<i>In vitro</i> model for chemoattractant properties of calcifying VSMCs on macrophages.

<p>The effect of calcifying VSMCs on the attraction of inflammatory cells was tested via invasion assays using PMA-stimulated THP-1 cells (macrophages). Conditioned medium of both control and calcifying VSMCs was used and calcium was added to control medium to obtain equal concentration of calcium in both conditions. Medium from calcifying VSMCs increased the invasion of macrophages significantly, indicating that VSMCs that calcify produce chemoattractants for inflammatory cells. Results were normalised to cell number at start. *P < 0.05, **P < 0.001 significance was assessed unpaired non-parametric t-test (Mann-Whitney).</p

Calcifying VSMCs <i>in Vitro</i> display a pro-inflammatory and not an osteochondrogenic phenotype.

<p>qPCR of calcifying human primary VSMCs show a significant decrease in MGP as compared to control VSMCs. No differences were found in the expression of the osteochondrogenic markers Runx2, BMP-2 and osteocalcin. The pro-inflammatory cytokines MCP1, IL1b and IFNy were significantly increased in calcifying VSMCs indicative that calcifying VSMCs can initiate local vascular inflammation and promote macrophage migration towards the vascular wall.</p

Regional differentiation of atherosclerotic lesion types.

<p>An overview of a human coronary artery section, depicting atherosclerotic stages I to IV, is shown in the left panel, with enlargements of the selected regions at the right panel. The regions of coronary lesions classified as types I, II, III, and IV, based on CD68 positivity, are shown in 1A, with corresponding regions in the calcium yield scan in 1B.</p

Model showing the potential mechanism of initiation and progression of calcification of the vascular wall.

<p>1) Contractile VSMCs in the thickened intima change phenotype towards synthetic VSMCs. Synthetic VSMCs start secreting extracellular vesicles into the extracellular environment. In case of shortage of vitamin K, a vitamin required for the conversion of ucMGP into the active form cMGP, extracellular vesicles are loaded with ucMGP which is unable to prevent nucleation of calcium-phosphate. 2) Calcifying vesicles provide the first nidus for mineralisation and microcalcifications will be formed. These microcalcifications induce an inflammatory response in VSMCs. 3) VSMCs start secreting pro-inflammatory cytokines that will attract macrophages. 4) Macrophages start fueling the inflammation process by phagocytosing mcirocalcifications and secreting pro-inflammatory cytokines. 5) Pro-inflammatory macrophages affect synthetic VSMCs which will in turn produce BMP2. Synthetic VSMCs will transdifferentiate towards osteochondrogenic VSMCs that subsequently will produce bone-forming proteins such as osteocalcin. 6) Macrocalcifications are the final result of the osteochondrogenic environment in the atherosclerotic plaque.</p

Representative images of type I, II, III and IV regions.

<p>Representative images of regions classified as type I, II, III and IV with the corresponding calcium yield scan and (immuno-)histochemical staining of von Kossa, αSMA, ucMGP, cMGP, BMP-2 and osteocalcin (OC) in adjacent sections. Arrows indicate calcification close to the internal elastic lamina. Scale bars are 200μm.</p

Baseline characteristics of patients not on VKA treatment.

<p>Continuous variables are presented as means ± SD, while categorical variables are expressed as number (percentage). BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein.</p

Warfarin treatment of apoE deficient mice on Western Type Diet does not influence plaque size but increases vascular calcification.

<p>ApoE^−/− mice developed atherosclerotic lesions in the aortic arch and carotid arteries when maintained on Western type diet (WTD) for 12 weeks (A). From baseline mice were fed with WTD plus vitamin K (VK₁) or WTD plus vitamin K and warfarin (VK₁&W) for the duration of one week or four weeks. Growth of intimal area was not significantly affected by warfarin. Vascular calcium was determined by AAS and revealed significant increase in calcium at 1 and 4 weeks warfarin treatment. Statistically significant differences were determined by the Kruskal Wallis test. *P<0.05.</p

Duration of VKA treatment significantly increases the amount of coronary calcification and number of calcified stenotic plaques.

<p>A non-enhanced scan was performed to measure the Coronary artery calcification (Agatston score). Panel A shows a significant increase in Agatston score in VKA users (p = 0.029), as patients use VKA for a longer time (T = tertile of VKA use). This increase is not visible in individually matched patients not on VKA (panel B; p = 0.965). Next, CT-angiography was performed. All coronary segments were assessed for plaque presence and plaque morphology. In patients using VKA, a significant trend (p = 0.009) was seen towards a higher percentage of calcified plaques in those patients treated longest with VKA (panel C). In contrast, this was not the case for the non-VKA users (panel D). Coronary plaques were identified and categorized as calcified (closed circles), mixed calcified (diamonds) and non-calcified (open circles). T1, T2 and T3 indicate 1^st, 2^nd and 3^rd tertile respectively.</p

Warfarin affects carboxylation of MGP in atherosclerotic plaque.

<p>To confirm action of warfarin locally in the plaque we stained plaques for ucMGP and cMGP. UcMGP is the result of vitamin K-deficiency. Within one week of warfarin treatment the amount of cMGP (A,E) decreased significantly and decreased even further after 4 weeks of warfarin treatment. The decrease in cMGP was accompanied by an increase in ucMGP (B,F). Immunohistochemistry for cMGP and ucMGP (E,F) showed increased amounts of ucMGP compared with cMGP in apoE^−/− mice on warfarin, whereas apoE^−/− mice on control diet had predominantly cMGP (C,D). cMGP indicates carboxylated MGP; ucMGP, uncarboxylated MGP. Statistically significant differences were determined by the Kruskal Wallis test. *P<0.05, **P<0.01. i, intima; m, media; l, lumen.</p

Weight and blood characteristics of apoE^−/− mice at baseline, and after one or four weeks of treatment.

<p>WTD, Western type diet.</p