Abstract
Fibrosis plays a central role in maladaptive remodelling processes, especially in the context of cardiovascular diseases. The fibrotic state depends on the balance between matrix metaloproteinases (MMPs) and their inhibitors, which alter the composition of extracellular matrix. Despite the necessity to localise and quantify fibrosis during cardiovascular diseases, only few biological markers of tissue fibrosis exist. Moreover none of them can evaluate fibrosis, in real time, in vivo. Therefore we developed an optical molecular probe (FR91) aimed at targeting cardiac matrix remodeling, with the innovative property of becoming fluorescent after cleavage by both MMP-2 and MMP-14. The present study was designed to validate this probe in cellular and animal models of MMP-2/MMP-14 activation and of fibrosis. Regarding cellular models, murine fibroblasts (3T3) were exposed to 20 ng/ml TGF-b 1 for 24h. RT-PCR and zymography demonstrated increased MMP-2 and MMP-14 expression and activity. Immunofluorescence (IF) showed that 3T3 cells were positive for both MMPs, and this MMP IF signal colocalized with FR91 labelling. Regarding animal models, C57BL/6J mice were subjected to Aortic Stenosis (AS ; constriction of the thoracic aorta to 30-35 % of its basal diameter). After 3 weeks, increased aortic pressure was confirmed and the heart taken out. We observed an increase by 60 % of the left ventricular (LV) mass, and a doubling of cardiomyocyte size. Histological analysis showed a 8 fold higher collagen content in AS mice compared to controls. MMP-2 expression (PCR) was doubled while its activity (zymography) was strongly increased. In parallel MMP-14 expression and activity were also increased. These changes were associated with a concomitant increase in LV fluorescence for FR91 (assessed ex vivo), which was significantly higher in AS vs. control mice. This increased FR91 fluorescence observed in AS mice disappeared after incubation with EDTA which inhibits MMPs activity. Thus, this study demonstrates that FR91 is a specific probe for the detection of increased MMP-2 and -14 activity and may be a unique tool to selectively detect cardiac fibrosis. Further experiments will test the capacity of FR91 to detect cardiac fibrosis in vivo.
Published Version
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