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- Supplementary methods and results sections
- Supplementary figure S1 - a) LPS dosage and timeline study: Mice were sacrificed at 4, 24, 48 hours post intratracheal LPS (25μg, 50μg, 100μg) administration (N=4 each). Saline-instilled mice that were sacrificed at these same time points were pooled as controls. The 48 hour post-LPS (100 μg) time point experiment was terminated prematurely because mice experienced sudden deaths or paralysis. Atheroma destabilisation was defined based on histological evidences of intraplaque hemorrhage and thrombus formation. Mice exposed to intratracheal LPS at dosage of 50 μg and 100 μg demonstrated similar rates of atheroma destabilisation at 4 hours and 24 hours. 24 hours post-LPS administration was chosen as the primary time point for investigation because LPS 24 hours versus saline 24 hours demonstrated significant change in plaque vulnerability (Fisher exact test P <0.005). b) A representative figure of different morphological components of a plaque: vessel lumen, plaque area, necrotic core and cap region in a cross section of BCT. Plaque area was determined by ascertaining the area enclosed by the internal elastic lamina (IEL) minus the area enclosed by the endothelial layer (lumen area). The necrotic core was defined as the component of the plaque that did not contain any matrix material (either collagen or elastin) or cells. This area was quantified by using color segmentation. The percent (%) necrotic core was calculated by dividing the necrotic core area by the total plaque area; and c) the method used to determine cap-plaque thickness ratio using Image Pro Plus software. A plaque image was overlaid with a known pattern ray originating from the mid portion of inner elastic lamina (IEL) of the plaque. The cap-plaque thickness ratio was calculated by determining the distance from the endothelium to the necrotic core (yellow line) and then dividing this number by the distance from the endothelium to the spectrum origin (yellow line + blue line). An arithmetic mean value of five measurements was calculated and used for statistical analysis.
- Supplementary figure S2 - a) H&E stained mouse left lung images 24 hours after intratracheal exposure to 50 μl sterile saline (left) and 100 μg LPS dissolved in 50 μl sterile saline (right). There were increased numbers of neutrophils, disrupted epithelial cells, blood congestion, and edema in the mouse lungs exposed to LPS; b) Quantitation of neutrophils in bronchoalveolar lavage (BAL) fluid from saline and LPS exposed mice at 24 hours were shown. Error bars represent standard error of the mean. Cells in the BAL were gently mixed and cytospun down to a glass slide and fixed with methanol before staining with H&E stain. PMNs were morphologically identified by evaluating size, shape, and presence of granules and multinucleation. Monocytes and alveolar macrophages were also determined by morphology; c) Examples of cytospin images of neutrophils in bronchoalveolar lavage (BAL) from LPS-Ctrl group (LPS-exposed mice injected with control antibodies; left) and LPS-ND (LPS-exposed mice injected with neutrophil specific anti-Ly6G antibodies, right); d) Quantitation of neutrophil count in the BAL. Error bars represent standard error of the mean. Neutrophil depletion in the LPS-exposed mice significantly attenuated the number of neutrophils in BAL.
- Supplementary figure S3 - Inflammatory cytokine concentration of a) keratinocyte chemoattractant (KC), b) monocyte chemotactic protein-1 (MCP-1), c) interleukin-6 (IL-6), and d) tumor necrosis factor alpha (TNF-α) in mouse plasma 24 hours after intratracheal exposure to saline (N=4) or LPS (N=4); error bars represent the standard errors.
- Supplementary figure S4 - Acute plaque complications due to LPS lung exposure: a) The edge region of plaque in LPS-exposed mice displayed blood aggregated on the surface of plaque and mixed with plaque tissue (H&E). Loss of endothelium integrity was observed at the edge of plaque. b) The necrotic core region in the plaque of LPS treated mice showed intraplaque hemorrhage. c) Immunofluorescent staining of thrombin (green) in the plaque is shown. d) Both intraplaque hemorrhage and luminal thrombus were detected in the BCT of LPS-exposed mice by OPT (high relative intensity signals are demarcated by a blue line, vessel wall is demarked by a pink line, and the perimeter of the plaque is demarcated by a green line). e) 3-D OPT cross-sectional image of a plaque containing hemorrhage (Green: lower intensity blood signal, Yellow: medium intensity blood signal; Red: highest intensity blood signal). f) Immunfluorescent staining of MMP-9 (green) of plaque in LPS-exposed mice (left) and saline-exposed mice (right). There was MMP-9 in the sub-endothelial region and in areas where plaque had been perturbed; in contrast, there was no MMP-9 signal detected in saline treated plaque. g) Luminal blood clots (high intensity signal) were detected on plaque shoulders of BCT and extended into aortic arch in the LPS lung exposed animal.
