Introduction: To support the validation of novel molecular drug targets in cardiovascular disease (CVD), molecular imaging and staining technologies are essential tools to provide documentation for the presence and localization of the targets in the affected tissue. The fact that the complex CVD etiology is linked to lipid metabolism and inflammation, both genetically and functionally emphasizes the need to study different types of molecular targets and markers directly in the diseased tissue. In the EU-funded CarTarDis project, we have had the unique opportunity to establish an infrastructure for in situ multimodal imaging and staining that enables visualization and localization of different types of molecular targets including protein, RNA and lipids.
Methods and Results: Tissue specimens of human coronary and carotid arteries with atherosclerotic lesions were supplied by the Socrates and BiKE biobanks at Leiden University Medical Center and Karolinska Institute, respectively. Conditions for imaging lipid metabolites in atherosclerotic tissue by mass spectrometry imaging (MSI) were optimized. Immunohistochemical (IHC) detection of protein targets and markers was performed using different kinds of assays, and conditions for detection of both non-coding and coding RNA targets were optimized using LNA-probe and branched-DNA probe technology for in situ hybridization (ISH). Conventional histology (H&E-staining) always supported the molecular staining. A number of examples will be presented, where we successfully have performed multimodal molecular imaging and staining including different types of molecular targets on neighboring sections or even multiplexing combining the methods on the same section. In addition, advantages and limitations of the different imaging and staining technologies will be addressed.
Conclusion: We have established an in situ multimodal molecular imaging and staining technology platform that enables obtaining in depth information about the (co)-localization of molecular targets and markers in atherosclerotic tissue. The individual technologies are provided across different geographic locations, accomplished through the exchange and sharing of samples and image documentation.