Figure 5 raw data. The raw values for each shear metric within the instrumented and control arteries of each mouse from Influence of shear stress magnitude and direction on atherosclerotic plaque composition

The precise flow characteristics that promote different atherosclerotic plaque types remain unclear. We previously developed a blood flow-modifying cuff for ApoE^−/− mice that induces the development of advanced plaques with vulnerable and stable features upstream and downstream of the cuff, respectively. Herein, we sought to test the hypothesis that changes in flow magnitude promote formation of the upstream (vulnerable) plaque, whereas altered flow direction is important for development of the downstream (stable) plaque. We instrumented ApoE^−/− mice (<i>n</i> = 7) with a cuff around the left carotid artery and imaged them with micro-CT (39.6 µm resolution) eight to nine weeks after cuff placement. Computational fluid dynamics was then performed to compute six metrics that describe different aspects of atherogenic flow in terms of wall shear stress magnitude and/or direction. In a subset of four imaged animals, we performed histology to confirm the presence of advanced plaques and measure plaque length in each segment. Relative to the control artery, the region upstream of the cuff exhibited changes in shear stress magnitude only (<i>p</i> < 0.05), whereas the region downstream of the cuff exhibited changes in shear stress magnitude and direction (<i>p</i> < 0.05). These data suggest that shear stress magnitude contributes to the formation of advanced plaques with a vulnerable phenotype, whereas variations in both magnitude and direction promote the formation of plaques with stable features

Validation of LTB4 measurements using ELISA.

<p>For validation of LTB4 measurements in tissue homogenates, six randomly chosen AAA tissue samples were used for subset analysis using LTB4 spiking and sample purification using lipid extraction (SPE (C-18) column). LTB4 levels were measured in six randomly chosen AAA samples in different dilutions (A). LTB4 spike recovery was performed in a pooled AAA sample (B) and Tris buffer only (control) (C) using different LTB4 concentrations. LTB4 spike recovery was also performed in individual AAA samples and compared to no spiking (D). Sample purification was performed on the same six randomly chosen AAA samples using the SPE (C-18) column purification method (E). LTB4 spike recovery was analyzed by adding a LTB4 spike (50 pg/ml) to the samples prior to column purification (n = 2) (F). LTB4 levels are expressed in pg/ml.</p

Validation of LTB4 measurements in plasma using ELISA.

<p>Validation of <i>ex vivo</i> LTB4 production after whole blood collection by EDTA (black bars, n = 4, duplo) or Sodium Citrate (white bars, n = 4, duplo) Vacutainers. Directly after collection, whole blood was incubated with a specific 5-LOX inhibitor Zileuton (100 µM for 30 minutes) and plasma was stored. LTB4 was measured using ELISA and compared with levels of CEA patients (n = 35). Data is expressed as mean ± standard deviation and in pg/ml.</p

Immunohistochemical staining of BLT1 and ALOX5 in human atherosclerotic plaques.

<p>BLT1 expression could mainly be observed in areas rich in (foamy) macrophages (A), in both a membranous and cytoplasmatic expression. A weaker expression of BLT1 could be observed in areas rich of SMCs (B). ALOX5 expression could predominantly be observed in areas rich in (foamy) macrophages (C), and around cholesterol clefts (D). Arrows indicate positive staining (brown color). Scale bar: 500 µm. The insert shows 5x extra magnification.</p

Comparison of LTB4 plaque expression levels and histological plaque composition of atherosclerotic carotid plaques.

<p>Bar graphs of LTB4 plaque expression levels in relation with semi-quantative histological plaque characteristics. All characteristics have been correlated with continuous LTB4 expression levels and analyzed by Pearson Bivariate correlation test. Differences with p-value <0.05 were regarded as being significant. All data is expressed as mean ± standard deviation. LTB4 levels are expressed in pg/mg. Abbreviations: SMC: smooth muscle cell content.</p

Clinical characteristics of patients undergoing open AAA repair in relation with LTB4 expression.

<p>LTB4 abdominal aortic aneurysm expression levels are expressed as median [IQR].</p><p>Abbreviations: AAA: Abdominal Aortic Aneurysm, ACE: Angiotensin Converting Enzyme, HDL: high-density lipoprotein, LDL: low-density lipoprotein, LTB4: Leukotriene B4, SD: standard deviation. Patient characteristics have been correlated with continuous LTB4 expression aneurysm levels. P-values were calculated using Kruskal-Wallis or Mann-Whitney U test. Differences with p-value <0.05 was regarded significant marked with an asterisk (*).</p

Uncorrected versus corrected LTB4 levels and secondary clinical outcome.

<p>LTB4 levels in atherosclerotic plaques and AAA tissue were measured using ELISA and corrected for total protein content. For validation purposes, also uncorrected data are depicted in these graphs. Uncorrected and corrected LTB4 levels were related to control versus events (no secondary outcome versus secondary outcome during follow-up). Both corrected and uncorrected LTB4 levels were not significantly different between controls and events for both CEA (A and B) and AAA (C and D) patients. Data is presented as mean ± standard deviation or median with interquartile range [IQR] and in pg/ul for uncorrected data and in pg/mg for corrected data.</p

Clinical characteristics of patients undergoing carotid endarterectomy in relation with LTB4 expression.

<p>LTB4 plaque expression levels are expressed as mean ± standard deviation.</p><p>Abbreviations: ACE: Angiotensin Converting Enzyme, HDL: high-density lipoprotein, LDL: low-density lipoprotein, LTB4: Leukotriene B4, SD: standard deviation. Patient characteristics have been correlated with continuous LTB4 expression plaque levels. P-values were calculated using Student’s T test. Differences with p-value <0.05 was regarded significant marked an with asterisk (*).</p

Characteristics with respect to follow-up and LTB4 tissue levels in CEA patients.

<p>LTB4 plaque expression levels are expressed as mean ± standard deviation and as pg/mg protein. P-values were calculated using Student’s T test. Differences with p-value <0.05 was regarded as being significant.</p><p>Abbreviations: CEA: carotid endarterectomy, FU: follow-up, LTB4: leukotriene B4, SD: standard deviation.</p

Comparision of LTB4 Abdominal Aotic Aneurysm (AAA) expression levels and histological tissue composition of AAA.

<p>Box-plots of LTB4 abdominal aortic aneurysm expression levels in relation with semi-quantative histological AAA characteristics. All characteristics have been correlated with continuous LTB4 expression levels and analyzed by Spearman’s Bivariate correlation test. Differences with p-value <0.05 were regarded as being significant. All data is expressed as median with interquartile range [IQR] and in pg/mg. Abbreviations: SMC: smooth muscle cell content.</p