Abstracts of the 15th Endothelial Cell Research Symposium Maastricht Auditorium, Maastricht, The Netherlands 23 November 2004

Angiogenesis, the formation of new blood vessels, is involved in many pathological processes like cancer, ischemic disease, atherosclerosis, and chronic inflammation [1]. Inhibition of angiogenesis in cancer is a novel strategy to stop tumor growth/progression. in the angiogenic cascade different cell surface receptors are expressed at the endothelium of the new vasculature. The avb3-integrin is such a receptor which is associated with angiogenesis. The non-invasive in vivo detection of this integrin would allow one to be able to monitor angiogenesis and to be able to follow the effect of anti-angiogenic therapies. Several studies have used the tripeptide sequence arginineglycine- aspartic acid (RGD) conjugated with a radiolabel to image the expression of this integrin with nuclear imaging methods [2,3]. In this study, we aim to non-invasively image the expression of avb3 in tumor bearing mice with Magnetic Resonance Imaging (MRI). Therefore, we developed and used MR-detectable and fluorescent liposomes[4], which carry over 300 RGD-moieties per liposome. The in vivo MRI findings were validated with ex vivo fluorescence microscopy. Material and Methods Paramagnetic and fluorescent liposomes were prepared as described previously [4]. The cyclic RGD and control RAD-peptide were conjugated to maleimide-PEG-DSPE incorporated in the liposomes. The specificity of the liposomes was determined on HUVEC with MRI and fluorescence microscopy. Tumor bearing mice were anesthetized with an isoflurane/airmixture and an infusion line was put into the tail vein. Next, the mice were placed in a 6.3 T MRI scanner. A T1-map and a T1-weighted image were generated before the contrast agent was injected and at 5 time points after the contrast agent was injected. Next, the mice were euthanized and the tumor was dissected and frozen in isopentane. Sections of tissue (10-lm thickness) were prepared and fluorescence microscopy was performed. Results Both MRI and fluorescence microscopy revealed a strong association of the RGD-liposomes to avb3 expressing HUVEC, while RADliposomes did not. MRI on tumor bearing mice howed contrast enhancement of the tumor after injecting the contrast agent, both for RGD-liposomes and RAD-liposomes. Fluorescence microscopy revealed a distinct difference between tumors of mice that were injected with RGD-liposomes or RAD-liposomes. In case of RADliposomes a diffuse pattern of fluorescence was found around tumor blood vessels in the tumor tissue. This can be ascribed to extravasation of the liposomes in the tumor tissue. In contrast, RGD-liposomes were exclusively found was associated with blood vessels. Conclusions: This study demonstrates that molecular MR imaging of angiogenesis is possible by using a targeted contrast agent specific for the avb3-intergin.