Nitric oxide donor molsidomine favors features of atherosclerotic plaque stability and reduces myocardial infarction in mice.

Nitric oxide (NO) donors are commonly used for the prevention and treatment of ischemic heart disease. Besides their effects on the heart, NO donors may also prevent hypoxic brain damage and exert beneficial effects on atherosclerosis by favoring features of plaque stability. We recently described that apolipoprotein E (ApoE) deficient mice with a mutation in the fibrillin-1 (Fbn1) gene (ApoE-/-Fbn1C1039G+/-) develop accelerated atherosclerosis, plaque rupture, myocardial infarction, cerebral hypoxia and sudden death. In the present study, we evaluated the effects of chronic treatment with the NO donor molsidomine on atherosclerotic plaque stability, cardiac function, neurological symptoms and survival in the ApoE-/-Fbn1C1039G+/- mouse model. Female ApoE-/-Fbn1C1039G+/- mice were fed a Western diet (WD). After 8 weeks of WD, the mice were divided into two groups receiving either molsidomine via the drinking water (1 mg/kg/day; n = 34) or tap water (control; n = 36) until 25 weeks of WD. Survival tended to increase after molsidomine treatment (68% vs. 58% in controls). Importantly, atherosclerotic plaques of molsidomine-treated mice had a thicker fibrous cap (11.1 ±â€¯1.2 vs. 8.1 ±â€¯0.7 µm) and showed an increased occurrence of plaque macrocalcifications (30% vs. 0%), indicative of a more stable phenotype. Molsidomine also improved cardiac function, as fractional shortening was increased (40 ±â€¯2% vs. 27 ±â€¯2%) combined with a decreased end diastolic (3.1 ±â€¯0.2 vs. 3.9 ±â€¯0.2 mm) and end systolic diameter (1.9 ±â€¯0.1 vs. 2.9 ±â€¯0.2 mm). Furthermore, perivascular fibrosis (23 ±â€¯2 vs. 30 ±â€¯2%) and the occurrence of myocardial infarctions (12% vs. 36%) was significantly reduced. Track width, a measure of the animal's hind limb base of support and representative of hypoxic brain damage, was also normalized as a result of molsidomine treatment (2.54 ±â€¯0.04 vs. 2.91 ±â€¯0.09 cm in controls). These findings demonstrate that the NO donor molsidomine improves cardiac function, reduces neurological symptoms and enhances atherosclerotic plaque stability.

Animal models of atherosclerosis.

An ideal animal model of atherosclerosis resembles human anatomy and pathophysiology and has the potential to be used in medical and pharmaceutical research to obtain results that can be extrapolated to human medicine. Moreover, it must be easy to acquire, can be maintained at a reasonable cost, is easy to handle and shares the topography of the lesions with humans. In general, animal models of atherosclerosis are based on accelerated plaque formation due to a cholesterol-rich/Western-type diet, manipulation of genes involved in the cholesterol metabolism, and the introduction of additional risk factors for atherosclerosis. Mouse and rabbit models have been mostly used, followed by pigs and non-human primates. Each of these models has its advantages and limitations. The mouse has become the predominant species to study experimental atherosclerosis because of its rapid reproduction, ease of genetic manipulation and its ability to monitor atherogenesis in a reasonable time frame. Both Apolipoprotein E deficient (ApoE-/-) and LDL-receptor (LDLr) knockout mice have been frequently used, but also ApoE/LDLr double-knockout, ApoE3-Leiden and PCSK9-AAV mice are valuable tools in atherosclerosis research. However, a great challenge was the development of a model in which intra-plaque microvessels, haemorrhages, spontaneous atherosclerotic plaque ruptures, myocardial infarction and sudden death occur consistently. These features are present in ApoE-/-Fbn1C1039G+/- mice, which can be used as a validated model in pre-clinical studies to evaluate novel plaque-stabilizing drugs.

Vulnerable plaque detection and quantification with gold particle-enhanced computed tomography in atherosclerotic mouse models.

Recently, an apolipoprotein E-deficient (ApoE-/-) mouse model with a mutation (C1039G+/-) in the fibrillin-1 (Fbn1) gene (ApoE-/-Fbn1C1039G+/- mouse model) was developed showing vulnerable atherosclerotic plaques, prone to rupture, in contrast to the ApoE-/- mouse model, where mainly stable plaques are present. One indicator of plaque vulnerability is the level of macrophage infiltration. Therefore, this study aimed to measure and quantify in vivo the macrophage infiltration related to plaque development and progression. For this purpose, 5-weekly consecutive gold nanoparticle-enhanced micro-computed tomography (microCT) scans were acquired. Histology confirmed that the presence of contrast agent coincided with the presence of macrophages. Based on the microCT scans, regions of the artery wall with contrast agent present were calculated and visualized in three dimensions. From this information, the contrast-enhanced area and contrast-enhanced centerline length were calculated for the branches of the carotid bifurcation (common, external, and internal carotid arteries). Statistical analysis showed a more rapid development and a larger extent of plaques in the ApoE-/-Fbn1C1039G+/- compared to the ApoE-/- mice. Regional differences between the branches were also observable and quantifiable. We developed and applied a methodology based on gold particle-enhanced microCT to visualize the presence of macrophages in atherosclerotic plaques in vivo.

Impaired gait pattern as a sensitive tool to assess hypoxic brain damage in a novel mouse model of atherosclerotic plaque rupture.

