Article Figures & Data
Figures
- FIG 1
HFD feeding promotes microbiota dysbiosis in Ldlr−/− mice as revealed by cecal 16S ribosomal DNA amplicon sequencing. (A) Principal coordinate analysis reveals distinct separation in microbiota composition due to diet (P = 0.003 for control diet [CD] versus high-fat diet [HFD]). LDLR, LDL receptor. (B) Linear discriminant analysis (LDA score) effect size (LEfSe) highlights differentially abundant microbial taxa (CD versus HFD). (C and D) Reduced alpha diversity in HFD versus CD using Chao richness (C) and Shannon entropy (D) metrics. (E) HFD-induced changes in microbiome composition on the phylum level. (F) Firmicutes/Bacteroidetes ratio in CD and HFD samples. In panels A, C, D, and F, the sex of the mice fed CD (triangles) and HFD (circles) is color coded (females [red] and males [blue]). In panels C, D, and F, means ± standard errors of the means (SEM) (error bars) are shown for the groups. Independent samples from mice fed HFD versus CD were compared by Student t tests, and values that were significantly different are indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.01; ****, P < 0.0001.
- FIG 2
Familial hypercholesterolemia is increased in GF Ldlr−/− mice on control diet (CD), but both cholesterol levels and atherosclerotic lesion size in the carotid artery are unchanged after 16 weeks on high-fat diet (HFD) for GF Ldlr−/− and CONV-R Ldlr−/− mice. (A and B) Total cholesterol level and fractioned cholesterol levels (VLDL, LDL, and HDL) of GF Ldlr−/− and CONV-R Ldlr−/− mice on CD (8 CONV-R mice; 6 GF mice) (A) and HFD (9 mice/group) (B). For the chromatogram, representative lipoprotein profiles detected in CONV-R (black lines) and GF (purple lines) mice are shown. OD, optical density. (C, left) Representative histology images showing hematoxylin-and-eosin-stained sections of carotid artery plaques in GF and CONV-R Ldlr−/− mice on HFD. Bars, 1 mm. (Right) Relative atherosclerotic plaque size (as a percentage) of the carotid artery of GF (17 mice [9 females and 8 males]) and CONV-R (17 mice [10 females and 7 males]) Ldlr−/− mice after 16 weeks on a HFD, split by sex. Means ± SEM (error bars) are shown for the groups. Independent samples were compared by Student t tests. Values that were significantly different are indicated by bars and asterisks as follows: *, P < 0.05; **, P < 0.01. Values that were not significantly different (n.s.) are indicated. (D and E) Correlation between age and the absolute plaque size (in square micrometers) (D) or relative plaque size (as a percentage) (E) at the carotid artery. For all panels, data for CONV-R mice are shown as gray dots, and data for GF animals are shown as white dots. The sex of the mice is color coded as follows: females in red and males in blue.
- FIG 3
GF Ldlr−/− mice show reduced vascular inflammation after 16 weeks on HFD. (A to D) Blood cell counts of total leukocytes (18 or 19 mice/group) (A) and percentage of CD45+ cells: monocytes (16 to 18 mice/group) (B), neutrophils (15 to 19 mice/group) (C), and lymphocytes (18 or 19 mice/group) (D) from GF and CONV-R Ldlr−/− mice on HFD for 16 weeks, analyzed by flow cytometry. Means ± SEM (error bars) are shown for the groups. (E) Multiplex cytokine ELISA quantification of mouse plasma samples (6 to 16 mice/group). (F) Intravital epifluorescence video microscopy of endothelial adherent and rolling leukocytes (green) in the common carotid artery of GF and CONV-R Ldlr−/− mice on HFD for 16 weeks (11 to 16 mice/group). Nucleated cells were visualized with acridine orange. Means ± SEM are shown for the groups. Independent samples were compared by Student t tests. Statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001. For all panels, data for CONV-R mice are shown in gray, and data for GF animals are shown in white. For panels A to D, the sex of the mice is color coded as follows: females in red and males in blue.
- FIG 4
Reduced FeCl3-induced carotid artery occlusion in GF C57BL/6J mice and reduced plaque rupture-induced thrombogenicity in GF Ldlr−/− mice. (A) Intravital epifluorescence video microscopy of thrombus formation (DCF-stained platelets [green]) at 0, 6, and 12 min in the 10% FeCl3-injured common carotid artery of GF (12 mice) and CONV-R (13 mice) C57BL/6J wild-type mice (representative images) with analysis of occlusion times. (B) Intravital epifluorescence video microscopy of thrombus formation (Rhodamin B-stained platelets [red]) in the common carotid artery of GF (7 mice) and CONV-R (13 mice) Ldlr−/− mice on HFD after plaque rupture induced by 5 min of ultrasound. Means ± SEM are shown for the groups. Independent samples were analyzed by Student t tests. Statistical significance was indicated by asterisks as follows: *, P < 0.05; **, P < 0.01. For all panels, CONV-R mice are shown in gray, and GF animals are shown as white dots.
- FIG 5
Standardized whole-blood flow chamber analysis for platelet deposition on collagen type I (A) and collagen type III (B). End-stage representative images of whole-blood platelet deposits after 3.5 min on collagen type I (A), and collagen type III (B). (C) Subtraction heatmap of HFD-fed GF Ldlr−/− mice (12 mice/group) compared to CONV-R Ldlr−/− mice (11 mice/group) (not shown). The degree of reduction relative to CONV-R Ldlr−/− mice is indicated in green (see scale panel). The analyzed parameters are as follows: 1, morphological score; 2, platelet surface area coverage; 3, thrombus contraction score; 4, multilayer score; 5, thrombus surface area coverage; 6, phosphatidylserine exposure; 7, P-selectin expression; 8, integrin αIIbβ3 (GPIIbIIIa) activation. (D and E) Descriptive statistics of HFD-fed GF and CONV-R Ldlr−/− mice on collagen type I (D) and collagen type III (E) are shown only for significant results. Means ± SEM are shown for the groups. Independent samples were tested by Student t tests. Statistical significance is indicated as follows: *, P < 0.05; ****, P < 0.0001. For all panels, data for CONV-R mice are shown as gray dots, and data for GF animals are shown as white dots. The sex of the mice is color coded as follows: females in red and males in blue.
