Advanced ex vivo analyses and multi-frequency ultrasound technology for improved evaluation and diagnosis of coronary plaque

Atherosclerosis is a disease which results in the build-up of plaques within the walls of arteries in certain lesion-prone areas. Atherosclerotic plaques can be grouped into stable and unstable plaques where the unstable plaques typically contain cholesterol and fatty substances. Myocardial infarction is a hallmark of coronary artery disease (CAD). The rupture of an unstable atherosclerotic plaque, with a resulting blockage of a coronary artery, is the most common cause of myocardial infarction. Classic statin treatment has led to a reduction of clinical events but a considerable residual risk of atherosclerotic cardiovascular-mortality still remains. The project uses an atherosclerotic rabbit model, with a lipoprotein metabolism similar to the human patient, with administration of cholesterol lowering agents both in the form of statins (well-known and documented) and PCSK9 inhibitors (new and promising). Using an experimental design that is mimicking the clinical situation, we aim to advance the knowledge related to atherosclerotic plaque instability and early detection of unstable plaque. Together with a Romanian consortium partner, a study on rabbits fed a high-fat diet was conducted. The animals were divided into different groups where some groups received hypolipidemic treatment (atorvastatin and PCSK9 siRNA-inhibitor). Mass spectrometry-based proteomic studies were complemented by immunologic and genomic studies. This is the first proteomic study focusing on a panel of cardiac stress-sensing molecules, termed alarmins, and their alteration in hyperlipidemia. We found that some specific alarmins could not be stabilized by the lipid-lowering therapy and may therefore be of special interest in further studies. Together with a French consortium partner, a new dual-frequency intravascular ultrasound catheter was designed and manufactured. The motivation was to test a new ultrasound method for improved imaging of microbubbles targeted to biomarkers at high imaging frequency. During the manufacturing process some challenges related to interconnection of the array elements were encountered. When we received the final catheter for characterization and testing, the catheter turned out to have problems related both to sensitivity and dead transducer elements. It was therefore decided not to use this catheter in an animal model. Important knowledge and experience that will be useful in future projects was gained even though the catheter could not be used for testing of the new ultrasound method in an animal model. To further test and validate the dual-frequency method for imaging of contrast agent media we therefore used a transcutaneous ultrasound probe. A method for local and non-infectious inflammation based on injection of the substance Zymosan was used to induce local inflammation in the hind limb of rabbits. A proof-of-concept study with molecular ultrasound imaging in a soft tissue inflammation model was then carried out using custom-made microbubbles targeted to inflammatory biomarkers. The new imaging technique showed enhanced accumulation of targeted microbubbles in inflamed tissue regions compared to normal tissues. The custom-made microbubles used were also fluorescent and the presence of targeted microbubbles in inflamed tissue regions was verified by confocal microscopy imaging of tissue sections post-mortem. This study demonstrated the potential of the new ultrasound method for imaging of targeted contrast agent media.

Alarmins are a class of endogenous proteins with a common characteristic of activating the innate immune system. We have studied alarmins in a relevant animal model for atherosclerosis in combination with lipid-lowering treatment. We found significant upregulation of specific alarmins and some of them did not respond to lipid-lowering treatment. These alarmins can potentially play an important role in the progression of silent atherosclerosis and they may possess an unexplored therapeutic potential for patients with atherosclerotic cardiovascular disease. Non-alcoholic fatty liver disease currently affects about 25% of the global adult population and its prevalence is continuing to increase at an alarming rate. Results with the experimental hyperlipidemic rabbit model used in this project has evidenced the presence and importance of increased oxidative stress in non-alcoholic fatty liver disease. Our results can contribute to improved understanding of this widespread and chronic liver disease. We have demonstrated the use of a new dual-frequency ultrasound method for imaging of microbubble contrast media targeted to biomarkers. This can impact and potentially facilitate the use of ultrasound in molecular imaging, first in a preclinical setting using animal models to explore mechanisms and later in a clinical setting.

Atherosclerosis is considered a multifactorial disease, with risk factors ranging from high fat diet, hypertension, smoking, diabetes, to genetic susceptibility and other factors. The general consensus is that myocardial infarction, a hallmark of coronary artery disease (CAD), can be prevented if these known risk factors are taken care of in due time. Classic statin treatment has led to a significant reduction of clinical events, but a considerable residual risk of cardiovascular-related mortality still remains. Alarmins are host biomolecules that can initiate and perpetuate a noninfectious inflammatory response. Myocardial ischemia, the result of coronary artery occlusion, initiates systemic inflammation through the release of such alarmins. Our hypothesis is that alarmins are critical players of CAD plaque progression, which can be directly used for targeted imaging. The XploreCAD project will identify potential markers of CAD that will be used with a novel multi-frequency technology for molecular imaging with intravascular ultrasound, to bring new insights into plaque development and mechanisms related to plaque instability. The methodology will include high performance nano-liquid chromatography mass spectrometric analysis and immunological assays of plaques and sera isolated from atherosclerotic animals and patients. Expected results will lead to a better understanding of the mechanisms of plaque progression and an improved imaging technique for coronary plaque diagnosis and personalized therapy.