Apolipoprotein E deficient (ApoE(-/-)) mice with a heterozygous mutation in the fibrillin-1 gene (Fbn1(C1039G+/-)) show spontaneous atherosclerotic plaque ruptures, disturbances in cerebral flow and sudden death when fed a Western-type diet (WD). The present study focused on motor coordination and spatial learning of ApoE(-/-) Fbn1(C1039G+/-) mice on WD for 20 weeks (n=21). ApoE(-/-) mice on WD (n=24) and ApoE(-/-) Fbn1(C1039G+/-) mice on normal diet (ND, n=21) served as controls. Starting from 10 weeks of diet, coordination was assessed every two weeks by the following tests: gait analysis, stationary beam, wire suspension and accelerating rotarod. The Morris water maze test was performed after 13 weeks of diet to study spatial learning. At the end of the experiment (20 weeks of WD), the mice were sacrificed and the brachiocephalic artery and brain were isolated. From 12 weeks onward, gait analysis of ApoE(-/-) Fbn1(C1039G+/-) mice on WD revealed a progressive increase in track width as compared to ApoE(-/-) mice on WD and ApoE(-/-) Fbn1(C1039G+/-) mice on ND (at 20 weeks: 29.8±0.6 mm vs. 25.8±0.4 mm and 26.0±0.5 mm). Moreover, the stationary beam test showed a decrease in motor coordination of ApoE(-/-) Fbn1(C1039G+/-) mice on WD at 18 and 20 weeks. The wire suspension test and accelerating rotarod could not detect signs of motor impairment. Spatial learning was also not affected. Histological analysis of the brachiocephalic artery showed larger and more stenotic plaques in ApoE(-/-) Fbn1(C1039G+/-) mice on WD. Furthermore, the parietal cortex of ApoE(-/-) Fbn1(C1039G+/-) mice on WD showed pyknotic nuclei as a sign of hypoxia and the percentage of pyknosis correlated with track width. In conclusion, gait analysis may be an efficient method for analyzing hypoxic brain damage in the ApoE(-/-) Fbn1(C1039G+/-) mouse model. This test could be of value to assess the effect of potential anti-atherosclerotic therapies in mice.

Dissecting abdominal aortic aneurysm in Ang II-infused mice: suprarenal branch ruptures and apparent luminal dilatation.

AIMS: In this work, we provide novel insight into the morphology of dissecting abdominal aortic aneurysms in angiotensin II-infused mice. We demonstrate why they exhibit a large variation in shape and, unlike their human counterparts, are located suprarenally rather than infrarenally. METHODS AND RESULTS: We combined synchrotron-based, ultra-high resolution ex vivo imaging (phase contrast X-Ray tomographic microscopy) with in vivo imaging (high-frequency ultrasound and contrast-enhanced micro-CT) and image-guided histology. In all mice, we observed a tear in the tunica media of the abdominal aorta near the ostium of the celiac artery. Independently we found that, unlike the gradual luminal expansion typical for human aneurysms, the outer diameter increase of angiotensin II-induced dissecting aneurysms in mice was related to one or several intramural haematomas. These were caused by ruptures of the tunica media near the ostium of small suprarenal side branches, which had never been detected by the established small animal imaging techniques. The tear near the celiac artery led to apparent luminal dilatation, while the intramural haematoma led to a dissection of the tunica adventitia on the left suprarenal side of the aorta. The number of ruptured branches was higher in those aneurysms that extended into the thoracic aorta, which explained the observed variability in aneurysm shape. CONCLUSION: Our results are the first to describe apparent luminal dilatation, suprarenal branch ruptures, and intramural haematoma formation in dissecting abdominal aortic aneurysms in mice. Moreover, we validate and demonstrate the vast potential of phase contrast X-ray tomographic microscopy in cardiovascular small animal applications.

Elastin fragmentation in atherosclerotic mice leads to intraplaque neovascularization, plaque rupture, myocardial infarction, stroke, and sudden death.

AIMS: There is a need for animal models of plaque rupture. We previously reported that elastin fragmentation, due to a mutation (C1039G(+/-)) in the fibrillin-1 (Fbn1) gene, promotes atherogenesis and a highly unstable plaque phenotype in apolipoprotein E deficient (ApoE(-/-)) mice on a Western-type diet (WD). Here, we investigated whether plaque rupture occurred in ApoE(-/-)Fbn1(C1039G+/-) mice and was associated with myocardial infarction, stroke, and sudden death. METHODS AND RESULTS: Female ApoE(-/-)Fbn1(C1039G+/-) and ApoE(-/-) mice were fed a WD for up to 35 weeks. Compared to ApoE(-/-) mice, plaques of ApoE(-/-)Fbn1(C1039G+/-) mice showed a threefold increase in necrotic core size, augmented T-cell infiltration, a decreased collagen I content (70 ± 10%), extensive neovascularization, intraplaque haemorrhage, and a significant increase in matrix metalloproteinase-2, -9, -12, and -13 expression or activity. Plaque rupture was observed in 70% of ascending aortas and in 50% of brachiocephalic arteries of ApoE(-/-)Fbn1(C1039G+/-) mice. In ApoE(-/-) mice, plaque rupture was not seen in ascending aortas and only in 10% of brachiocephalic arteries. Seventy percent of ApoE(-/-)Fbn1(C1039G+/-) mice died suddenly, whereas all ApoE(-/-) mice survived. ApoE(-/-)Fbn1(C1039G+/-) mice showed coronary plaques and myocardial infarction (75% of mice). Furthermore, they displayed head tilt, disorientation, and motor disturbances (66% of cases), disturbed cerebral blood flow (73% of cases; MR angiograms) and brain hypoxia (64% of cases), indicative of stroke. CONCLUSIONS: Elastin fragmentation plays a key role in plaque destabilization and rupture. ApoE(-/-)Fbn1(C1039G+/-) mice represent a unique model of acute plaque rupture with human-like complications.