Sandra Donnini

Linomide blocks angiogenesis by breast carcinoma vascular endothelial growth factor transfectants

The blocking of angiogenesis provides a novel therapeutic target to inhibit tumour spreading. In this study, we investigated the effect of linomide on angiogenesis induced in vivo by highly angiogenic breast carcinoma cells. The rabbit cornea was used to assess neovascular growth in the absence of a tumour mass. MCF-7 cells stably transfected with the cDNA encoding for vascular endothelial growth factor 121 (VEGF121) (V12 clone) were used to elicit a potent VEGF-dependent corneal angiogenesis. After tumour cell implant, albino rabbits received 100 mg kg(-1) day(-1) linomide for 5 consecutive days. Daily observation of neovascular progression indicated that linomide blocked angiogenesis. The antiangiogenic effect of linomide was apparent within 48 h from the beginning of the treatment and was both angiosuppressive and angiostatic. The block of neovascular growth lasted over 10 days from treatment suspension, and preformed vessels, which had regressed, remained dormant, suggesting the persistence of unfavourable conditions for capillary progression. Linomide (50-200 microg ml[-1]) was not cytotoxic in vitro on resting capillary endothelial cells but blocked endothelial cell replication induced by VEGF. Our data indicate that linomide can efficiently and persistently block VEGF-dependent angiogenesis in vivo in the absence of a growing tumour mass. These data suggest that linomide could be a chemopreventive drug in breast cancer patients and a valuable tool in clinical settings in which metastatic spreading occurs in the absence of a detectable tumour mass.

The effect of linomide on the migration and the proliferation of capillary endothelial cells elicited by vascular endothelial growth factor

In order to assess the mechanism of action of the quinoline‐3‐carboxyamide linomide as an antiangiogenic drug, the effect of linomide was studied in vitro on postcapillary endothelial cells exposed to vascular endothelial growth factor (VEGF). Linomide did not block the spontaneous replication of endothelial cells, but significantly suppressed endothelial cell growth and migration elicited by VEGF. It is concluded that linomide appears to be an effective tool to inhibit VEGF‐dependent angiogenesis.

Endostatin: a promising drug for antiangiogenic therapy.

Angiogenesis, the formation of new blood vessels from existing capillaries, is critical for tumors to grow beyond a few in size. Tumor cells produce one or more angiogenic factors including fibroblast growth factor and vascular endothelial growth factor. Surprisingly, antiangiogenic factors or angiogenesis inhibitors have been isolated from tumors. Some angiogenesis inhibitors, such as angiostatin, are associated with tumors while others, such as platelet-factor 4 and interferon-alpha are not. Endostatin, a C-terminal product of collagen XVIII, is a specific inhibitor of endothelial cell proliferation, migration and angiogenesis. The mechanism by which endostatin inhibits endothelial cell proliferation and migration is unknown. Endostatin was originally expressed in a prokaryotic system and, late, in a yeast system, thanks to which it is possible to obtain a sufficient quantity of the protein in a soluble and refolded form to be used in preclincial and clinical trials.

Nitric oxide modulates the angiogenic phenotype of middle-T transformed endothelial cells

The role of nitric oxide (NO) in the induction of angiogenesis was evaluated in a murine heart endothelioma cell line (H.end.FB) carrying the mT oncogene. Two clonal derivatives of H.end.FB, H80 and H73, exhibiting different NO synthase (NOS) activities were selected and used in the study. The relationship among NOS activity and tumor cell behaviour (growth, and angiogenic capacity) and the molecular control of gene expression were investigated. H.end.FB and H80 on one side and H73 on the other side exhibited the highest and lowest NOS activity, respectively. Cell growth was inversely correlated to the amount of NO produced by the cell lines. Conversely, in the avascular rabbit cornea assay, H.end.FB and H80 cells were strongly angiogenic, while H73 were poorly angiogenic, indicating that the ability of the cells to induce neovascularization was associated with the extent of NO produced. Consistently, systemic administration to rabbits of the NOS inhibitor N(w)-nitro-L-arginine methyl ester (L-NAME) significantly reduced the angiogenicity of H.end.FB cells. RT-PCR evidenced that H.end.FB expressed mRNA for TGF-beta1 and all VEGF isoforms, VEGF165 being predominantly expressed. NOS inhibition reduced the basal expression of VEGF isoforms, while it markedly potentiated TGF-beta1 expression. These results indicate that the endogenous production of NO in tumor cells can serve as an autocrine/paracrine signalling mechanism of progression, by controlling angiogenic factor/modulator expression.

Differential contribution of bradykinin receptors in angiogenesis.

Bradykinin (BK) contributes to the inflammatory response inducing vasodilation of postcapillary venules and has been demonstrated to induce neovascular growth in subcutaneous rat sponges. In this study the ability of BK to stimulate cell growth and migration in cultured endothelium from coronary postcapillary venules (CVEC) has been investigated. BK promotes growth of endothelial cells and its mitogenic activity involves c-Fos expression. Only the Bl receptor appears to be responsible for the proliferation induced by BK and suggests that this type of receptor might be implicated in favouring angiogenesis of coronary venules. Phospholipase C (PLC) activation and the endogenous upregulation of nitric oxide synthase (NOS) were monitored in response to Bl and B2 receptor activation. BK-induced IP turnover was triggered by B2 activation, while NOS activation was linked to the Bl receptor. NOS inhibition in CVEC prevented BK/B1 induced cell growth. These results substantiate the protective and trophic role of BK on microvascular endothelium by demonstrating that Bl receptor stimulation on venular endothelium is mandatory for the angiogenic effect of BK through its coupling to NOS activation.

Angiogenic Polypeptides in Breast Cancer: Expression of Mrna’s in Primary Human Tumours, MCF-7 Cell Transfection and Xenograft Models

Screening of 84 primary human breast carcinomas for the mRNA expression of seven angiogenic polypeptides showed that the most commonly expressed and/or the most highly expressed when compared to normal breast tissue were vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor/thymidine Phosphorylase (PDECGF/TP)1. The neurokines midkine (MK) and pleiotrophin were also fairly commonly expressed in tumour but not normal tissue (unpublished data from this group and ref. 1).