- FIG 6
Effects of gut microbiota on late carotid artery atherosclerosis and atherothrombosis. (A) For this study, either GF or CONV-R Ldlr−/− mice on a conventional diet (CD) were fed for 16 weeks with a high-fat diet (HFD), thus resulting in the onset and progression of atherosclerosis. For CONV-R Ldlr−/− mice, the HFD yielded a reduced diversity of the commensal microbiota, with an increase in the Firmicutes/Bacteroidetes ratio. Interestingly, when fed with a CD, GF Ldlr−/− mice presented higher cholesterol levels with respect to CONV-R counterparts. (B) After cholesterol accumulation, resulting from deposition of VLDL and LDL lipoproteins, an atherosclerotic plaque presents a subendothelial lipid core with infiltrating leukocytes characterized by the accumulation of smooth muscle cells (SMC) from the tunica intima and tunica adventitia of the blood vessels. Although the relative plaque sizes at the carotid artery were unchanged between the two groups, GF animals showed altered vascular inflammatory parameters and immune cell populations. EC, endothelial cells. (C) During atherothrombosis, the plaque rupture yields to platelet deposition to the exposed subendothelial collagen, and subsequently to a bloodstream-circulating thrombus growing on the platelet plug. In this context, GF Ldlr−/− mice fed with a HFD presented lower yields of plaque rupture, collagen adhesion, and thrombus growth. Arterial occlusion time was increased in CD-fed GF C57BL/6J mice compared to CONV-R controls.
Supplemental Material
FIG S1
Sterility controls on the feces from GF Ldlr−/− mice. Lane 1, molecular weight DNA marker; lane 2, feces from GF Ldlr−/− mice; lane 3, positive 16S rDNA control; lane 4, negative (empty) control. Download FIG S1, PDF file, 0.03 MB.
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FIG S2
Taxon summary on the genus level. Download FIG S2, PDF file, 0.2 MB.
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TABLE S1
Differences in microbial taxa between HFD and CD mice. Download Table S1, PDF file, 0.03 MB.
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FIG S3
High-frequency small animal ultrasound of both carotids of GF and CONV-R mice 8 weeks after HFD or CD. (A) Representative ultrasound B-images of the carotid arteries of HFD mice. (B) Measurement of the wall thickness of the anterior carotid wall from the right (RCC) and left common carotid (LCC) arteries. (C) Cumulative atherosclerotic plaque size of RCC (right) and LCC (left) in B-mode. Mean plus SEM (error bars) are shown for the groups (2 to 5 mice per group). For panels B and C, CONV-R mice are shown by gray bars, and GF animals are shown in white bars. Independent samples were tested by Student t test. **, P < 0.01. Download FIG S3, PDF file, 0.3 MB.
Copyright © 2019 Kiouptsi et al.
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TABLE S2
Absolute (μm2) and relative (%) values for atherosclerotic plaque area at the carotid artery and aortic root (zero-level) of the 40 CONV-R (gray) and GF (white) animals fed for 16 weeks with HFD. Animals are color coded as in Fig. 2C and D: males are shown in blue, while females are shown in red. Download Table S2, PDF file, 0.04 MB.
Copyright © 2019 Kiouptsi et al.
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FIG S4
(A) Standardized whole-blood flow chamber analysis for platelet deposition on collagen type I and collagen type III. (A) Subtraction heatmap of control diet (CD)-fed GF Ldlr−/− mice (14 mice/group) compared to CONV-R Ldlr−/− mice (12 mice/group). The degree of reduction relative to CONV-R Ldlr−/− mice is indicated in green. The numbers below the panels indicate the following parameters: 1, morphological score; 2, platelet surface area coverage; 3, thrombus contraction score; 4, multilayer score; 5, thrombus surface area coverage; 6, phosphatidylserine exposure; 7, P-selectin expression; 8, integrin αIIbβ3 (GPIIbIIIa) activation. Independent samples were tested by Student t tests. *, P < 0.05. (B and C) End-stage representative images of whole-blood platelet deposits after 3.5 min on collagen type I (B) and collagen type III (C). Download FIG S4, PDF file, 0.7 MB.
Copyright © 2019 Kiouptsi et al.
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FIG S5
Descriptive statistics on the standardized whole-blood flow chamber analysis for platelet deposition of HFD-fed GF Ldlr−/− and CONV-R Ldlr−/− mice on collagen type I and collagen type III. Means ± SEM are shown for groups. For all the panels, CONV-R animals are shown as black dots, while GF animals are shown as white dots. Download FIG S5, PDF file, 0.1 MB.
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FIG S6
Descriptive statistics on the standardized whole-blood flow chamber analysis for platelet deposition of CD-fed (control diet fed) GF Ldlr−/− and CONV-R Ldlr−/− mice on collagen type I and collagen type III. Means ± SEM are shown. Independent samples were tested by Student t tests. **, P < 0.01. For all the panels, CONV-R animals are shown as black triangles, while GF animals are shown as white triangles. Download FIG S6, PDF file, 0.1 MB.
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TEXT S1
Commands. Download TEXT S1, DOCX file, 0.1 MB.
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