Published Online
on November 26, 2007

A more recent version of this article appeared on December 11, 2007

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Submitted on August 7, 2007
Accepted on October 1, 2007

Osteogenesis Associates With Inflammation in Early-Stage Atherosclerosis Evaluated by Molecular Imaging In Vivo

Elena Aikawa MD, PhD^*, Matthias Nahrendorf MD, Jose-Luiz Figueiredo MD, Filip K. Swirski PhD, Timur Shtatland PhD, Rainer H. Kohler PhD, Farouc A. Jaffer MD, PhD, Masanori Aikawa MD, PhD, and Ralph Weissleder MD, PhD

From the Center for Molecular Imaging Research (E.A., M.N., J.-L.F., F.K.S., T.S., R.H.K., F.A.J., R.W.), Massachusetts General Hospital, Harvard Medical School; Donald W. Reynolds Cardiovascular Clinical Research Center on Atherosclerosis at Harvard Medical School (E.A., M.N., F.K.S., F.A.J., M.A., R.W.); Cardiovascular Division, Department of Medicine (M.A.), Brigham and Women’s Hospital, Harvard Medical School; and Cardiology Division, Department of Medicine (F.A.J.), Massachusetts General Hospital, all in Boston, Mass.

^* To whom correspondence should be addressed. E-mail: eaikawa{at}mgh.harvard.edu.

Background—Arterial calcification is associated with cardiovascular events; however, mechanisms of calcification in atherosclerosis remain obscure.

Methods and Results—We tested the hypothesis that inflammation promotes osteogenesis in atherosclerotic plaques using in vivo molecular imaging in apolipoprotein E^-/- mice (20 to 30 weeks old, n=35). A bisphosphonate-derivatized near-infrared fluorescent imaging agent (excitation 750 nm) visualized osteogenic activity that was otherwise undetectable by x-ray computed tomography. Flow cytometry validated the target specifically in osteoblast-like cells. A spectrally distinct near-infrared fluorescent nanoparticle (excitation 680 nm) was coinjected to simultaneously image macrophages. Fluorescence reflectance mapping demonstrated an association between osteogenic activity and macrophages in aortas of apolipoprotein E^-/- mice (R²=0.93). Intravital dual-channel fluorescence microscopy was used to further monitor osteogenic changes in inflamed carotid arteries at 20 and 30 weeks of age and revealed that macrophage burden and osteogenesis concomitantly increased during plaque progression (P<0.01 and P<0.001, respectively) and decreased after statin treatment (P<0.0001 and P<0.05, respectively). Fluorescence microscopy on cryosections colocalized near-infrared fluorescent osteogenic signals with alkaline phosphatase activity, bone-regulating protein expression, and hydroxyapatite nanocrystals as detected by electron microscopy, whereas von Kossa and alizarin red stains showed no evidence of calcification. Real-time reverse-transcription polymerase chain reaction revealed that macrophage-conditioned media increased alkaline phosphatase mRNA expression in vascular smooth muscle cells.

Conclusions—This serial in vivo study demonstrates the real-time association of macrophage burden with osteogenic activity in early-stage atherosclerosis and offers a cellular-resolution tool to identify preclinical microcalcifications.

Key words: atherosclerosis • calcification • inflammation • imaging

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