Scientists at the University of Tsukuba say they have developed a tool that can image the development of atherosclerotic plaque in the body and follow its progression over time, enabling accurate evaluation of drugs to treat atherosclerosis and potentially analysis of the likely risk posed by such plaque in individual patients.
Their study (“A Novel iRFP-Incorporated in vivo Murine Atherosclerosis Imaging System”), published in Scientific Reports, reported that the team induced atherosclerosis in mice by inactivating a fat and cholesterol-related receptor and feeding them on a high-cholesterol diet. They also exposed these mice to x-rays to wipe out the native cells of their immune system, and then transplanted them with genetically engineered immune cells exhibiting fluorescence.
“By using near-infrared fluorescent protein (iRFP)-expressing hematopoietic cells, we established a novel, quantitative, in vivo, noninvasive atherosclerosis imaging system. This murine atherosclerosis imaging approach targets macrophages expressing iRFP in plaques. Low-density lipoprotein receptor-deficient (LDLR−/−) mice transplanted with beta-actin promoter-derived iRFP transgenic (TG) mouse bone marrow (BM) cells (iRFP → LDLR−/−) were used. Atherosclerosis was induced by a nonfluorescent 1.25% cholesterol diet (HCD). Atherosclerosis was compared among the three differently induced mouse groups. iRFP → LDLR−/−mice fed a normal diet (ND) and LDLR−/− mice transplanted with wild-type (WT) BM cells were used as controls. The in vivo imaging system (IVIS) detected an enhanced iRFP signal in the thoracic aorta of HCD-fed iRFP → LDLR−/− mice, whereas iRFP signals were not observed in the control mice,” write the investigators.
“Time-course imaging showed a gradual increase in the signal area, which was correlated with atherosclerotic plaque progression. Oil red O (ORO) staining of aortas and histological analysis of plaques confirmed that the detected signal was strictly emitted from plaque-positive areas of the aorta. Our new murine atherosclerosis imaging system can noninvasively image atherosclerotic plaques in the aorta and generate longitudinal data, validating the ability of the system to monitor lesion progression.”
“A main advantage of our approach is that the introduced immune cells, as macrophages, congregate in atherosclerotic plaque, so the level of fluorescence emitted by them strongly correlates with the amount of plaque that has formed,” says Yoshihiro Miwa, Ph.D., assistant professor at the University of Tsukuba. “Because the expressed fluorescent proteins emit light in the near-infrared part of the spectrum, they can be detected at deeper locations within the body, such as the thoracic aorta.”
To confirm that this method can be used to identify the amount of atherosclerotic plaque within the mice, rather than just whether or not such plaque is present, the team established three different groups with differing feeding patterns. Mice were fed the high-cholesterol diet every day or the high-cholesterol diet and a normal diet on alternate weeks, or just the normal diet. The findings based on the intensity of the fluorescent signal confirmed the expected stepwise differences in plaque quantity among these three groups and also showed clear increases with a longer time spent consuming the unhealthy diets.
“Because we can now clearly analyze the amount of plaque present and its change over time, our work should lead to more effective monitoring of how well anti-atherosclerotic drugs work,” says Michito Hamada, Ph.D., assistant professor at the University of Tsukuba. “This method can also reduce the number of experimental animals used because there's no need to sacrifice them and remove tissues for analysis at each time point within an experiment.”
The team hopes to further increase the sophistication of this tool, which could potentially lead to accurate analysis of the risk associated with the buildup of plaque in human patients and produce a range of associated medical benefits.