Very Low Molecular Weight Heparins Retain the Capacity to Inhibit Endothelial Cell Migration and Capillary-Like Tube Formation Induced by Tumor Cells.

A beneficial effect of low molecular weight heparins (LMWH) on the survival of cancer patients is suggested by clinical data and has been attributed to a broad range of activities, including the capacity to affect the hemostatic and angiogenic properties of endothelial cells. We have previously observed that unfractionated heparin and LMWHs with a mean molecular weight (MW) of 4.5 to 5.7 KDa can effectively antagonise tumor cell-induced angiogenesis in a microvascular endothelium-based Matrigel assay (Marchetti et al, Thromb Res 2008).

Since this anti-angiogenic activity of heparins may depend on the mean MW and the composition of their polysaccharidic chains, this study was designed to evaluate the capacity of ‘second generation’ LMWHs, with a lower MW, to counteract the pro-angiogenic stimulus of tumor cells: i.e. the LMWH Bemiparin, that possesses the lowest MW among commercially available LMWH (i.e. 3.6 KDa), and of its ultra low MW derivative RO-14 (MW=2.5 KDa, Laboratorios Farmaceuticos Rovi S.A., Madrid, Spain).

Tumor cell conditioned media (TCM) were obtained from three human cell lines of different tumor types, i.e. H69 (small cell lung cancer), MDA.MB.231 (breast cancer), and NB4 (acute promyelocytic leukemia). Human microvascular endothelial cells (HMEC-1) were incubated for 24h in Matrigel with TCM, or purified proangiogenic factors (VEGF, FGF-2), in the absence or presence of increasing concentrations (0.01-0.1-1 IU/ml) of Bemiparin or RO-14. Tubule formation was photographed under inverted microscopy and quantified by an image analysis software. The same heparins were tested for their capacity to counteract endothelial migration elicited by TCM in the wound healing assay. The levels of the pro-angiogenic factors VEGF, FGF-2 and IL-1beta in TCM were quantified by ELISA.

All three TCM induced a significant (p<0.05) increase in total tube length (43-61% mean increase) compared to control medium. These increases were dose-dependently inhibited by both Bemiparin and RO-14, with about 75-100% inhibition at the heparin concentration of 1 IU/ml. In the same experimental condition the anti-VEGF antibody inhibited by 67-98% the TCM-induced capillary tube formation. Both heparins significantly inhibited tube formation induced by standard VEGF and FGF-2. In the wound healing assay, Bemiparin and RO-14 prevented the endothelial migration induced by all three different TCM. The evaluation of proangiogenic cytokine content in TCM showed that VEGF was the main product in all of the three cancer cell lines.

Bemiparin and RO-14 counteract the proangiogenic stimulus of tumor cells and of standard proangiogenic factors on microvascular endothelium. Interestingly, the Ultra-low MWH RO-14 presents an anti-angiogenic activity similar to that of the LMWH bemiparin. This suggests that the anti-angiogenic action can be retained by heparins with shorter polysaccharidic chains, which may be of interest in patients with cancer receiving LMWH for prophylaxis or therapy of thrombosis.

No relevant conflicts of interest to declare.