Gene expression levels of matrix metalloproteinases in human atherosclerotic plaques and evaluation of radiolabeled inhibitors as imaging agents for plaque vulnerability.
BACKGROUND
Atherosclerotic plaque rupture is the primary cause for myocardial infarction and stroke. During plaque progression macrophages and mast cells secrete matrix-degrading proteolytic enzymes, such as matrix metalloproteinases (MMPs). We studied levels of MMPs and tissue inhibitor of metalloproteinases-3 (TIMP-3) in relation to the characteristics of carotid plaques. We evaluated in vitro two radiolabeled probes targeting active MMPs towards non-invasive imaging of rupture-prone plaques.


METHODS
Human carotid plaques obtained from endarterectomy were classified into stable and vulnerable by visual and histological analysis. MMP-1, MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MMP-14, TIMP-3, and CD68 levels were investigated by quantitative polymerase chain reaction. Immunohistochemistry was used to localize MMP-2 and MMP-9 with respect to CD68-expressing macrophages. Western blotting was applied to detect their active forms. A fluorine-18-labeled MMP-2/MMP-9 inhibitor and a tritiated selective MMP-9 inhibitor were evaluated by in vitro autoradiography as potential lead structures for non-invasive imaging.


RESULTS
Gene expression levels of all MMPs and CD68 were elevated in plaques. MMP-1, MMP-9, MMP-12 and MMP-14 were significantly higher in vulnerable than stable plaques. TIMP-3 expression was highest in stable and low in vulnerable plaques. Immunohistochemistry revealed intensive staining of MMP-9 in vulnerable plaques. Western blotting confirmed presence of the active form in plaque lysates. In vitro autoradiography showed binding of both inhibitors to stable and vulnerable plaques.


CONCLUSIONS
MMPs differed in their expression patterns among plaque phenotypes, providing possible imaging targets. The two tested MMP-2/MMP-9 and MMP-9 inhibitors may be useful to detect atherosclerotic plaques, but not the vulnerable lesions selectively.
Center for Radiopharmaceutical Sciences, ETH Zurich, Zurich, Switzerland. Electronic address: adrienne.mueller@pharma.ethz.ch.
Adrienne
A
Müller
Center for Radiopharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.
Stefanie D
SD
Krämer
Center for Radiopharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.
Romana
R
Meletta
Center for Radiopharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.
Katharina
K
Beck
Center for Radiopharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.
Svetlana V
SV
Selivanova
Clinic for Cardiovascular Surgery, University Hospital Zurich, Zurich, Switzerland.
Zoran
Z
Rancic
Department of Nuclear Medicine, University Hospital Zurich, Zurich, Switzerland.
Philipp A
PA
Kaufmann
Global Drug Discovery, Bayer Healthcare, Berlin, Germany.
Bernhard
B
Vos
Global Drug Discovery, Bayer Healthcare, Berlin, Germany.
Jörg
J
Meding
Global Drug Discovery, Bayer Healthcare, Berlin, Germany.
Timo
T
Stellfeld
Global Drug Discovery, Bayer Healthcare, Berlin, Germany.
Tobias K
TK
Heinrich
Global Drug Discovery, Bayer Healthcare, Berlin, Germany.
Marcus
M
Bauser
Global Drug Discovery, Bayer Healthcare, Berlin, Germany.
Joachim
J
Hütter
Global Drug Discovery, Bayer Healthcare, Berlin, Germany.
Ludger M
LM
Dinkelborg
Center for Radiopharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.
Roger
R
Schibli
Center for Radiopharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.
Simon M
SM
Ametamey
D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000235:genetics / Q000378:metabolism; D001158:Arteries / Q000378:metabolism; D019817:Benzoic Acid / Q000737:chemistry; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007553:Isotope Labeling; D008264:Macrophages / Q000378:metabolism; D008297:Male; D061965:Matrix Metalloproteinase Inhibitors / Q000737:chemistry; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008875:Middle Aged; D057054:Molecular Imaging / Q000379:methods*; D058226:Plaque, Atherosclerotic / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics / Q000378:metabolism; D014316:Tritium; D000097382:CD68 Molecule
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D019817:Benzoic Acid; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases; D019717:Tissue Inhibitor of Metalloproteinase-3; D014316:Tritium; C067980:CD68 antigen, human; D000097382:CD68 Molecule
Atherosclerosis; Autoradiography; Carotid artery plaque; Inflammation; Matrix metalloproteinases; Tissue inhibitor of metalloproteinase-3