Echogenic perfluorohexane-loaded macrophages adhere in vivo to activated vascular endothelium in mice, an explorative study

Abstract

Background: Macrophages may concentrate ultrasound contrast agents and exhibit selective adhesion to activated endothelium. The present study investigates in mice the potential of perfluorohexane (PFH) loaded macrophages to act as ultrasound contrast agent with high reflectivity and specifically targeted at (atherosclerotic) vascular lesions.

Methods: Lung passage was evaluated with a mouse echo scanner after injection, at a slow pace or as a bolus, of varying doses of PFH-loaded and unloaded bone marrow macrophages (BMM) into the jugular vein. The interaction of PFH-loaded and unloaded BMM with TNF-α stimulated carotid artery endothelium after tail vein injection was assessed by means of intravital microscopy.

Results: High doses of jugular vein injected PFH-loaded BMM were visible with ultrasound in the pulmonary artery and detectable in the carotid artery. At intravital microscopy, tail vein injected BMM exhibited rolling and adhesion behavior at the TNF-α stimulated carotid endothelium, similar to that of native blood leukocytes. Rolling behavior was not different between PFH-loaded and unloaded BMM (p = 0.38).

Conclusion: In vivo, perfluorohexane loaded macrophages pass the pulmonary circulation and appear on the arterial side. Moreover, they roll and adhere selectively to activated endothelium under physiological flow conditions. These findings indicate that perfluorohexane loaded BMM could be used to study processes in vivo where endothelial activation plays a role, such as atherosclerosis.

Figure 1

Diameter distribution of mouse blood leukocytes (N, neutrophil; L, lymphocyte; M, monocyte) and cultured bone marrow macrophages (BMM). Histograms show a broad diameter distribution for both PFH loaded (2%) and unloaded (0%) BMM compared to blood leukocytes. The bracket on the x-axis indicates the approximate diameter range of blood monocytes. n = 200 cells, for each group.

Figure 2

Ultrasound images of a mouse heart before (left) and after (right) jugular vein injection of 7 million PFH (2% v/v) loaded BMM. Right panel shows a clear enhancement (indicated with an arrow) in the pulmonary artery (PA) compared to the aorta (Ao) and the left ventricle (LV).

Figure 3

Left: blood enhancement in the pulmonary artery (PA) and aorta (Ao) after a slow injection (30 s) of 15 million 4% v/v loaded BMM into the jugular vein. Right: transient increase of blood echogenicity enhancement in the carotid artery just after a bolus injection of 15 million of PFH (4% v/v) loaded BMM into the jugular vein.

Figure 4

Intravital fluorescence microscopy images of the mouse carotid artery. Interactions of leukocytes are virtually absent in unstimulated mice (left). TNF-α stimulation effectively increases the number of leukocytes interacting with the endothelium (right). In the right picture the left wall of the artery is out of focus. Leukocytes are Rhodamine labeled. Scale bar = 50 μm.