Non-linear optical microscopy in clinical translation : imaging of postinterventional endothelial regeneration

The endothelium plays an important role, not only in the progression of atherosclerosis, but also vasculitis and other inflammatory diseases. Endothelial activation triggered by abnormal shear stress, local inflammation and chemokine release is followed by the upregulation of surface adhesion molecules, such as intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and selectins. These surface adhesion molecules facilitate the migration of immune cells, responsible for the elicitation of inflammation. Similarly, injury related inflammation is induced during interventional surgery such as balloon angioplasty with stent implantation and endoarterectomy. During the process of endothelial regeneration, the blood vessel is vulnerable to thrombus formation, immune cell accumulation and restenosis. Disease- and injury-associated biomarkers are expressed on the luminal surface of major arteries and pose a potential target for imaging and early diagnosis. In this study, the main aim was to establish a vascular contrast agent that can withstand physiological flow and shear stress conditions in major arteries while specifically binding to a molecular target long enough for imaging. Clinical imaging modalities lack sufficient sensibility and resolution for tracking single particles. Two-photon laser scanning microscopy (TPLSM) was applied as a deep-tissue imaging modalities for the characterization of surface marker expression and binding kinetics of rhodamine-loaded microbubbles as a preclinical step towards translation. Microbubbles, a polymer-based air-filled particles with the diameter of 1-2 µm (MB) have been previously established in tumor imaging, which are able to attach to the vascular endothelial growth factor receptor at relatively low flow rates. Fluorescent loading of the MB shell promotes the bimodal detection of the particles both using TPLSM and molecular ultrasound. Using TPLSM imaging on TNFα stimulated explanted murine carotid arteries in an ex vivo flow chamber system, the shear stress resistance of intercellular adhesion molecule 1 targeted MB were characterized at shear rates matching and exceeding physiological conditions, showing its potential imaging application in inflamed major arteries in this first proof-of-concept study. During vascular intervention, the endothelium is severely injured and inflamed, resulting in dysfunction of the regulatory mechanisms of the endothelium, increasing thrombosis and restenosis risk. Endothelial regeneration following intervention is a crucial step towards full recovery of patients. Vascular cell adhesion molecule 1 is expressed after endothelial denudation on the medial smooth muscle cells, during the acute inflammation phase on both the smooth muscle layer and regenerating endothelial cells, but disappears from the endothelial layer after recovery, making it the perfect molecular marker to track endothelial healing. Using in vivo TPLSM, single MB bound to the vascular lumen was imaged and quantified showing specific retention. Translation into molecular ultrasound confirmed injury-specific MB retention and accurate prediction of the endothelial state in correlation with immunohistology and TPLSM imaging of the luminal surface