Silencing of Anticoagulant Protein C Evokes Low-Incident but Spontaneous Atherothrombosis in Apolipoprotein E–Deficient Mice—Brief ReportHighlights
Objective—Murine atherosclerosis models do not spontaneously develop atherothrombotic complications. We investigated whether disruption of natural anticoagulation allows preexisting atherosclerotic plaques to progress toward an atherothrombotic phenotype.
Approach and Results—On lowering of plasma protein C levels with small interfering RNA (siProc) in 8-week Western-type diet–fed atherosclerotic apolipoprotein E–deficient mice, 1 out of 4 mice displayed a large, organized, and fibrin- and leukocyte-rich thrombus on top of an advanced atherosclerotic plaque located in the aortic root. Although again at low incidence (3 in 25), comparable thrombi at the same location were observed during a second independent experiment in 9-week Western-type diet–fed apolipoprotein E–deficient mice. Mice with thrombi on their atherosclerotic plaques did not show other abnormalities and had equally lowered plasma protein C levels as siProc-treated apolipoprotein E–deficient mice without thrombi. Fibrinogen and thrombin–antithrombin concentrations and blood platelet numbers were also comparable, and plaques in siProc mice with thrombi had a similar composition and size as plaques in siProc mice without thrombi. Seven out of 25 siProc mice featured clots in the left atrium of the heart.
Conclusions—Our findings indicate that small interfering RNA–mediated silencing of protein C in apolipoprotein E–deficient mice creates a condition that allows the occurrence of spontaneous atherothrombosis, albeit at a low incidence. Lowering natural anticoagulation in atherosclerosis models may help to discover factors that increase atherothrombotic complications.
Atherothrombosis, characterized by superimposed luminal thrombus formation on a ruptured or eroded atherosclerotic plaque, is a major cause of acute coronary syndromes and cardiovascular death in humans.1 Pathological changes of the atherosclerotic plaque, such as thinning of the fibrous cap and the development of a large necrotic core, are known to make the plaque prone to rupture and, thereby, expose triggers for thrombosis.2
Murine models of atherosclerosis, such as hyperlipidemic apolipoprotein E–deficient (Apoe−/−) mice, have been instrumental to study atherosclerosis pathophysiology and the search for highly needed novel therapeutic targets. However, in these murine models, the final stage of atherosclerosis, that is, plaque rupture and the subsequent induction of atherothrombosis, does not occur spontaneously. Factors suggested to underlie the absence of spontaneous atherothrombosis include resistance of murine plaques to rupture because of a different plaque composition and differences in hemodynamics.3 Moreover, species differences in anticoagulation could contribute to the absence of progression to atherothrombosis in mice. The half-life of active coagulation factor IIa in mouse plasma is significantly shorter as compared with its human counterpart, pointing toward more potent natural anticoagulation in mice.4 In line, atherosclerotic plaques in hyperlipidemic mice with impaired hemostasis revealed markers of thrombotic events in carotid artery plaques.5,6
In the present study, we tested whether small interfering (si) RNA–mediated lowering of natural anticoagulants antithrombin (Serpinc1) and protein C (Proc) in Apoe−/− mice allowed plaques to progress toward an atherothrombotic phenotype. We found that lowering of Proc evokes low-incident spontaneous atherothrombosis in Apoe−/− mice.
Materials and Methods
Materials and Methods are available in the online-only Data Supplement.
Mouse (C57BL/6J) hepatic Serpinc1 and Proc expression can be effectively lowered using specific siRNAs.7 To investigate whether this siRNA approach also allowed successful downregulation of both anticoagulants in a hyperlipidemic background, Apoe−/− mice fed a Western-type diet for 2 weeks were injected with siRNA targeting Serpinc1 (siSerpinc1), Proc (siProc), or negative control siRNA (siNEG; 9 mice per group) and monitored for 7 days (experiment 1; median plasma total cholesterol levels 7.2 mg/mL; range, 4.4–12.7; Figure I in the online-only Data Supplement). Within 4 days after siRNA injection, several siSerpinc1 mice demonstrated lethargy, weight loss (1.5 g; 0.1–2.4), exophthalmos, periocular hemorrhages, and swelling of the mandibular area. Two mice died within this time frame. This phenotype has been described before in wild-type C57BL/6J mice treated with siSerpinc1 and siProc and is likely spontaneous venous thrombosis.7 In contrast to the siSerpinc1 group, all Western-type diet–fed Apoe−/− mice that received only siProc remained grossly healthy for ≤7 days. Liver transcript analysis showed that the siSerpinc1 mice had a Serpinc1 transcript level of 6.0% (4.4–11.4; day 3; n=3) of the level in siNEG mice and 3.4% (3.2–3.7; n=3) compared with that in non–siRNA-treated mice. Proc transcript levels in the siProc mice were 21.2% of siNEG (17.3–27.5; day 7; n=4) and 10.7% (3.8–17.4, n=3) of non–siRNA-treated mice.
