Evaluation of a soft atherosclerotic lesion in the rabbit aorta by an invasive IVUS method versus a non-invasive MRI technology.

The intravascular ultrasound (IVUS) modality has rapidly gained acceptance for the measurement of arterial plaque thickness and for anatomical characterization. In view, however, of the growing interest in the direct assessment of plaque size after therapeutic modalities directly reducing plaque burden, a non-invasive method such as magnetic resonance imaging (MRI) may be of help for repeated evaluations. The two methods were compared directly on a focal plaque developed at the abdominal aortic level by a combination of local electric lesion followed by a hypercholesterolemic diet. The plaque was fully characterized histopathologically at intervals up to 120 days from lesion induction, and maximal plaque formation was detected at 90 days from electrical injury. Plaques could be well assessed by IVUS at each time point analyzed and data correlated very well to histopathologic findings (r = 0.969, P = 0.0014). The MRI technology provided reliable determinations only at 90 days after lesion induction, i.e. at maximal plaque formation, with excellent correspondence to IVUS determinations (r = 0.989, P = 0.0111). Altogether these findings indicate that the non-invasive MRI technology, when applied to the analysis of arterial plaques of adequate size, can be used successfully for plaque determination, with results comparable to the invasive IVUS technique.

Development of a lipid-rich, soft plaque in rabbits, monitored by histology and intravascular ultrasound.

Lipid rich, soft plaques in the clinic are a common forerunner to occlusive thrombus formation, even with modest arterial stenosis. Animal models of atherosclerosis, obtained by various methods, do not generally allow direct in vivo evaluation of the lesion and, furthermore, cannot be examined more than once. The aim of the study was the generation of a rabbit model of atherosclerosis, with morphological characteristics similar to human lipid-rich, soft atheromatous plaques, and the evaluation of the reliability of intravascular ultrasound (IVUS) technology in the study of the development of atherosclerotic lesions in this model. Briefly, New Zealand white rabbits undergo perivascular electrical injury at both common carotid arteries, together with a 1.5% cholesterol diet for up to 90 days. The lesioned arterial segments show progressive changes, from diffuse cellular mortality, to macrophage infiltration in the media, up to the final migration of macrophages to the neointima, resulting in bulky, eccentric, macrophage and lipid-rich lesions. At IVUS, the produced lesions clearly resemble those described as 'soft plaques' in the clinical setting, with minimal calcification and reduced echo-reflectivity versus the adventitial layer. Quantitative and morphometric analysis of plaques shows a significant correlation between histological and IVUS measurements at each time point. In conclusion, vascular injury in the common carotids of rabbits generates atherosclerotic lipid-rich, soft plaques, that can be properly assessed by the IVUS methodology. The easy accessibility of the arterial lesion allows serial IVUS investigations and the direct evaluation of a number of locally or generally delivered therapeutic agents.

Phospholipid distribution in murine mammary adenocarcinomas induced by activated neu oncogene.

The aim of this study was to analyze possible changes in the total phospholipid distribution in murine mammary adenocarcinomas induced in transgenic mice by the tissue-specific expression of the neu oncogene, as compared with normal tissues. To understand whether the altered phospholipid profile might be specifically tissue-related to the oncogene expression, phospholipid composition also has been analyzed in liver, kidney, lung, and spleen. The data indicate that only tumor mammary tissues show a drastic increase of the total phospholipid content (P < 0.0001) associated with a significant increment of phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin (P < 0.05). Moreover, gas-chromatography analysis of total phospholipid-derived fatty acids shows a decrease in the percentage content of linoleic acid in tumor tissues, suggesting an altered metabolism of this fatty acid related to the enhanced epithelial proliferation. We conclude that neu transgenic mice provide a good model to clarify the involvement of phospholipids in neu-induced neoplastic transformation and to study in vivo the metabolic pathways related to the intracellular signaling.

Oncogene transgenic mice: an useful model to study in vivo the relationships between gangliosides and oncogenes.

