Hyaluronan, a high molecular weight, negatively charged polysaccharide, is a major constituent of the extracellular matrix. High molecular weight hyaluronan is antiangiogenic, but its degradation by hyaluronidase generates proangiogenic breakdown products. Thus, by expression of hyaluronidase, cancer cells can tilt the angiogenic balance of their microenvironment. Indeed, hyaluronidase-mediated breakdown of hyaluronan correlates with aggressiveness and invasiveness of ovarian cancer metastasis and with tumor angiogenesis. The goal of this work was to develop a novel smart contrast material for detection of hyaluronidase activity by magnetic resonance imaging (MRI). Gadolinium-diethylenetriaminepentaacetic acid (GdDTPA) covalently linked to hyaluronan on the surface of agarose beads showed attenuated relaxivity. Hyaluronidase, either purified from bovine testes or secreted by ES-2 and OVCAR-3 human epithelial ovarian carcinoma cells, activated the hyaluronan-GdDTPA-beads by rapidly altering the R 1 and R 2 relaxation rates. The change in relaxation rates was consistent with the different levels of biologically active hyaluronidase secreted by those cells. Hyaluronan-GdDTPAbeads were further used for demonstration of MRI detection of hyaluronidase activity in the proximity of s.c. ES-2 ovarian carcinoma tumors in nude mice. Thus, hyaluronan-GdDTPAbeads could allow noninvasive molecular imaging of hyaluronidase-mediated tilt of the peritumor angiogenic balance. (Cancer Res 2005; 65(22): 10316-23)
One of the attractions of molecular imaging using 'smart' bioactive contrast agents is the ability to provide non-invasive data on the spatial and temporal changes in the distribution and expression patterns of specific enzymes. The tools developed for that aim could potentially also be developed for functional imaging of enzyme activity itself, through quantitative analysis of the rapid dynamics of enzymatic conversion of these contrast agents. High molecular weight hyaluronan, the natural substrate of hyaluronidase, is a major antiangiogenic constituent of the extracellular matrix. Degradation by hyaluronidase yields low molecular weight fragments, which are proangiogenic. A novel contrast material, HA-GdDTPA-beads, was designed to provide a substrate analog of hyaluronidase in which relaxivity changes are induced by enzymatic degradation. We show here a first-order kinetic analysis of the time-dependent increase in R 2 as a result of hyaluronidase activity. The changes in R 2 and the measured relaxivity of intact HAGdDTPA-beads (r 2B ) and HA-GdDTPA fragments (r 2D ) were utilized for derivation of the temporal drop in concentration of GdDTPA in HA-GdDTPA-beads as the consequence of the release of HA-GdDTPA fragments. The rate of dissociation of HA-GdDTPA from the beads showed typical bell-shaped temperature dependence between 7 and 36 °C with peak activity at 25°C. The tools developed here for quantitative dynamic analysis of hyaluronidase activity by MRI would allow the use of activation of HA-GdDTPA-beads for the determination of the role of hyaluronidase in altering the angiogenic microenvironment of tumor micro metastases.
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