Fig. 4

Effect of NGAL silencing on calcifying VICs from men donors undergoing elective surgical AV replacement. NGAL silencing was evidenced at transcript levels in calcifying male-derived VICs. Scramble (Scr) control group was used as calibrator for the fold-change calculations. Scatter dot plots for the relative expression of NGAL codifying gene (LCN2) in calcifying male-derived VICs (A). GAPDH, 18S, HPRT and ACTA2 were used as housekeeping genes. Scatter dot plot of calcium deposits in calcifying Scr and siNGAL male-derived VICs (B). Markers of inflammation CCL-2, RANTES, myeloperoxidase and galectin-3 (C); ECM remodelling markers MMP-2, MMP-9, TIMP-1 and TIMP-2 (D); and osteogenesis (E) were assessed by ELISA in cell supernatants from siNGAL VICs under HP conditions. Osteogenic markers RUNX2 and BMP2 were analysed at the mRNA transcript level as well as the quiescent VIC marker LECT1 for chondromodulin (Chm)-I (F). AU arbitrary units; *p < 0.05; **p < 0.01; ***p < 0.001; p < 0.0001. Scr scramble, siNGAL small interfering RNA against NGAL, LCN2 NGAL codifying gene, CCL2 chemokine (C–C motif) ligand 2 (CCL2) or monocyte chemoattractant protein 1 (MCP1), MPO myeloperoxidase, Gal-3 galectin-3, MMP metalloproteinase, TIMP tissue inhibitor of metalloproteinases, BMP bone morphogenetic protein, LECT1 chondromodulin (Chm)-I codifying gene; periostin; RUNX2 Runt-related transcription factor 2