm). The measurement from the suprasternal notch to the umbilicus and then to the middle of the cuff was recorded, in addition. The distance from the suprasternal notch diagonally to the middle of the neck cuff was also recorded as a separate measurement. For carotid to radial PWV, path length was defined from the distance from the suprasternal notch to the middle of the radial cuff.
Measurement of far wall carotid intima-media thickness (cIMT) with B-mode ultrasound is a noninvasive and reproducible technique for identifying and quantifying vascular disease and for evaluating cardiovascular risk. For this study, experienced vascular technicians carried out all cIMT measurements following a standardised imaging protocol (see acknowledgments). The Zonare ultrasound scanner (Zonare Medical System) with a high resolution probe, was used to image both the right (RCCA) and left (LCCA) common carotid arteries longitudinally 1cm proximal to the carotid bifurcation. Images were focussed on the posterior (far) wall of the artery and the zoom function was used to magnify the area. Ten second cineloops were recorded in DICOM format and downloaded for offline analysis. Three end-diastolic frames were selected and analysed for mean cIMT, defined as the interface between lumen-intima and media-adventitia, for both right and left carotid arteries using an automated carotid analyser (Carotid Analyser, M.I.A). The images were analysed by accredited readers and the mean of both the left and right-sided readings was used for the analysis.