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Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Químicas, TRUE, pub

Background: Elastolysis and ineffective elastogenesis favor the accumulation of tropoelastin, rather than cross-linked elastin, in atherosclerotic plaques. We developed gadolinium-labeled tropoelastin-specific magnetic resonance contrast agents (Gd-TESMAs) for tropoelastin imaging in animal models. Methods and Results: Two peptides, VVGSPSAQDEASPLS and YPDHVQYTHY were selected to target tropoelastin. In vitro binding, relaxivity, and biodistribution experiments enabled characterization of the probes and selecting the best candidate for in vivo MRI. MRI was performed in atherosclerotic apolipoprotein E-deficient (ApoE-/-) mice and New Zealand white rabbits with stable and rupture-prone plaques using Gd-TESMA. Additionally, human carotid endarterectomy specimens were imaged ex vivo. The VVGSPSAQDEASPLS-based probe discriminated between tropoelastin and cross-linked elastin (64±7% vs 1±2%, P=0.001), had high in vitro relaxivity in solution (r1-free=11.7±0.6mM-1s-1, r1-bound to tropoelastin = 44±1mM-1s-1) and favorable pharmacokinetics. In vivo mice vascular enhancement (4wks=0.13±0.007mm2, 8wks=0.22±0.01mm2, 12wks=0.33±0.01mm2, P<0.001) and R1 relaxation rate (4wks=0.90±0.01 s-1, 8wks=1.40±0.03 s-1, 12wks=1.87±0.04s-1, P[removed], British Heart Foundation, Chilean Agency of Technology and Science, EU’s H2020 research and innovation program, EPSRC Centre for Medical Engineering at King’s College London, Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Químicas, TRUE, pub

OBJECTIVE: Evidence from preclinical and clinical studies has demonstrated that myocardial infarction promotes atherosclerosis progression. The impact of focal vascular inflammation on the progression and phenotype of remote atherosclerosis remains unknown. APPROACH AND RESULTS: We used a novel ApoE−/− knockout mouse model of sustained arterial inflammation, initiated by mechanical injury in the abdominal aorta. Using serial in vivo molecular MRI and ex vivo histology and flow cytometry, we demonstrate that focal arterial inflammation triggered by aortic injury, accelerates atherosclerosis in the remote brachiocephalic artery. The brachiocephalic artery atheroma had distinct histological features including increased plaque size, plaque permeability, necrotic core to collagen ratio, infiltration of more inflammatory monocyte subsets, and reduced collagen content. We also found that arterial inflammation following focal vascular injury evoked a prolonged systemic inflammatory response manifested as a persistent increase in serum IL-6 (interleukin 6). Finally, we demonstrate that 2 therapeutic interventions—pravastatin and minocycline—had distinct anti-inflammatory effects at the plaque and systemic level. CONCLUSIONS: We show for the first time that focal arterial inflammation in response to vascular injury enhances systemic vascular inflammation, accelerates remote atheroma progression and induces plaques more inflamed, lipid-rich, and collagenpoor in the absence of ischemic myocardial injury. This inflammatory cascade is modulated by pravastatin and minocycline treatments, which have anti-inflammatory effects at both plaque and systemic levels that mitigate atheroma progression., Engineering and Physical Sciences Research Council (EPSRC), British Heart Foundation (BHF), King’s BHF Centre for Research Excellence, EPSRC Centre for Medical Engineering, Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Químicas, TRUE, pub

Cardiovascular disease (CVD) remains the leading cause of death in Western countries despite improvements in prevention, diagnosis and treatment. Atherosclerosis is a chronic inflammatory disease that remains clinically silent for many decades. Sudden rupture of “high-risk/vulnerable” plaques has been shown to be responsible for the majority of acute cardiovascular events, including myocardial infarction and stroke. Therefore, early detection of biological processes associated with atherosclerosis progression and plaque instability may improve diagnosis and treatment and help to better monitor the effectiveness of therapeutic interventions. Molecular magnetic resonance imaging (MRI) is a promising tool to detect molecular and cellular changes in the carotid, aortic and coronary vessel wall including endothelial dysfunction, inflammation, vascular remodelling, enzymatic activity, intraplaque haemorrhage and fibrin deposition and thus may allow early detection of unstable lesions and improve the prediction of future coronary events. Evaluation of atherosclerosis at both, the preclinical and clinical level includes non-contrast-enhanced (NCE) and contrast-enhanced (CE) MRI with and without the use of MR contrast agents. To increase the biological information obtained by MRI a variety of targeted-specific molecular probes have been developed for the non-invasive visualization of particular biological processes at the molecular and cellular level. This review will discuss the recent advances in molecular MRI of atherosclerosis, covering both pulse sequence development and also the design of novel contrast agents, for imaging atherosclerotic disease in vivo., Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Químicas, TRUE, pub

