Abstract
BackgroundThe inflammatory nature of atherosclerosis provides a broad range of potential molecular targets for atherosclerosis imaging. Growing interest is focused on targets related to plaque vulnerability such as the co-stimulatory molecules CD80 and CD86. We investigated in this preclinical proof-of-concept study the applicability of the CD80/CD86-binding fusion protein belatacept as a probe for atherosclerosis imaging.MethodsBelatacept was labeled with indium-111, and the binding affinity was determined with CD80/CD86-positive Raji cells. In vivo distribution was investigated in Raji xenograft-bearing mice in single-photon emission computed tomography (SPECT)/CT scans, biodistribution, and ex vivo autoradiography studies. Ex vivo SPECT/CT experiments were performed with aortas and carotids of ApoE KO mice. Accumulation in human carotid atherosclerotic plaques was investigated by in vitro autoradiography.Results111In-DOTA-belatacept was obtained in >70 % yield, >99 % radiochemical purity, and ~40 GBq/μmol specific activity. The labeled belatacept bound with high affinity to Raji cells. In vivo, 111In-DOTA-belatacept accumulated specifically in Raji xenografts, lymph nodes, and salivary glands. Ex vivo SPECT experiments revealed displaceable accumulation in atherosclerotic plaques of ApoE KO mice fed an atherosclerosis-promoting diet. In human plaques, binding correlated with the infiltration by immune cells and the presence of a large lipid and necrotic core.Conclusions111In-DOTA-belatacept accumulates in CD80/CD86-positive tissues in vivo and in vitro rendering it a research tool for the assessment of inflammatory activity in atherosclerosis and possibly other diseases. The tracer is suitable for preclinical imaging of co-stimulatory molecules of both human and murine origin. Radiolabeled belatacept could serve as a benchmark for future CD80/CD86-specific imaging agents.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-015-0157-4) contains supplementary material, which is available to authorized users.
Highlights
The inflammatory nature of atherosclerosis provides a broad range of potential molecular targets for atherosclerosis imaging
Conjugation, radiolabeling, and quality control of 111In-DOTA-belatacept The bifunctional chelating agent p-SCN-Bn-DOTA was successfully conjugated to belatacept under aqueous conditions by reaction with lysine amino groups of the protein according to a published procedure [14]
Due to technical limitations with available antibodies, we were not able to counterstain the atherosclerotic tissues from single-photon emission computed tomography (SPECT) and autoradiography for CD80 and CD86, the proteins that we found upregulated in human vulnerable atherosclerotic plaques [7]
Summary
The inflammatory nature of atherosclerosis provides a broad range of potential molecular targets for atherosclerosis imaging. They observed a higher CD86 mRNA expression in human carotid plaques of symptomatic than asymptomatic patients Their immunohistochemical analysis revealed that virtually all mature DCs were CD86-positive and in close proximity to activated T cells. Further evidence for an involvement of mature APCs in atherosclerosis was presented in an expression analysis of human carotid endarterectomized plaques [9]. The increase in vulnerable plaques was more pronounced for the mature subpopulation of DCs than the total number including immature DCs [9] These studies indicate that advanced atheroma contain a population of fully maturated DCs and macrophages expressing co-stimulatory molecules and that these APCs could be efficient regulators of T cell activity in atherosclerosis. Antigen presentation is not restricted to lymph nodes but occurs in atherosclerotic plaques as supported by co-localization data of DCs and T cells [7, 8, 10]
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