Imaging Tropoelastin in Atherosclerosis

The cardiovascular imaging field has evolved from detecting plaque by assessing luminal stenosis to plaque phenotyping by imaging vessel wall morphology and composition.¹ From pathology studies, we have learned the relevance of differentiating plaque based on phenotype. Some plaques, independent of their size and luminal protrusion, are fibrous plaques and unlikely to cause clinical events. Plaques with a large lipid rich necrotic core, abundant inflammatory activity, and a thin fibrous cap are at high risk for rupture.² Plaques with superficial erosions and abundance of proteoglycan and hyaluronan matrix are also ominous, allegedly responsible for 30–40% of atherothrombotic events.³

If we aim to unravel what drives atherosclerosis and causes plaque rupture, depicting plaque size, morphology and composition does not suffice. To enhance our insight in atherosclerosis biology and investigate therapeutic efficacy, imaging applications that allow for visualization of cellular and molecular processes are being developed.¹ The adoption of positron emission tomography (PET) for vessel wall imaging is a luminary example of the progress that has been made.⁴ For example, ¹⁸F-fludeoxyglucose (¹⁸F-FDG) PET, quantifies glucose metabolism and reflects inflammatory activity in plaques. A different approach is provided by ⁶⁸Ga-DOTATATE PET/CT, which also facilitates detection of plaque inflammation, however in this case by imaging the somatostatin receptor subset-2, an epitope expressed by activated macrophages.⁴ With ¹⁸F-sodium fluoride (¹⁸F-NaF) PET/CT a different atherosclerosis hallmark of vulnerability can be visualized. By utilizing the property of Fluoride ions that incorporate into hydroxyapatite in the vessel wall, ¹⁸F-NaF PET/CT imaging allows imaging the vascular calcification process.⁴

In addition to PET imaging, magnetic resonance imaging (MRI) has also proved a utile and versatile imaging platform. The most commonly used agents for contrast enhancement in MRI are based on gadolinium (Gd).⁵ In routine clinical practice, Gd is administered as a complex with a chelating agent such as DOTA, or DTPA.⁶ These complexes are not tissue specific and are rapidly cleared from the blood stream. To manipulate the Gd chelates’ properties, their structure can be adjusted. Clinically relevant examples include Gd agents that bind to albumin to enhance their circulation time in the blood, or gadoxetate disodium which has a high hepatic clearance and allows for specific use in liver imaging.^5,6 The functionalization of Gd contrast agents for cardiovascular imaging is rapidly evolving and can be achieved by attaching them to a variety of molecules⁷, incorporating them in nanoparticle structures⁸, or combining them with target-specific proteins⁹. The purpose is to facilitate tissue specific, cell specific or even molecule specific MR imaging.

In this issue of Circulation Cardiovascular Imaging, Phinikaridou et al.¹⁰ report on the development of a gadolinium-labeled tropoelastin-specific magnetic resonance contrast agent (Gd-TESMAs). They show in human endarterectomy specimens that ruptured plaques have higher tropoelastin content than stable plaques, suggesting that this epitope may serve as a marker of lesion instability.

Their work starts out by investigating Gd-TESMAs binding specificity for tropoelastin. The contrast agent they selected contains a tropoelastin binding peptide (referred to as VVGS) which is attached to a Gd³⁺ DOTA complex. In vitro, Gd-TESMA showed excellent discriminating capability between tropoelastin (>60% binding) and mature elastin (<5% binding). Although binding to albumin was not observed, the approximatley 40% binding to collagen I and fibronectin indicates that binding is not exclusively for tropoelastin. To corroborate tropoelastin binding in plaques in vivo, rhodamine-labelled VVGS was infused in atherosclerotic mice (Apoe^−/−). Fluorescence microscopy of plaque sections revealed rhodamine co-localization with tropoelastin. Furthermore, ex vivo MRI of human endarterectomy specimens soaked in Gd-TESMA showed retention of the agent, indicative of tropoelastin binding.

