Gene expression levels of matrix metalloproteinases in human atherosclerotic plaques and evaluation of radiolabeled inhibitors as imaging agents for plaque vulnerability.

INTRODUCTION: 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.

Design, synthesis, and initial evaluation of a high affinity positron emission tomography probe for imaging matrix metalloproteinases 2 and 9.

The activity of matrix metalloproteinases (MMPs) is elevated locally under many pathological conditions. Gelatinases MMP2 and MMP9 are of particular interest because of their implication in angiogenesis, cancer cell proliferation and metastasis, and atherosclerotic plaque rupture. The aim of this study was to identify and develop a selective gelatinase inhibitor for imaging active MMP2/MMP9 in vivo. We synthesized a series of N-sulfonylamino acid derivatives with low to high nanomolar inhibitory potencies. (R)-2-(4-(4-Fluorobenzamido)phenylsulfonamido)-3-(1H-indol-3-yl)propanoic acid (7) exhibited the best in vitro binding properties: MMP2 IC50 = 1.8 nM, MMP9 IC50 = 7.2 nM. Radiolabeling of 7 with no carrier added (18)F-radioisotope was accomplished starting from iodonium salts as precursors. The radiochemical yield strongly depended on the iodonium counteranion (ClO4(-) > Br(-) > TFA(-) > tosylate). (18)F-7 was obtained in up to 20% radiochemical yield (decay corrected), high radiochemical purity, and >90 GBq/µmol specific radioactivity. The radiolabeled compound showed excellent stability in vitro and in mice in vivo.

Vascular enhancement in early dynamic liver MR imaging in an animal model: comparison of two injection regimen and two different doses Gd-EOB-DTPA (gadoxetic acid) with standard Gd-DTPA.

OBJECTIVES: To investigate the influence of 2 different doses and injection rates on the enhancement of liver vasculature in Gd-EOB-DTPA enhanced liver MRI compared with the standard dose Gd-DTPA. MATERIALS AND METHODS: This animal experimental study has been approved by the local authorities. In 5 pigs, a time-resolved T1w 3D GRE sequence, and in a second experiment, a single-slice turbo FLASH T1w sequence with 3 frames/s were started each with contrast agent application of Gd-EOB-DTPA and Gd-DTPA. Three sets with different doses were performed with an injection rate of 1 and 2 mL/s which are as follows: A, Standard dose: 25 [mu]mol Gd-EOB-DTPA/kg body-weight (bw); B, Double dose: 50 [mu]mol Gd-EOB-DTPA/kg bw; and C, Standard dose: 100 [mu]mol Gd-DTPA/kg bw. Signal intensities were measured in aorta, vena cava inferior, portal vein, and liver parenchyma, and time-signal-to-noise (SNR)-curves were generated. The increase in SNR from baseline and several perfusion parameters were calculated. Statistical significance was tested with the Wilcoxon rank test at a significance level of P = 0.05. RESULTS: Mean peak enhancement (SNR) in the aorta was not significantly different for set A, B, and C with the same injection rate. Mean peak enhancement in the aorta was significantly higher for set A at 1 mL/s than at 2 mL/s (159.1 vs. 144.4, P = 0.043). Mean peak enhancement in the PV, the vena cava inferior, and the liver parenchyma was significantly lower for set A compared with set B and set C, at both injection speeds. The full width at half max was significantly longer for injection protocols with 1 as compared with 2 mL/s for sets A and set B. Set A with 1 mL/s reached similar values as compared with set C with 2 mL/s for the full width at half max (8.92 vs. 9.40; P = 0.35), for the area-under-the curve (1736.64 vs. 1912.74; P = 0.69) and the maximal signal-to-noise ratio (25.21 vs. 25.37; P = 0.69). CONCLUSION: Despite the lower amount of gadolinium in the standard dose of Gd-EOB-DTPA, the results showed that the arterial enhancement in Gd-EOB-DTPA-enhanced dynamic liver MRI was comparable to Gd-DTPA. This result can be explained mainly by the higher relaxivity. Choosing a lower injection rate additionally supported to compensate for the lower injection volume by stretching the bolus without decreasing the peak. In this respect, an injection rate of 1 mL/s showed better results with regard to the arterial enhancement compared with 2 mL/s.