To investigate whether inhibition of anticoagulation affects the atherosclerotic phenotype, Apoe−/− mice were fed Western-type diet for 8 weeks to induce advanced atherosclerosis in the aortic root, prior to siSerpinc1 or siProc treatment. In this experiment (experiment 2; 4 mice per group; siSerpinc1, siProc, and siNEG), mice were euthanized 2 days after siRNA injection, which enabled us to include also siSerpinc1-treated mice to study the impact on the atherosclerotic phenotype. Moreover, the siRNA dosage was increased to achieve a higher knockdown of Proc. Already within 2 days, 1 mouse in the siSerpinc1 group demonstrated the characteristic venous thrombotic coagulopathy, while siProc and siNEG mice again remained healthy. Histological analysis of the head, heart, kidneys, lungs, and liver of the remaining healthy siSerpinc1 mice revealed no abnormalities (including no signs of coagulopathy). The early onset of spontaneous venous thrombosis in Apoe−/− mice precluded studies using siSerpinc1 to inhibit Serpinc1 for a longer time period.
On histological analysis, siProc and siNEG mice appeared identical to the healthy siSerpinc1 mice. However, 1 mouse in the siProc group revealed an organized and large structure superimposed on 1 of the advanced atherosclerotic plaques in the aortic root, which was identified as a fibrin-positive thrombus (Martius Scarlet staining; Figure 1). The thrombus consisted of layers of eosin-positive structures and was infiltrated by leukocytes typically at the luminal side. Serial sections demonstrated that this thrombus was superimposed on the plaque for at least 320 μm (Figure II in the online-only Data Supplement).
Although at a low incidence, the presence of an organized and large thrombus superimposed on an aortic root atherosclerotic plaque is unique and has, to our knowledge, not been reported before. To investigate the reproducibility of this finding, 25 mice were treated solely with siProc (experiment 3). Silencing of Proc for 7 days in atherosclerotic Apoe−/− mice was again well tolerated, and no (macroscopic) abnormalities were seen. Importantly, 3 mice had organized and large fibrin-rich thrombi superimposed on atherosclerotic plaques in the aortic roots, with a similar size and composition as in experiment 2 (Figure 1). Combining this result with experiment 2, a total of 4 out of 29 mice in 2 independent experiments showed atherothrombosis, resulting in a proportion of 13.8% with 95% confidence limits of 5.5% to 30.5%.
Longitudinal sections of the aorta (arch, abdominal, and descending) demonstrated limited advanced atherosclerotic plaques at this location. In 1 mouse (out of 25), we observed Martius Scarlet–positive structures (indicative for fibrin) within an advanced atherosclerotic plaque in the aortic arch (Figure III in the online-only Data Supplement). Lungs, kidneys, spleen, and liver of all siProc mice were subjected to detailed microscopic analysis and did not exhibit any abnormalities or signs of thrombosis. Of note, in 7 out of 25 siProc-treated mice, the left atrium of the heart featured clots (28% with 95% confidence limits of 14.3%–47.6%), composed of fibrin (red) and erythrocytes (green/yellow) without a thrombus-like layered organization (Martius Scarlet staining; Figure IV in the online-only Data Supplement).
The 3 mice with an atherosclerotic plaque–associated thrombus had comparable knockdown of plasma protein C compared with siProc mice without such features (10.1% [4.4–16.5] versus 9.7% [7.6–10.2] of normal pool plasma; P=0.87; Figure 2A). In addition, for the mice with and without plaque-associated thrombosis, blood platelets numbers, plasma fibrinogen, and thrombin–antithrombin levels were comparable and in the normal range (Figure 2B through 2D), indicating that the atherothrombotic events in the aortic root did not coincide with, or are part of, a consumptive coagulopathy. Furthermore, plaques with a superimposed thrombus were of similar size and composition (collagen, necrotic core, and CD68-positive area; Figure 2E through 2H) as plaques without a thrombus.
