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. 2018 Oct 10;8(1):15106.
doi: 10.1038/s41598-018-32584-y.

Predilection of Low Protein C-induced Spontaneous Atherothrombosis for the Right Coronary Sinus in Apolipoprotein E deficient mice

Marco Heestermans  1   2 , Amber B Ouweneel  3 , Jasmin Hassan  3 , Meander Kloosterman  1   2 , Pieter H Reitsma  1   2 , Marion J J Gijbels  4   5 , Bart J M van Vlijmen  1   2 , Miranda van Eck  6
Affiliations

Predilection of Low Protein C-induced Spontaneous Atherothrombosis for the Right Coronary Sinus in Apolipoprotein E deficient mice

Marco Heestermans et al. Sci Rep. .

Abstract

Silencing of anticoagulant protein C using RNA interference (siProc) evokes low incident but spontaneous atherothrombosis in the aortic root of apolipoprotein E-deficient (Apoe-/-) mice. The aims of the current study were (1) to analyze if plaque characteristics or circulating factors could be linked to atherothrombosis susceptibility, (2) to increase the incidence of atherothrombosis by transiently increasing blood pressure, and (3) to direct atherothrombosis to an additional predefined vascular site by applying a semi-constrictive collar around the carotid artery. siProc-driven spontaneous atherothrombosis in the aortic root of Apoe-/- mice was reproduced and occurred at an incidence of 23% (9 out of 39 mice), while the incidence of collar-induced atherothrombosis in the carotid artery was 2.6% (1 out of 39 mice). Treatment with phenylephrine, to transiently increase blood pressure, did not increase atherothrombosis in the aortic root of the Apoe-/- mice nor in the carotid arteries with collars. Plaques in the aortic root with an associated thrombus were lower in collagen and macrophage content, and mice with atherothrombosis had significantly more circulating platelets. Plasma protein C, white blood cell counts, total cholesterol, fibrinogen, serum amyloid A, and IL-6 were not different amongst siProc treated mice with or without thrombosis. Remarkably, our data revealed that thrombus formation preferably occurred on plaques in the right coronary sinus of the aortic root. In conclusion, there is a predilection of low protein C-induced spontaneous atherothrombosis in Apoe-/- mice for the right coronary sinus, a process that is associated with an increase in platelets and plaques lower in collagen and macrophage content.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Atherothrombosis in the aortic root of siProc treated Apoe−/− mice. (A) Representative section of the aortic root of a mouse treated with siNEG. (B) Representative section of the aortic root of a mouse treated with siProc, without a thrombus (siProc − THR). (C and D) Representative section of the aortic root of a mouse treated with siProc, with a thrombus associated with an atherosclerotic plaque (siProc + THR, panel D is a magnification of the black square in panel C). (E) Representative section of the common carotid artery containing an atherosclerotic lesion upon collar placement, derived from an siProc treated mouse. (F) A thrombus-like structure associated with an atherosclerotic lesion in the common carotid artery. All sections are hematoxylin and eosin (HE) stained. The scattered black lines in panels A, B, and C mark borders of the plaque. LCS: Left coronary sinus, NCS: Non-coronary sinus, RCS: Right coronary sinus. *Thrombus. Black bars represent 200 μm.
Figure 2
Figure 2
Overview of all siProc associated atherothrombotic events in the aortic root of Apoe−/− mice. (A) In 5 mice in the siProc group without phenylephrine (siProc − PE) treatment atherothrombosis in the aortic root was observed. (B) In 4 mice in the siProc group with phenylephrine (siProc + PE) treatment atherothrombosis in the aortic root was observed. All sections were HE stained. Black bars represent 500 μm. LCS: Left coronary sinus, NCS: Non-coronary sinus, RCS: Right coronary sinus. *Thrombus.
Figure 3
Figure 3
Proc liver transcript and plasma protein C levels of siNEG and siProc treated Apoe−/− mice. (A) Proc transcript in the liver upon sacrifice (7 days after siProc treatment) compared to the mean value of siNEG treated mice (100%). (B) Plasma protein C levels, measured by ELISA, compared to the mean value of siNEG treated mice (100%). Black bars indicate the median. **P < 0.01 for siNEG vs. siProc. $P < 0.05 for siProc − THR vs. siProc + THR.
Figure 4
Figure 4
Blood cell populations of siNEG and siProc treated Apoe−/− mice. Blood cell numbers from the siProc treated group is divided in plaques without a thrombus (siProc − THR) and plaques containing a thrombus (siProc + THR). (A) Total white blood cells, (B) neutrophils, (C) lymphocytes, (D) monocytes, (E) eosinophils, and (F) red blood cells are displayed. Black bars indicate the median. No significant differences between the groups were scored.
Figure 5
Figure 5
Blood platelet levels and plasma analysis of siNEG and siProc treated Apoe−/− mice. (A) Total blood platelet levels, (B) plasma total cholesterol levels, (C) plasma fibrinogen levels, measured by ELISA, and expressed as % of normal pool plasma. We did not have any reason to exclude outliers with a low value (e.g. due to coagulation upon blood withdrawal). (D) Serum Amyloid A levels, and (E) plasma IL-6 levels (negative measurements were set to 0 pg/mL). Black bars indicate the median. **P < 0.01 for siNEG vs. siProc. $P < 0.05 for siProc − THR vs. siProc + THR.
Figure 6
Figure 6
Plaque composition of siNEG and siProc treated Apoe−/− mice. (A,B) Representative photomicrograph of a Masson’s Trichrome stained plaque in the aortic root (RCS) of mice treated with siProc, without (A) and with (B) a thrombus associated to a plaque from the aortic root. Blue areas were assessed as collagen-positive, non-stained areas as necrotic core, and pink nucleated areas as macrophage content/foam cells. The scattered black lines mark borders of the plaque. Black bars represent 200 μm. (C–F) Individual plaques from the siProc treated group are divided in plaques without a thrombus (siProc − THR) and plaques containing a thrombus (siProc + THR). (C) Total plaque area, (D) collagen, (E) cellular content, (F) necrotic core. For all panels, the indicated values represent an average measurement of three sections. Black bars indicate the median. $P < 0.05 for siProc − THR vs. siProc + THR.
Figure 7
Figure 7
siProc induced atherothrombosis in the different sinuses of the aortic root of Apoe−/− mice. (A) HE stained section of the aortic root and surrounding tissues. The non-coronary sinus (NCS) of the aortic root is covered by the two atria of the heart (atria are indicated with HA). Left and right of the NCS the left and right coronary sinuses (LCS and RCS, respectively) are present, which are both partly covered by a muscle-rich area, from which the largest part is the right ventricle (indicated with RV). *Thrombus. The scattered black lines mark borders of the plaque. Black bar represents 500 μm. (B) Overview of three independent studies, where atherothrombosis in the aortic root occurred upon siProc treatment. *P < 0.05, **P < 0.01.
Figure 8
Figure 8
Plaque composition of the LCS, NCS, and LCS. Individual plaques from all groups were pooled and analyzed for (A) total plaque area, (B) collagen, (C) cellular content, (D) and necrotic core. For all panels, the indicated values represent an average measurement of three sections. Black bars indicate the median. LCS: Left coronary sinus, NCS: Non-coronary sinus, RCS: Right coronary sinus. **P < 0.01.
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