The in vivo Gd-TESMA performance was subsequently investigated in the Apoe^−/− atherosclerosis mouse model and compared to a contrast agent that is known to bind to both tropoelastin as well as elastin (Gd-ESMA). Delayed enhancement (DE) MRI of the brachiocephalic arteries was performed 30–40 min after Gd-TESMA infusion. In control mice with healthy vessels, which contain elastin but lack tropoelastin, vessel wall enhancement was observed with Gd-ESMA but not Gd-TESMA, as expected. In atherosclerotic mice, in which lesions do contain tropoelastin, vessel wall enhancement was seen with Gd-TESMA as well as Gd-ESMA. Furthermore, enhancement by Gd-TESMA increased as lesions severity advanced. These findings were mirrored by increased plaque tropoelastin content as assessed by immunohistology and Western blotting. Moreover, a strong correlation between the percentage of plaque area enhancement and plaque tropoelastin percentage, assessed with histology, was observed. The increase in tropoelastin content could in part be mitigated when animals were treated with statins, as observed by DE-MRI, immunohistology as well as Western blotting. It is interesting to note that in Apoe^−/− mice statins do not affect lipid levels, suggesting this effect is lipid independent. Perhaps this can be attributed to direct effects of statins on inflammatory activity, but this was not the focus of the study. Altogether, these data indicate that Gd-TESMA DE-MRI of the vessel wall reflects plaque tropoelastin content.

Subsequently, the authors went on to test their hypothesis that Gd-TESMA DE-MRI can discriminate between stable and rupture-prone plaques. For this purpose, they used a rabbit model of controlled plaque rupture. In short, this model consists of New Zealand White rabbits fed a high cholesterol diet and receiving an abdominal aorta balloon injury to induce plaque development. By administration of Russell’s viper venom and histamine, plaque rupture can be induced ref. DE-MRI was performed prior to and after induction of plaque rupture. The authors observed that rupture prone plaques had markedly less Gd-TESMA uptake when compared to stable plaques. Gd-ESMA uptake however could not discriminate stable from unstable plaques. These data suggest that tropoelastin MR imaging with Gd-TESMA may potentially serve as an imaging biomarker of plaque stability.

Phinikaridou et al. provide an appealing proof-of-concept study on tropoelastin imaging in atherosclerosis with Gd-TESMA DE-MRI. The rationale for tropoelastin imaging pertains to the fact that the expression of this peptide is associated with atherosclerotic plaque development. In atherosclerosis, both elastolysis and elastogenesis take place as a consequence of macrophage-driven chronic inflammatory process.¹¹ Tropoelastin is a peptide synthesized and secreted in different isoforms (62, 65, and 67.5 kDa) as the soluble precursor for elastin.¹² Subsequently, cross-linking of tropoelastin by lysyl oxidase (LOX) gives rise to insoluble elastin.¹³ Healthy arteries contain elastin but little tropoelastin. In atherosclerotic lesions, however, tropoelastin is continuously produced by macrophages and vascular smooth muscle cells, but its maturation to elastin is impaired.^11,14 Imaging tropoelastin therefore facilitates visualization of the derailment of extracellular matrix production that goes on in atherosclerosis. This work adds new insights to existing literature that has mainly focused on elastin imaging.^15,16

Definite conclusions on the value and applicability of this technique for detecting high risk plaques cannot be drawn. Limitations include Gd-TESMA’s binding to collagen I and fibronectin, which are both present in atherosclerotic lesions. Binding to other proteins that were not tested cannot be excluded. Furthermore, experiments were conducted in a small number of animals, and the results of the plaque rupture rabbit model cannot easily be extrapolated to the clinical setting of plaque rupture in humans. Nonetheless, this paper reports on an interesting and innovative concept, and the data warrant further exploration of the potential of Gd-TESMA in experimental studies, possibly followed by a clinical study to ultimately assess its diagnostic value. As for now, tropoelastin imaging with Gd-TESMA may be an interesting and valuable new tool in experimental studies to decipher the role of the distorted extracellular matrix production in atherosclerosis development and plaque rupture, and possibly assess effects of novel anti-atherosclerotic drugs.