Aims Dysfunctional matrix turnover is present at sites of abdominal aortic aneurysm (AAA) and leads to the accumulation of monomeric tropoelastin rather than cross-linked elastin. We used a gadolinium-based tropoelastin-specific magnetic resonance contrast agent (Gd-TESMA) to test whether quantifying regional tropoelastin turnover correlates with aortic expansion in a murine model. The binding of Gd-TESMA to excised human AAA was also assessed. Methods and results We utilized the angiotensin II (Ang II)-infused apolipoprotein E gene knockout (ApoE−/−) murine model of aortic dilation and performed in vivo imaging of tropoelastin by administering Gd-TESMA followed by late gadolinium enhancement (LGE) magnetic resonance imaging (MRI) and T1 mapping at 3 T, with subsequent ex vivo validation. In a cross-sectional study (n = 66; control = 11, infused = 55) we found that Gd-TESMA enhanced MRI was elevated and confined to dilated aortic segments (control: LGE=0.13 ± 0.04 mm2, control R1= 1.1 ± 0.05 s−1 vs. dilated LGE =1.0 ± 0.4 mm2, dilated R1 =2.4 ± 0.9 s−1) and was greater in segments with medium (8.0 ± 3.8 mm3) and large (10.4 ± 4.1 mm3) compared to small (3.6 ± 2.1 mm3) vessel volume. Furthermore, a proof-of-principle longitudinal study (n = 19) using Gd-TESMA enhanced MRI demonstrated a greater proportion of tropoelastin: elastin expression in dilating compared to non-dilating aortas, which correlated with the rate of aortic expansion. Treatment with pravastatin and aspirin (n = 10) did not reduce tropoelastin turnover (0.87 ± 0.3 mm2 vs. 1.0 ± 0.44 mm2) or aortic dilation (4.86 ± 2.44 mm3 vs. 4.0 ± 3.6 mm3). Importantly, Gd-TESMA-enhanced MRI identified accumulation of tropoelastin in excised human aneurysmal tissue (n = 4), which was confirmed histologically. Conclusion Tropoelastin MRI identifies dysfunctional matrix remodelling that is specifically expressed in regions of aortic aneurysm or dissection and correlates with the development and rate of aortic expansi, British Heart Foundation, Chilean Agency of Technology and Science, Centre for Medical Engineering, Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Químicas, TRUE, pub

Background: Compromised structural integrity of the endothelium and higher microvessel density increase vascular permeability. We investigated whether vascular permeability measured in vivo by magnetic resonance imaging using the albumin-binding contrast agent, gadofosveset, is a surrogate marker of rupture-prone atherosclerotic plaque in a rabbit model. Methods and results: New Zealand white rabbits (n=10) were rendered atherosclerotic by cholesterol-diet and endothelial denudation. Plaque rupture was triggered with Russell's viper venom and histamine. Animals were imaged pre-triggering, at 3 and 12 weeks, to quantify plaque area, vascular permeability, vasodilation, and stiffness and post-triggering to identify thrombus. Plaques identified on the pretrigger scans were classified as stable or rupture-prone based on the absence or presence of thrombus on the corresponding post-trigger magnetic resonance imaging, respectively. All rabbits had developed atherosclerosis, and 60% had ruptured plaques. Rupture-prone plaques had higher vessel wall relaxation rate (R1; 2.30±0.5 versus 1.86±0.3 s-1; P<0.001), measured 30 minutes after gadofosveset administration, and higher R1/plaque area ratio (0.70±0.06 versus 0.47±0.02, P= 0.01) compared with stable plaque at 12 weeks. Rupture-prone plaques had higher percent change in R1 between the 3 and 12 weeks compared with stable plaque (50.80±7.2% versus 14.22±2.2%; P<0.001). Immunohistochemistry revealed increased vessel wall albumin and microvessel density in diseased aortas and especially in ruptured plaque. Electron microscopy showed lack of structural integrity in both luminal and microvascular endothelium in diseased vessels. Functionally, the intrinsic vasodilation of the vessel wall decreased at 12 weeks compared with 3 weeks (18.60±1.0% versus 23.43±0.8%; P<0.001) and in rupture-prone compared with stable lesions (16.40±2.0% versus 21.63±1.2%; P<0.001). Arterial stiffness increased at 12 weeks compared with 3 weeks (5.00±, Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Químicas, TRUE, pub