Metabolic syndrome is characterized by a clustering of metabolic abnormalities in conjunction with underlying insulin resistance and is closely associated with the onset of diabetes and accelerated cardiovascular disease. The corpulent JCR:LA-cp rat model of metabolic syndrome exhibited obesity, severe insulin resistance, hyperinsulinemia, and hypertriglyceridemia, as reported by others (13
). Vasa vasorum expansion and neovasculature proliferation, previously unreported in this model, was demonstrated and longitudinally assessed with MR molecular imaging at 3.0-T with ανβ3
-targeted paramagnetic nanoparticles, whereas benfluorex-treated and lean rats had significantly lower MR signal enhancement throughout the study. The MR enhancement observed in the aortic wall of the lean animals was perhaps a result of circulating nanoparticles. In an atherosclerotic rabbit model, the MR enhancement in the aortic wall persisted up to 8 and 12 h for nontargeted and ανβ3
-targeted nanoparticle formulations, respectively (22
). Noninvasive MR molecular imaging results were closely corroborated by microscopic estimates of microvessel density within the aortic wall. Both MR and histology objectively measured overall neovascularity by encompassing all regions of the abdominal aorta, including the entire vessel circumference and all imaged slices and not only subjectively selected areas identified as “hot spots” of plaque development.
Neovascularization is a prominent feature of human atherosclerotic lesions characterized as unstable lipid-rich plaques, but only a minor component of stable fibrocalcific lesions (6
). Angiogenesis not only provides a portal of entry for blood-borne cells and nutrients into the inflamed tissue (23
), but these fragile neovessels are also prone to shear-induced disruption, resulting in intraplaque hemorrhage. Intraplaque hemorrhage induces amplification of intramural inflammatory and immune processes, which may ultimately progress to plaque rupture (8
-integrin is a heterodimeric transmembrane glycoprotein and biomarker that is differentially up-regulated in proliferating versus quiescent endothelial cells, but it is also expressed by numerous cell types prominently represented in atherosclerotic plaques, including endothelial cells (26
), macrophages (28
), platelets (29
), lymphocytes (29
), and smooth muscle cells (30
). We have shown that ανβ3
-targeted paramagnetic nanoparticles specifically detect and quantify angiogenesis in atherosclerotic plaques of hypercholesterolemic rabbits (31
). Microvessel counts have been closely correlated with the MR imaging signal enhancement, which increased monotonically at higher adventitial microvessel counts and decreased rapidly as neovessel counts decreased (32
). Moreover, MR signal enhancement has been clearly shown to reflect a neovascular response to targeted antiangiogenic treatment given alone and in synergistic combination with atorvastatin therapy (32
). Histological staining of the ανβ3
-integrin itself was not performed in this study because numerous cell types, including macrophages, platelets, lymphocytes, and smooth muscle cells (32
), express this integrin. Earlier publications have demonstrated that ανβ3
-targeted nanoparticles are confined to the vasculature (12
) where they can only interact with endothelial cells. Therefore, histological staining of an endothelial marker, such as von Willebrand factor, is more likely to reflect possible sites of nanoparticle binding than staining for the integrin itself, which would greatly overestimate particle binding.
Dietary benfluorex, which increases neurotransmitter serotonin levels to produce satiety and loss of appetite, decreased diet intake to the lean control consumption. Body weight and circulating levels of insulin and leptin were reduced relative to the untreated obese animals, but were still elevated compared with the lean controls, in agreement with previous reports (18
). In earlier studies, the pairing of obese rat food intake to control animals and the feeding of benfluorex or d
-fenfluramine have been found to diminish cardiovascular disease and myocardial lesions in this corpulent rat model (14
). Ad libitum feed consumption in benfluorex-treated obese rats was comparable to the intake of their lean control counterparts, which was mirrored by a low aortic neovascular MR signal in both groups of animals. Collectively, these findings support the contention that the progression of cardiovascular disease is correlated with increasing intramural angiogenesis, which can be noninvasively and serially interrogated and quantified with MR molecular imaging. Other changes in body weight and hormonal profiles of the obese rats were directionally similar to the appetite suppression effects of benfluorex, but only noninvasive MR assessments of angiogenesis closely paralleled the diminished food consumption effect of the lean control group.
One limitation of the present study and other animal model studies is the focus on very early atherosclerotic disease because many of the key features of human atherosclerotic plaque reported to amplify neovascular response, such as intraplaque hemorrhage and associated T cell–mediated immune response, are not present. Although the ameliorative effect of benfluorex on dietary intake, body weight increase, and aortic neovascular expansion in early plaque was evident, the regression or stabilization of advanced plaque remains to be demonstrated.
This study measured the MR imaging enhancement arising from only the ανβ3
-integrin targeted nanoparticle formulation. Although we did not use nontargeted nanoparticles to determine the nonspecific accumulation of the contrast agent due to the hyperpermeability of angiogenic vessels, this issue has been explored in previous studies of animal models of cardiovascular disease and cancer (10
). Typically, the nontargeted formulation produces approximately 50% less image enhancement compared with the targeted particles. Although it is reasonable to assume that the nontargeted agent would also yield 50% less enhancement in the JCR rat, further studies will be required to assess the nonspecific contribution and the dependence of particle dose and post-injection time point on the nontargeted component.
The clinical use of benfluorex has been discontinued in the U.S. due to safety concerns. However, the molecular imaging techniques described in this study are sensitive to neovascular proliferation in the aortic wall and are not limited to only detecting the response to benfluorex treatment. This method could be applied to monitoring the end-organ effects of a vast array of therapeutic approaches, such as lifestyle modifications, surgical interventions, lipid-lowering medications, and glucose normalization.