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

<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

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

Incorporation of Disulfide Containing Protein Modules into Multivalent Antigenic Conjugates: Generation of Antibodies against the Thrombin-Sensitive Region of Murine Protein S

Antigenic peptide conjugates can be used as vaccines and for the production of antibodies for clinical and research use. A method is presented here for the construction of conjugates incorporating oxidatively folded protein domains in their native conformation. This method was used to prepare multiple antigenic peptide constructs of the thrombin-sensitive loop region of murine anticoagulant protein S

Habitual dietary intake per vitamin K-containing food component assessed by food frequency questionnaires.

<p>Data are expressed as median (IQR).</p

Patient characteristics stratified for normal vs poor vitamin K intake.

<p>Patient characteristics stratified for total vitamin K intake. Normal intake was defined as ≥120 µg/day (men) or ≥90 µg/day (women) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047991#pone.0047991-National1" target="_blank">[20]</a>.</p>*<p>P value for comparison of subjects with normal vs poor vitamin K intake by Mann Whitney test. Nutritional goals apply to ages 51 years and older.</p>a<p>Percentage of total energy intake.</p><p>Abbreviations: BMI, body mass index; SBP, systolic blood pressure; ADPKD, autosomal dominant polycystic kidney disease; MCKD, medullary cystic kidney disease; CVD, cardiovascular disease; mTOR, mammalian target of rapamycin; hsCRP, high-sensitivity C-reactive protein; NT-proBNP, N terminal-pro brain natriuretic peptide; MGP, matrix gla protein.</p

Hr-anxA1 has no significant effect on early plaque development.

<p>12 weeks old mice were fed WTD during 6 weeks. Hr-anxA1 treatment started at start of WTD. (A) Representative H&E staining of aortic arches after 6 weeks of treatment. (B) Treatment with hr-anxA1 did not affect plaque burden in the arch and subclavian (SA) brachiocephalic (BCA) and left common carotid artery (CA). (C) Individual plaque stability and progression was scored on following parameters: neutrophil and macrophage content, apoptosis and necrosis, cap thickness and calcification status (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130484#pone.0130484.s007" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130484#pone.0130484.s008" target="_blank">S2</a> Tables). Hr-anxA1 treatment had no effect on early plaque stability and progression. (D, E) Hr-anxA1 treatment had no effect on endothelial ICAM-1 expression of IX and SL lesions. IX: intimal xanthoma; SL: small lesion; IL: intermediate lesion; AL: advanced lesion. All values are represented as mean ±SEM, n = 12 animals per group. Panel A: 40x magnification, scale bar represents 500μm. Panel D: 200x magnification, scale bar represents 100μm.</p

Hr-anxA1 does not affect macrophage polarization in atherosclerotic plaque.

<p>(A) Representative staining for M1 macrophage specific marker iNOS. Black arrows indicate fully commited M1 macrophages, white arrows indicate iNOS negative macrophages. (B) Quantification of iNOS-staining indicates no difference between control and anxA1 treated animals. (C) Representative staining of M2 specific marker Ym1. Both control and hr-anxA1 treated mice were negative for Ym1. (D) Staining of Ym1 positive cells in the spleen indicating functionality of anti-Ym1 antibody. All values are represented as mean ±SEM, n = 12 animals per group. Panel A: 200x magnification, scale bar represents 100μm; panel D: 40x and 400x magnification, scale bar represent 500μm and 50μm respectively.</p