The role of Ca2+/NFAT in Dysfunction and Inflammation of Human Coronary Endothelial Cells induced by Sera from patients with Kawasaki disease

Figure 1. Levels of NFAT mRNA and protein were higher in the KD group. (a,b) HCAECs were treated with medium containing 15% sera from HC (n = 8) and KD (n = 29) subjects. After 6 hours, NFATc1 and NFATc3 mRNA levels were measured by qPCR (a: all samples were normalized to HCAECs with 15% FBS treatment), and protein levels in HCAECs were measured by Western blot (b; HC, n = 5 and KD, n = 12). The NFATc1 and GAPDH blots in one group were cropped from different parts of the same gel. The upper group and lower group of blots were cropped from different gels. The right panel of (b) is the statistical analysis of immunoblots. NFATc1 protein levels were measured by Western blotting. Full-length blots are presented in Supplementary Fig. S1. The bar graphs are mean ± SEM. *P < 0.05 and **P < 0.01 compared with respective control group.

Figure 2. Ca 2+/NFAT were activated in coronary artery endothelial cells induced by KD sera. (a,b) HCAECs were incubated with HC sera (pooled from 8 individuals), KD sera (pooled from 12 individuals) and febrile control (FC) sera (pooled from 10 individuals) for 6 hours. The cytoplasm proteins and nucleus proteins were extracted by Nuclear and Cytoplasmic Protein Extraction Kit. Levels of NFATc1 and NFATc3 proteins were measured using Western blotting (in a and b). The NFATc1, b-tubulin and Histone H3 blots in one group were cropped from different parts of the same gel. Full-length blots are presented in Supplementary Fig. S1. Data in (a,b) are mean ± SEM from 3 independent experiments involving different batches of cells but the s ame pooled HC or KD sera. *P < 0.05 and **P < 0.01 when compared with respective controls or between indicated groups.

Figure 3. Disruption of endothelial cell homeostasis in the KD group. (a) HCAECs were treated with medium containing 15% sera from HC (n = 17), CAL- and IVIG-responsive KD (n = 25), CAL+ KD (n = 7), IVIG-resistant KD (n = 11) and convalescence KD (n = 13) subjects. After 24 hours and 48 hours, the proliferation of HCAECs were detected by CCK8 (all samples were normalized to HCAECs with 15% FBS treatment and each sample was examined in triplicate). (b) HCAECs were treated with medium containing 15% sera from HC (n = 3) and KD (n = 8) subjects. Cells proliferation was monitored for 140 h by RT-CES system. Green curve and red curve represent mean cell numbers of KD group and HC group respectively. SR CI indicates slope rate cell index; 85h-CI indicates cell index at 85 h. (c) In vitro angiogenesis. HCAECs were plated on growth factor-reduced Matrigel to migrate or join together in the presence of 15% sera from HC (pooled from 8 individuals) and KD (pooled from 12 individuals) subjects. After 4 hours, cells were photographed at a magnification of 40×. The lower panel of (c) is the statistical analysis of angiogenesis. (d) HCAECs were treated with medium containing 15% sera from HC (n = 8) and…

Figure 4. CsA inhibited inflammation and attenuated dysfunction via Ca2+/NFAT. (a,b) HCAECs were pretreated with KD sera (pooled from 12 individuals) or HC sera (pooled from 8 individuals) for 6 hours and treated with or without CsA (500 ng/ml) for 2 h. VCAM-1, ICAM-1, TF, P-selectin, E-selectin, MCP-1 and IL8 mRNA and protein were detected and quantified. (b) The VCAM-1 and GAPDH blot in one group were cropped from different parts of the same gel. The NFATc1 and GAPDH blot in one group were cropped from different parts of the same gel. Full-length blots are presented in Supplementary Fig. S1. (c) HCAECs were pretreated with KD sera (pooled from 12 individuals) for 24 hours and treated with different concentration of CsA for 24 h. Cells proliferation were detected and quantified. (d) HCAECs were plated on Matrigel in the presence KD sera (pooled from 12 individuals) or co-cultured with KD sera and CsA. After 4 hours, cells were photographed and quantified. Data are mean ± SEM from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 compared with the control or indicated group.