E — PROLIFERATION, APOPTOSE, MICROPARTICULES
Apoptosis plays a central role in cardiovascular diseases, and thus constitutes an attractive target for molecular imaging of the severity of these diseases. We developed and characterized non nuclear molecular probes, specifically targeting Caspase –3, one of the key regulatory enzyme of apoptosis. For this purpose, we generated quenched near-infrared fluorescent probes which become fluorescent after being cleaved by caspase –3 and freely penetrate in cells. In this study we evaluated the selectivity/ specificity of one of these probes, CP220, to detect myocardial caspase –3 activity in vitro and ex vivo.
For in vitro evaluation, we induced apoptosis/caspase-3 activation by incubating cultured heart microvascular endothelial cells with 1μm staurosporine (STAU) for 6hours. No fluorescent signal of cleaved CP220 was observed in the absence of STAU. In contrast, STAU-treated cells displayed an increased fluorescent signal for cleaved CP220, which colocalized with the immunofluorescent (IF) signal obtained with an anti caspase –3 antibody, and was reduced by a caspase –3 inhibitor. Figure 1 shows FACS analysis and demonstrates a similar profile between cleaved CP220 and Annexin V (% of positive cells: Annexin V: control 11±2, STAU 34±2; STAU+caspase inhibitor 11±2–CP220: control 9±1; STAU 26±4; STAU+inhibitor 9±3)
For ex vivo data, cardiac apoptosis was induced by administration of doxorubicin (20mg/kg i.p.). Heart sections obtained four days after doxorubicin displayed a more than 3 fold increase in caspase-3 activity, and a marked increase in the fluorescent signal for cleaved CP220, which colocalized with that of the antibody against cleaved caspase –3.
These data demonstrate the capacity of these novel probes to detect apoptosis in vitro and ex vivo, allowing us to plan tests of in vivo detection of cardiac apoptosis.Le texte complet de cet article est disponible en PDF.