- Supplementary figure S5 - a) A representative auto-fluorescence intensity map of mouse BCT from LPS-Ctrl group demonstrating intraplaque high intensity signal consistent with intraplaque hemorrhage (left) and that from LPS-ND group demonstrating lower signal intensity consistent with a stable plaque (right). b) A representative OPT cross-sectional image of mouse BCT from LPS-Ctrl group that shows high intensity signal at the shoulder region consistent with intraplaque hemorrhage (left) and that from LPS-ND group that shows a stable phenotype (right). c) An OPT cross-sectional image from the proximal end of BCT from a LPS-Ctrl mouse that shows a luminal blood clot at the edge of a plaque. d) OPT cross-sectional images from the proximal end of BCT of a mouse injected with control antibodies (Ctrl, left) and of a mouse injected with neutrophil-specific antibody (ND, right). Both images show a stable plaque phenotype with no features of vulnerability. e) Immunofluorescent staining of MPO (green) and CD68 (red) in cross-sections of BCT from LPS-exposed mice that shows MPO+ polymorphonuclear cells and CD68+ macrophages localized at the edges of a plaque, and f) these MPO+ polymorphonuclear cells (green) also observed in the plaques of LPS-Ctrl mice (mouse treated with control antibodies) g) An confocal image of representative neutrophil stained with MPO (green) found in an area of plaque rupture.
- Supplementary figure S6 - Inflammatory cell counts from the bronchoalveolar lavage (BAL); Error bars represent standard error of the mean: a) BAL neutrophil counts from saline group, Ctrl group (with injections of control IgG antibodies), and ND group (injections of neutrophil-specific antibodies) are shown. Injections of antibodies into mice in vivo did not induce neutrophil lung infiltration. b) Quantitation of macrophage in the BAL of mice in the saline group, Ctrl group, ND group, LPS group, LPS-Ctrl group (LPS group with injections of control IgG antibodies), and LPS-ND group (LPS group with injections of neutrophil specific antibodies. c) Comparisons of BAL neutrophil counts between IT (intratracheal) route and IP (intraperitoneal) route at the 8 hour time point. LPS exposure via IT route induced neutrophil lung inflammation, but not LPS exposure via IP route. d) There were no significant changes in the macrophage counts between these groups. Eosinophil and basophil counts were consistently rare (less than 1% of the total cell counts); thus, they were not included in these data.
- Supplementary figure S7 - Circulating white blood cell counts from IT (intratracheal) and IP (intraperitoneal) groups are shown. Error bars represent the standard errors: a) Total white blood cells, b) neutrophils, c) lymphocytes, and d) monocytes in mouse blood 8 hours after saline and LPS administration demonstrated a similar pattern change. Eosinophil and basophil counts were consistently rare (less than 1% of the total cell counts); thus, they were not included in these data.
- Supplementary video S1 - 3D presentation of mouse aortic arch containing brachiocephalic trunk (BCT) from a LPS-exposed mouse: Blood (red color) was auto-fluorescent 10 times higher than the vessel wall (blue color). Blood clot in LPS mice was associated with shoulders of ruptured plaque in BCT.
- Supplementary video S2 - 3D presentation of mouse aortic arch and brachiocephalic trunk (BCT) from a saline-exposed mouse: No blood clot or intraplaque hemorrhage was observed in the atheroma of BCT.
- Supplementary video S3 - 3D presentation of aortic arch containing brachiocephalic trunk (BCT) from another LPS-exposed mouse that displayed intraplaque hemorrhage (red color) inside atheroma.
- Supplementary video S4 - 3D presentation of aortic arch containing brachiocephalic trunk (BCT) from a different LPS-exposed mouse: Blood clots (color red) localized in the plaque shoulders at the proximal end of BCT and extended into the aortic arch.
- Supplementary video S5 - 3D presentation of brachiocephalic trunk (BCT) from LPS-exposed mice treated with neutrophil-specific antibodies (LPS-ND, left) and control antibodies (LPS-Ctrl, right): Blood clots (color red) localized at the plaque shoulders of BCT in the LPS-Ctrl group but not in the LPS-ND group.