Fig 2: Plasma and tissue osteomodulin (OMD) protein analyses in chronic kidney disease (CKD) and calcific aortic valve disease (CAVD) patients. (A) Spearman correlation between plasma OMD levels and aortic valve calcification (in Agatston scoring units) in CKD patients (n = 65). (B) OMD protein measurements in plasma from CKD patients stratified in groups according to the medial calcification grade/score (CS) of epigastric arteries from these patients (ranging from 0 to 3, where 0 signifies no arterial calcification, 1 and 2 refer to moderate calcification and 3 refers to extensive arterial calcification). Number of patients per group: n = 25 for CS = 0, n = 25 for CS = 1, n = 24 for CS = 2, n = 24 for CS = 3. One‐way ANOVA multiple comparison test; data presented as mean with SD. (C) Representative histological images of epigastric arteries from CKD patients with the four different grades of calcification, immunostained for OMD (red signal) and α‐SMA (green). Arrows point to OMD positive signal in the tissues. (D) Representative images from consecutive human aortic valve leaflet slides stained with Alizarin red and von Kossa to visualise calcification, or immunostained for α‐SMA, OMD and RUNX2. Scale bar as indicated in all images. Insets show corresponding isotype negative control. Differences between groups were considered significant at p‐values < .05 (*p < y.05)

Fig 3: Extracellular osteomodulin (OMD) attenuates smooth muscle cell osteogenic transition and represses calcification. (A) Quantification of the in vitro calcification of human coronary smooth muscle cells (HCoSMCs) treated with siRNA for OMD or scramble control in osteogenic medium consisting of 0.1 mM l‐ascorbate phosphate, 10 mM β‐glycerophosphate and 10 nM dexamethasone for 14 days (n = 3 independent experiments in duplicates). Statistical significance between groups was assessed by one‐way ANOVA multiple comparison test; data expressed as mean with standard error of mean (SEM). Representative images of the calcification assay as it was visualised by Alizarin Red staining. (B) Quantification of the in vitro calcification of human aortic smooth muscle cells (HAoSMCs) treated with 2.6 mM Pi for 12 days in the absence or presence of 50 ng/ml human recombinant OMD (rhOMD) protein. The experiment was performed in triplicate with cells from human biopsies. Statistical significance between groups was assessed by one‐way ANOVA multiple comparison test; data expressed as mean with SEM. Representative images of the calcification assay where calcification was visualised by an Alexa Fluor 546 coupled fetuin A probe. Scale bar 1000 μm. (C) Volcano plot showing the top significantly upregulated (red) and downregulated (blue) genes comparing HCoSMCs treated with siRNA for OMD (n = 3) versus scramble control (n = 3) in osteogenic medium for 14 days. (D) Volcano plot showing the top significantly upregulated (red) and downregulated (blue) genes comparing HAoSMCs treated with rhOMD (n = 3) versus control (n = 3) in osteogenic medium for 6 days. Differences between groups were considered significant at p‐values < .05 (*p < .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001)

Fig 4: Schematic representation of the mechanism by which osteomodulin (OMD) could mediate macro‐calcification formation by smooth muscle cells

Fig 5: Osteomodulin (OMD) is induced in smooth muscle cells by inflammatory and osteogenic stimuli. (A) OMD mRNA expression levels in human aortic smooth muscle cells (HAoSMCs) treated with vehicle (control, CTR), IL‐4, IFNγ, IL‐6, BMP2 or TGFβ1 for 12, 24, 48 or 72 h. An increase of OMD gene expression was detected only after 48 h of BMP2 treatment. The experiment was performed in triplicate. Statistical significance between groups was assessed by Student's t‐test; data expressed as mean with standard error of mean (SEM). (B) Gene expression analysis of typical smooth muscle cell markers, osteochondrogenic, inflammatory markers of HAoSMCs treated with 50 ng/ml human recombinant OMD (rhOMD) for 24 h. The values for genes of interest in rhOMD treated HAoSMCs were normalised with the corresponding values of non‐treated control cells (green dotted line). Statistical significance between groups was assessed by Student's t‐test; data expressed as mean with SEM. (C) OMD mRNA expression levels in HAoSMCs treated with 2.6 mM Pi for up to 12 days. The experiment was performed in triplicate with cells from human biopsies. Statistical significance between groups was assessed by Student's t‐test and one‐way ANOVA multiple comparison test; data expressed as mean with SEM. (D) OMD mRNA expression levels in human coronary smooth muscle cells (HCoSMCs) treated with osteogenic medium consisting of 0.1 mM ascorbic acid, 10 mM β‐glycerophosphate and 100 nM dexamethasone, for promoting the osteoblast phenotype. Statistical significance between groups was assessed by one‐way ANOVA multiple comparison test; data expressed as mean with SEM. (E) Correlations between OMD mRNA levels and the expression of typical smooth muscle cell markers (left) and osteochondrogenic markers in the same cells (right). Differences between groups were considered significant at p‐values < .05 (*p < .05, **p ≤ .01, ***p ≤ .001, ****p ≤ .0001). Data in (D and E) were extracted from Alves et al. public microarray dataset (GEO accession no. GSE37558)