(A) VSMC were pretreated with IL-6 antibody (10 g/mL) for 2 h and CRAMP (100 ng/mL) for 2 h and then stimulated with PDGF-BB (20 ng/mL) for 24 h

(A) VSMC were pretreated with IL-6 antibody (10 g/mL) for 2 h and CRAMP (100 ng/mL) for 2 h and then stimulated with PDGF-BB (20 ng/mL) for 24 h. Results 3.1. CRAMP Inhibits PDGF-BB-Induced VSMC Phenotypic Transformation, Proliferation and Migration VSMC phenotypic change to dedifferentiated state was a key step in arterial neointimal hyperplasia during the formation of Cspg2 restenosis [29]. To investigate the function of CRAMP on VSMC phenotypic transformation, we first detected the cytotoxity of CRAMP on VSMC. The MTT assay showed that CRAMP have almost no effects on VSMC at the maximum dose at 1000 ng/mL (Figure 1A). Furthermore, the western blot results showed that CRAMP concentration-dependently reversed PDGF-BB-mediated the decrease of -SMA and SM22 expression (Figure 1B). These results suggested that CRAMP could inhibit PDGF-BB-induced VSMC phenotypic transformation. Open in a separate window Figure Chaetocin 1 Cathelicidin-related antimicrobial peptides (CRAMP) inhibits platelet-derived growth factor-BB (PDGF-BB)-induced vascular smooth Chaetocin muscle cells (VSMC) Phenotypic transformation. (A) Measurement of changes in cell viability of VSMC after 48 h incubation with a range of concentrations (0, 1, 10, 100 and 1000 ng/mL) of CRAMP. (B) VSMC were pretreated with CRAMP (100 ng/mL) for 2 h and then stimulated with PDGF-BB (20 ng/mL) for 24 h followed by immunoblotting with -SMA and SM22 antibodies. Data of 3 independent experiments is presented as mean SEM. ** 0.01; *** 0.001 compared with control. We then detected the effects of CRAMP on VSMC proliferation and migration. As showed in Figure 2A,B CRAMP significantly inhibited PDGF-BB-enhanced cell viability of VSMC. The EdU assay also showed that CRAMP could decrease PDGF-BB-mediated VSMC proliferation. Followingly, we detected the wound healing assay and transwell assay, and the results showed that CRAMP could significantly inhibit both PDGF-BB-induced VSMC migration and invasion. Above data suggested that CRAMP could inhibit PDGF-BB-elevated VSMC proliferation and migration. Open in a separate window Figure 2 CRAMP inhibits PDGF-BB elevated VSMC proliferation and migration. (A,B) VSMC were pretreated with CRAMP (100 ng/mL) for 2 h and then stimulated with PDGF-BB (20 ng/mL) for 24 h or 48 h. Chaetocin Cell viability was detected using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Data of 3 independent experiments is presented as mean SEM. ** 0.01 compared with control, = 8. (C) VSMC were pretreated with CRAMP (100 ng/mL) for 2 h and then stimulated with PDGF-BB (20 ng/mL) for 24 h. Proliferation of VSMC was detected using EdU assay. Data of 3 independent experiments is presented as mean SEM. *** 0.001 compared with control, = 6. (D,E) VSMC were pretreated with CRAMP (100 ng/mL) for 2 h and then stimulated with PDGF-BB (20 ng/mL) for 24 h. Proliferation of VSMC was detected using wound healing assay (D) and transwell assay (E). Data of 3 independent experiments is presented as mean SEM. ** 0.01, *** 0.001 compared with control, = 3. 3.2. CRAMP Inhibited PDGF-Mediated IL-6/STAT3 Activation Activation of ERK1/2 and STAT3 plays an effective role in VSMC phenotypic switching [30,31,32,33,34,35]. To find out the mechanisms of CRAMP in regulating VSMC phenotypic modulation, we first examined the effects of CRAMP on ERK1/2 and STAT3 activation. As showed in Figure 3A, the phosphorylation of ERK1/2 and STAT3 were significantly enhanced when treated with PDGF-BB, while the level of p-STAT3 but not p-ERK1/2 was inhibited when treated with both PDGF-BB and CRAMP..