Osteomodulin attenuates smooth muscle cell osteogenic transition in vascular calcification

Abstract Rationale Vascular calcification is a prominent feature of late‐stage diabetes, renal and cardiovascular disease (CVD), and has been linked to adverse events. Recent studies in patients reported that plasma levels of osteomodulin (OMD), a proteoglycan involved in bone mineralisation, associate with diabetes and CVD. We hypothesised that OMD could be implicated in these diseases via vascular calcification as a common underlying factor and aimed to investigate its role in this context. Methods and results In patients with chronic kidney disease, plasma OMD levels correlated with markers of inflammation and bone turnover, with the protein present in calcified arterial media. Plasma OMD also associated with cardiac calcification and the protein was detected in calcified valve leaflets by immunohistochemistry. In patients with carotid atherosclerosis, circulating OMD was increased in association with plaque calcification as assessed by computed tomography. Transcriptomic and proteomic data showed that OMD was upregulated in atherosclerotic compared to control arteries, particularly in calcified plaques, where OMD expression correlated positively with markers of smooth muscle cells (SMCs), osteoblasts and glycoproteins. Immunostaining confirmed that OMD was abundantly present in calcified plaques, localised to extracellular matrix and regions rich in α‐SMA+ cells. In vivo, OMD was enriched in SMCs around calcified nodules in aortic media of nephrectomised rats and in plaques from ApoE −/− mice on warfarin. In vitro experiments revealed that OMD mRNA was upregulated in SMCs stimulated with IFNγ, BMP2, TGFβ1, phosphate and β‐glycerophosphate, and by administration of recombinant human OMD protein (rhOMD). Mechanistically, addition of rhOMD repressed the calcification process of SMCs treated with phosphate by maintaining their contractile phenotype along with enriched matrix organisation, thereby attenuating SMC osteoblastic transformation. Mechanistically, the role of OMD is exerted likely through its link with SMAD3 and TGFB1 signalling, and interplay with BMP2 in vascular tissues. Conclusion We report a consistent association of both circulating and tissue OMD levels with cardiovascular calcification, highlighting the potential of OMD as a clinical biomarker. OMD was localised in medial and intimal α‐SMA+ regions of calcified cardiovascular tissues, induced by pro‐inflammatory and pro‐osteogenic stimuli, while the presence of OMD in extracellular environment attenuated SMC calcification.


Computed tomography (CT) angiography image analysis 27
Carotid plaques in BiKE cohort were assessed in pre-operative CT angiographies using a novel semi-28 automated, histopathologically validated software as previously described (vascuCAP,Elucid 29 Bioimaging, Boston, MA) 1 , rendering tissue and structural characteristics of the plaque, e.g.,

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Cardiac CT scans in CKD cohort were performed using a 64-channel detector scanner (Lightspeed 33 VCT; General Electric (GE) Healthcare, Milwaukee, WI). CAC scores were expressed in Agatston 34 units 2 , the protocol and measurements as described previously in detail 2 . Total CAC score was 35 calculated as the sum of CAC scores in the left main artery, the left anterior descending artery, the left 36 circumflex artery, and the right coronary artery.

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Animal material and studies

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In general, the simple randomization method was applied in all animal studies and group results were 40 analyzed in a blinded fashion. Male animals were used to ensure better control over the possible 41 variability of data related to sex. There was no exclusion of animals from the study and analyses were

Rat model of medial vascular calcification 61
Sprague-Dawley rats (Charles River, Ecully, France) (n=90) 10 weeks of age and 220-250 gram were 62 maintained in a controlled laboratory setting with water and food ad libitum (Teklad Diets, Madison 63 WI, USA) and 12-hour day/night cycle. ¾ nephrectomy was performed to induce kidney failure 4 . One

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week later the animals were put on diet containing calcium 0.76%, phosphate 0.45%, 3 mg/g warfarin 65 (Sigma) and 1.5 mg/g vitamin K1 (Sigma) to prime the rats for calcification and deplete the levels of 66 vitamin K. Vitamin K1 was co-administrated with warfarin to avoid bleeding, yet having no effect in 67 extra-hepatic tissues 5 . After three weeks the diet was changed to a purified diet consisting of calcium 68 1.34%, phosphate 1.2% and equal number of rats were allocated into high (100 μg/gram) and low (5 and used between passage 5-8 for calcification assays. Briefly, intima, fat and connective tissue were 84 carefully removed before cutting the sample into small fragments and placing into laminin (#L2020,

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HAoSMCs cytokine stimulation assays 92 HAoSMCs between passage 8-10 were plated on 6-well plates and left to adhere over-night in SmGM.

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Migration assay 105 To assess HAoSMC migration, an in vitro scratch assay was conducted as previously described 7 .

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Thereafter, a straight scratch was created in a monolayer using a 1000 μL pipette tip and cells were 108 washed once with PBS. Immediately, 50 ng/ml rhOMD was added for 48 hours in 5% FBS SmGM.

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Wound closure was quantified by measuring the distance between the migration fronts at 3 random 111 locations of 3 wells per time point and condition. Images were processed with Fiji Image J software.

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The full data set is available from Gene Expression Omnibus (accession number GSE21545). The BiKE 227 study cohort demographics (

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In general, prior to all stainings, tissue specimens were deparaffinized in 2 changes of xylene (VWR,

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To enable further processing for histology, macro-calcified plaques were de-calcified after fixation in

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For quantification of the cell culture AR staining, 100 mM cetylpyridinium chloride (Sigma-Aldrich)

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in 10 mM sodium phosphate buffer (Roth) was added to each well and incubated for 20 min at 37°C.

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The eluted stain was measured at 570 nm using a spectrophotometer.

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Antibodies 286 The following primary antibodies were used in the study: anti-OMD (ab154249, Abcam and AF3308,

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For staining of mouse, rat and human aortic valve sections, sequential 5 µm slides were rehydrated, and 302 antigens were retrieved by boiling in TriSodiumCitrate buffer (pH 6.0). Primary antibodies were applied 303 and incubated over-night at 4 o C and then visualized with a Nova-RED substrate (Vector #SK-4800,

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Prior to tissue analyses, whole tissue reference images were taken with the VENTANA iScan HT slide

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where 0 signifies no arterial calcification, 1 and 2 refer to mild and moderate calcification, while 3 400 refers to extensive arterial calcification). Number of patients per group: n=25 for CS=0, n=25 for CS=1, 401 n=24 for CS=2, n=24 for CS=3. One-way ANOVA multiple comparison test; data presented as mean 402 with SD. Differences between groups were considered significant at P values < 0.05 (*P < 0.05).

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N=3 independent experiments in duplicates. Statistical significance between groups was assessed by 414 Student's t-test; data expressed as mean with SEM. Differences between groups were considered 415 significant at P values < 0.05 (*P < 0.05).

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Quantification of the in vitro calcification of HAoSMCs treated with 2.6mM Pi for 6 days in 475 combination with 150ng/ml BMP2 and 50ng/ml OMD proteins. Representative images of the 476 calcification assay. Quantification performed with Image J and calcified nodules were quantified as 477 percentage of calcification per optical field area. Statistical difference between groups assessed by one-478 way ANOVA; data expressed as mean with SEM. Differences between groups were considered 479 significant at P values < 0.05 (*P < 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). Tables   481  482  483 Supplementary