In the present study, we have demonstrated that silencing the natural anticoagulant Proc in Apoe−/− mice evokes low incidence atherothrombosis in the aortic root. While our brief report was reviewed, data from another independent mouse experiment became available, again showing the unique spontaneous, low-incident atherothrombosis phenotype in the aortic root of Apoe−/− mice after Proc silencing (25%), highlighting that our original observations are reproducible. Our findings concur with other studies in which atherosclerotic plaques in hyperlipidemic mice with a stronger coagulation or an impaired anticoagulation were positive for markers of thrombotic events.5,6 Altogether, these data indicate that a strong natural anticoagulation system protects mice against atherothrombosis. For now, the low incidence of low protein C–associated atherothrombosis precludes (1) detailing the mechanism why some mice and some plaques trigger thrombosis formation and (2) using the current concept as a mouse model for validating antiatherothrombotic drugs or genes. We speculate that similar as in humans, the plaques that are associated with atherothrombosis in low protein C mice are those that have a thin fibrous cap, demonstrate plaque erosion, and are highly inflamed. Possible ways to increase the incidence of atherothrombosis may be to study the effects of siProc treatment in mice with even more advanced atherosclerosis or introduce a second hit which is thought to predispose to atherothrombosis (eg, higher blood pressure, stress, and genetic modifications). Future studies should clarify this.
Lowering antithrombin resulted in spontaneous venous thrombosis, which was unexpected given studies in normal C57BL/6 mice,7 but no atherothrombosis. Although it is tempting to speculate on different roles in vivo for antithrombin and protein C in atherothrombosis, the extensive venous thrombosis in the siSerpinc1 group precluded the study of this important anticoagulant in the potential protection against thrombosis in the arterial system, like protein C.
In several siProc-treated mice, signs of clotting were observed in the left atrium, a phenotype that has not been reported before in hyperlipidemic or hemostasis mouse models. The absence of clots in major organs and normal levels of blood coagulation parameters in siProc mice indicates that this clot formation is a local cardiac event and does not represent siProc-mediated disseminated intravascular coagulopathy. Interestingly, Pepler et al reported cardiac clotting, albeit in the ventricle, when providing a procoagulant challenge to endothelial protein C receptor (Epcr) mutant mice.8 In addition, lethal perinatal thrombosis in FvQ/Q (mice with a homozygous R504Q mutation in the Fv gene) mice on a 129Sv genetic background has been reported, including features of thrombus formation in the left atrium of the heart.9 Altogether, these data suggest that protein C plays a role in the prevention of cardiac clotting.
In conclusion, our findings indicate that siRNA-mediated silencing of Proc in Apoe−/− mice creates a condition that allows the formation of spontaneous atherothrombosis, albeit at low incidence. Our unique approach may be of value as a tool to identify factors that increase atherothrombotic complications.
Sources of Funding
This work was supported by VICI grant 91813603 from the Netherlands Organization for Scientific Research awarded to M. Van Eck. M. Van Eck is an Established Investigator of the Dutch Heart Foundation (grant number 2007T056).
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.117.309188/-/DC1.
- Received October 14, 2016.
- Accepted March 1, 2017.
- © 2017 American Heart Association, Inc.
- Davies MJ
- Jackson CL,
- Benbow U,
- Galley DJ,
- Karanam S
- Borissoff JI,
- Otten JJ,
- Heeneman S,
- et al
- Safdar H,
- Cheung KL,
- Salvatori D,
- Versteeg HH,
- Laghmani el H,
- Wagenaar GT,
- Reitsma PH,
- van Vlijmen BJ
- Pepler L,
- Yu P,
- Dwivedi DJ,
- Trigatti BL,
- Liaw PC
- Cui J,
- Eitzman DT,
- Westrick RJ,
- Christie PD,
- Xu ZJ,
- Yang AY,
- Purkayastha AA,
- Yang TL,
- Metz AL,
- Gallagher KP,
- Tyson JA,
- Rosenberg RD,
- Ginsburg D
Synthetic small interfering RNAs targeting anticoagulants protein C and antithrombin allows transient lowering of these plasma proteins in Apoe−/− mice.
Small interfering RNA–mediated silencing of protein C in atherosclerotic Apoe−/− mice induces spontaneous, low incidence thrombus formation associated with atherosclerotic plaques.
Our data indicate that protein C protects mice from atherothrombosis.