Additional file 5: Table S4. of Characterization and clinical significance of right ventricular mechanics in pulmonary hypertension evaluated with cardiovascular magnetic resonance feature tracking

Cox proportional adjusted hazard ratio for final multivariate alternative model of each RV strain parameter. (DOCX 45 kb

Additional file 1: Table S1. of Characterization and clinical significance of right ventricular mechanics in pulmonary hypertension evaluated with cardiovascular magnetic resonance feature tracking

Inter- and intra-observer agreement. (DOCX 48 kb

Additional file 3: Table S2. of Characterization and clinical significance of right ventricular mechanics in pulmonary hypertension evaluated with cardiovascular magnetic resonance feature tracking

Correlation coefficients of right ventricular strain indices (absolute values) with CMR and RHC-derived parameters. (DOCX 82 kb

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^−/− mice increases apoptosis in atherosclerotic plaque.

<p>Vascular calcification is closely linked to apoptosis. Therefore we stained sections for caspase-3. Moreover, all animals were injected with annexin A5-biotin, a protein with high affinity for phosphatidylserine (PS). Both cleaved caspase-3 (A,C) and annexin A5-biotin (B,D) increased in the warfarin treatment, with annexin A5 accumulation significantly increased after 4 weeks of warfarin treatment (B). Annexin A5 staining using AP vector blue confirmed PS externalization at different sites in the atherosclerotic plaques. Activated and apoptotic macrophages are known to express PS on their surface. We found co-localization of macrophages in the shoulder of the atherosclerotic plaque (Mac3; F) and PS (annexin A5 stained with AP vector red; E), indicating activated and apoptotic macrophages. In the inset annexin A5 (blue) localizes with Mac-3 staining (red/brown), indicating activated macrophages. Moreover, at baseline annexin A5 was only incidentally present at the surface of the plaque (G), whereas at the 4 weeks warfarin treatment, annexin A5 stained significantly positive (H). Statistically significant differences were determined by the Kruskal Wallis test. *P<0.05. i, intima; m, media; l, lumen.</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

Warfarin treatment rapidly increases medial and intimal plaque calcification in apoE<i>^−/−</i> mice.

<p>ApoE−/− mice received WTD for three month (baseline) and subsequently control diet (WTD plus VK₁) or warfarin (WTD plus VK₁&W). Von Kossa stained calcified plaques were scored for medial (A,C) and intimal plaque calcification (B,D). In addition calcification was categorized as microcalcification (E, arrow heads) and macrocalcification (F, arrows). Microcalcifications occur either alone or in conjunction with macrocalcification. Statistically significant differences were determined by the Kruskal Wallis test. *P<0.05, **P<0.01, ***P<0.001. i, intima; m, media; l, lumen; a, adventitia.</p