Background Kawasaki disease (KD) is now the most common cause of acquired cardiac disease in children due to permanent coronary artery damage with unknown etiology. observations were verified using miR\223 knockout mice and the chimeric mice generated by transplantation of bone marrow from miR\223 knockout mice into wild\type mice. Conclusions In KD Tegaserod maleate IC50 patients, the levels of blood cellCderived miR\223 in ECs are significantly increased. The increased miR\223 in ECs could work as a novel endocrine genetic signal and participate in vascular injury of KD. MiR\223 may provide a novel mechanism and a new therapeutic target for vascular complication of KD. for 15?minutes at 4C. This technique removed all cells, but the microparticles or exosomes remained; removal of these smaller entities requires centrifuging at a higher speed for a longer time, as we showed previously.6, 7, 12 The levels of miR\223 in collected medium were determined and were increased with the increased THP\1 macrophages. 6 The medium collected after 12?hours of THP\1 macrophage culture was added into the cultured medium of ECs. After 12?hours, levels of miR\223 in ECs were determined. Western Blot Analysis Proteins were isolated from cultured ECs, and vessels were determined by Western blot analysis using antibodies. IGF1R antibody was from Cell Signaling Technology. Tegaserod maleate IC50 GAPDH antibody (1:2000 dilution; Cell Signaling Technology) was used as a loading control. Cell Proliferation and Apoptosis EC proliferation was induced by platelet\derived growth factor (20?ng/mL) and was determined by MTT assay (Roche) and 5\ethynyl\2\deoxyuridine assay (EdU kit; RiboBio).16 EC apoptosis in cultured cells was induced by H2O2 (100?mol/L) for 24?hours and measured by terminal deoxynucleotidyl transferase dUTP nick end labeling assay (Roche).16 Luciferase Assay The reporter plasmid, a firefly luciferase reporter construct (psiCHECK\2; Promega), was inserted in a fragment of the 3 untranslated region of IGF1R mRNA containing the putative miR\223 binding sequence. The construct with the mutated fragment of the 3 untranslated region of IGF1R mRNA without the putative miR\223 binding sequences was used as the mutated control. ECs were transfected with the construct or the mutated control construct. These ECs were then treated with vehicle, scramble control, or miR\223 mimics (multiplicity of infection 30). Cell extract was isolated to measure the luciferase expression on a scintillation counter using a dual luciferase reporter system. Animals The male C57BL/6 mice and miR\223 knockout mice were from the CCNF Jackson Laboratory (Bar Harbor, ME). The chimeric mice were generated in our laboratory by transplantation of bone marrow from miR\223 knockout mice into lethally irradiated wild\type mice, as described previously.14 The well\established leukocyte depletion model in male wild\type C57BL/6 mice (aged 3?months) was applied in some mice, as described previously, in which neutrophils in blood were depleted by vinblastine (2.5?mg/kg IP).12 Platelets in blood were depleted by antiCmouse thrombocyte serum (50?L IP).12 All animal protocols were approved by the Tegaserod maleate IC50 institutional animal care and use committee and were consistent with the Guide for the Care and Use of Laboratory Animals (2011 version; National Institutes of Health). Human Serum Samples and Blood Cell Data This study was approved by the by the institutional review committee of the Second Affiliated Hospital and Yuying Children’s Hospital, and the participants gave informed consent, which conformed with the Declaration of Helsinki. Human serum samples were from age\ and sex\matched healthy control participants (n=103) and from patients with KD (n=78). Among the KD patients, 12 had coronary artery lesions. In addition, 22 serum samples from patients with KD after treatment with high\dose intravenous immunoglobulin were also collected. In addition, total white blood cell and circulating platelet counts for all participants were obtained from clinical data. The serum samples were Tegaserod maleate IC50 prepared as described in our previous studies.6 In brief, peripheral venous blood was collected and was placed for 1?hour at room temperature (26C). The blood samples were centrifuged at 1600for 15?minutes at 4C. All residual blood cells were removed via centrifugation before storage. Serum samples were then carefully transferred into plain propylene tubes and stored at ?80C until miRNA isolation. Serum miRNAs were isolated in 200?L serum using the solution miRNAs Isolation Kit (RNA Bioscience), according to the kit procedures. In addition to volume control (200?L serum), we used an exogenous spiked\in control probe (cel\miR\39). Statistics The blood cell count data were presented as meanSD. All other data were presented as meanSE. For relative gene expression, the mean value of the vehicle control group is defined as 100%.