Univariate and multivariate analysis of factors predicting all-cause mortality.

1<p>p value from Kaplan Meier log-rank test.</p>2<p>p value from Cox-regression analysis.</p>3<p>No patient with less than 25 g/dl albumin.</p>4<p>CKD is not independent risk factor as creatinine level included in GRACE score.</p

AR deletion from vascular smooth muscle, but not from endothelial cells, altered vascular density in ischaemic gastrocnemius.

<p>(A) Detection of endothelial cells (IB4-positive) and smooth muscle cells (SMA-positive) in vascular structures was achieved using immunohistochemistry on cross-sections of gastrocnemius muscle. High resolution tile-scanning (@ x200) using slide scanner Axio Scan.Z1 (Zeiss) allowed quantification of vascular phenotype and density of the whole muscle section. (B) Quantitation of vessel density indicated that deletion of AR from neither vascular smooth muscle nor endothelium altered the rise in density of IB4-positive vessels in ischaemic hindlimb. **, p<0.01 by two way ANOVA. (C) Deletion of AR from vascular smooth muscle, but not endothelium, prevented the ischaemia-induced increase in SMA-positive vessels. * p<0.05; ** p<0.01; by two way ANOVA plus Tukey post hoc test. ## P<0.01, by Student’s t-test (n = 7–10).</p

Clinical characteristics and outcomes of Gold standards Framework (GSF) positive (defined as meeting 1 general criterion and 2 heart disease criteria) versus Gold Standards Framework negative patients.

<p>Clinical characteristics and outcomes of Gold standards Framework (GSF) positive (defined as meeting 1 general criterion and 2 heart disease criteria) versus Gold Standards Framework negative patients.</p

Kaplan-Meier survival curves for factors independently predicting all-cause mortality.

<p>Kaplan-Meier survival curves for factors independently predicting all-cause mortality.</p

Baseline patient characteristics and alive versus dead by 12 months comparison.

<p>P values for alive versus dead comparison, GSF – Gold Standards Framework, SD standard deviation, ST – electrocardiogram ST segment, COPD – chronic obstructive pulmonary disease, CKD – chronic kidney disease.</p

Deletion of AR from smooth muscle, but not from endothelium, reduced post-operative reperfusion of the ischaemic hindlimb.

<p>(A) Laser Doppler imaging demonstrated partial restoration of hindlimb perfusion over 21 days following femoral artery removal. The blood flow in the foot pad was quantified and normalised to flow in the tail. (Areas of interest are indicated by yellow boxes. The grey area is excluded by software automatically.) (B) Deletion of AR from smooth muscle impaired this recovery, whereas deletion of AR from endothelial cells did not. $ p<0.01 difference among genotypes; * p<0.01 versus WT; # p<0.01 versus VE-ARKO. Data were analysed by two way ANOVA plus Tukey post hoc test (n = 14–16).</p

Localisation of vascular ARKO in murine ischaemic gastrocnemius.

<p>(A) AR was detected in the vascular smooth muscle (AR = green, SMA = red, DAPI = blue) in the ischaemic gastrocnemius muscle at day 21 from WT and VE-ARKO, but not SM-ARKO and SM/VEARKO. No positive staining of AR could be identified on the luminal (endothelial) side of the vessel in any genotypes. (Yellow arrows indicate AR-positive nuclei) (B) Double staining of AR and CD31 in WT confirmed that AR was not expressed in vascular endothelium in the ischaemic hindlimb. (White arrows indicate nuclei of endothelial cells.) Some AR positive cells were also found in the perivascular connective tissue.</p

Impact of vascular AR deletion on pre- and post-operative hindlimb perfusion.

<p>(A) Laser Doppler was used to monitor perfusion of the foot pad immediately before and after femoral artery ligation, which was quantified by software and normalised to tail. (Areas of interest are indicated by yellow boxes. The grey area is excluded by software automatically.) (B) Perfusion in the un-operated hindlimb was unaffected by selective deletion of vascular AR. Femoral artery removal produced a dramatic reduction in hindlimb perfusion that was similar in wild type and ARKO mice. ** p<0.01 compared with corresponding pre-operative measurements (n = 14–16).</p

Sensitivity and specificity of prognostic scores.

<p>NPV – negative predictive value, PPV – positive predictive value, GSF – Gold Standards Framework criteria positive, GRACE – Global Registry of Acute Coronary Events.</p

Gold Standards Framework criteria.

<p>Gold Standards Framework criteria.</p