Several studies have demonstrated that transfer of oncogenes in cultured cells reproducibly induces transmissible alterations in their ganglioside profile; the transfection of the same oncogene into different cell lines and the different localization of the oncogene product result in a different ganglioside expression. In the present study the modifications of the ganglioside pattern in mammary carcinomas induced in transgenic mice by the activated form of the rat neu oncogene have been investigated. Whereas control mammary tissues contain quite exclusively GM3, all neoplastic samples show a substantial decrease of this ganglioside, an accumulation in variable amount of GM3-derived species (GM1, GD3, GD1a, GD1b, GT and GQ) and the appearance of new, not yet identified, sialic acid containing molecules. Interestingly, three out of 10 tumors analyzed, even if histologically comparable to the others but with a larger dimension, show a significative difference as regard to the GM1, GD3 and GD1a content. Our data suggest that an activated oncogene may induce also in vivo a specific and transmissible alteration in the ganglioside pattern, but this distribution could be susceptible to further modifications during the tumor progression.

Glycosphingolipid expression in solid tumours and transformed cell lines.

Glycosphingolipids are assumed to play a crucial role in cell-cell and cell-substrate interactions, including cell adhesion, proliferation, differentiation and apoptosis. Furthermore, cell surface glycolipid profile changes in the so called "social disorders", such as malignant transformation. To better investigate these modifications, the ganglioside composition in different solid tumours and in two transformed cell lines was analyzed. In some of these models we also tried to correlate the pattern of gangliosides to the key enzymes involved in their metabolism. The results we obtained can be summarized as follows:(1), meningiomas with or without chromosome 22 deletion: predominance of ganglioside GD3 in the former and of ganglioside GM3 in the latter. Correlation between GM3/GD3 ratio and SAT-2 activity; (2), mammary carcinomas developed in MMTV/c-neu transgenic mice: accumulation of GM3-derived species. The different ganglioside distribution seems to correlate with the tumour size; (3), Sarcoma Galliera-strain cells SGS/3A and normal syngenic murine fibroblasts FG: transformed cells exhibit a lower activity of sialyltransferases (SAT-1, SAT-2, SAT-4) compared to normal fibroblasts, suggesting a possible correlation with the ganglioside pattern. The neuraminidase activity seems to correlate to the glycoprotein sialic acid content; (4), 3T3 normal murine fibroblasts and SVT2 transformed cells: GM3 is absent in 3T3, while it accounts for the main ganglioside species in SVT2. On the contrary, GM2 present in a large amount in normal fibroblasts, is practically absent in transformed cells. No correlation has been observed between ganglioside profile and glycosyltransferase activities so far examined.

Local regression of breast tumors following intramammary ganciclovir administration in double transgenic mice expressing neu oncogene and herpes simplex virus thymidine kinase.

Females from a mouse lineage transgenic for the activated rat neu oncogene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) all develop breast tumors with high reproducibility within the first 2-3 months of life. These animals were crossed with mice from a lineage transgenic for the herpes simplex virus thymidine kinase gene (HSVtk) under the control of its own promoter and polyoma enhancer. Double transgenic mice (for both neu and tk) developed breast neoplasias with the same kinetics as the neu-only mice. Tumor-bearing double transgenic mice, treated intratumorally with the antiviral agent ganciclovir (GCV), showed an inhibiting effect on tumor growth. However, this effect was not seen either on GCV-treated neu-only transgenic mice or on saline-injected controls. This suggests that tk-engineered breast tumors are susceptible to GCV administered locally, and implies that neu-mice could be a useful model for testing the effectiveness of HSVtk-bearing vectors followed by systemic GCV on breast cancer cells.

The use of pre-implantation mouse embryos cultured in vitro in toxicological studies.

The preimplantation mouse embryo has been found to be a good model for various toxicological investigations. This communication deals with two examples of research activities carried out in our laboratory to detect the embryotoxic properties of tritium and of 1,2:3,4-diepoxybutene (DEB). Exposures of blastocysts for 24 hr to concentrations as high as 0.296 kBq/ml tritiated amino acids or nucleoside induced a statistically significant reduction in the percentage of embryos that reached the stage of two-layer inner cell mass (ICM). The same quantity of tritiated arginine, but not of thymidine or tryptophan, also induced a lower percentage of differentiating ICM than the control when added to culture medium during the second cleavage division. These findings support the idea that tritium released by nuclear power plants as HT or as tritiated water (HTO) could become a radiotoxicological problem since it can be converted easily into organic compounds by living organisms. DEB was found to be highly embryotoxic in preimplantation mouse embryos in vitro at micromolar concentrations. This compound is formed in mammalian cells by the oxidative metabolism of 1,3-butadiene, a chemical used in rubber industries and present in tobacco smoke. Again, the most sensitive stages of preimplantation development were found to be the two- and the four-cell embryos. These results were confirmed by in vivo measurements.