Transforming growth factor-beta1 induces microvascular abnormalities through a down-modulation of neural cell adhesion molecule in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a very angiogenic and malignant cancer. Conventional chemotherapy is poorly effective because of the abnormal structural organization of HCC-infiltrating vessels. In previous work, we demonstrated that HCC angiogenesis is driven by transforming growth factor beta-1(TGF-\u3b21)/CD105 axis, stimulating liver-derived microvascular endothelial cells (Ld-MECs) migration. As TGF-\u3b21 also affects mural cells (MCs) recruitment and maturation, we asked whether it may contribute to HCC-induced vascular abnormalities. HCC and adjacent non-neoplastic liver (nNL) biopsies obtained from 12 patients were analyzed by immunohistochemistry for angiogenic markers CD105, TGF-\u3b21, CD44 and vascular endothelial growth factor-a (VEGFa) and for MC markers NG2, \u3b1-smooth muscle actin (\u3b1SMA) and neural cell adhesion molecule (NCAM). The same markers were also investigated by immunocytochemistry on cultured HCC-derived stromal cells (HCC-StCs) and nNL-derived StCs (nNL-StCs) isolated from the same liver biopsies. Angiogenic factors released by StCs were analyzed by ELISA and the interaction between StCs and Ld-MECs by adhesion assay. Compared with nNL, HCC biopsies showed increased angiogenic markers and \u3b1SMA that was localized in vessels. By contrast, NG2 and NCAM were substantially localized in tumor cells but absent in vessels and stroma. Cultured HCC-StCs showed less expression of NG2, \u3b1SMA and NCAM. They also demonstrated a lower capacity to release angiogenic factors and adhered on Ld-MECs. HCC-StCs and nNL-StCs treated with TGF-\u3b21 or with of HepG2 (a human hepatoma cell line) derived conditioned medium (CM), down-modulated NCAM expression, whereas anti-NCAM antibodies significantly reduced the adhesion of StCs to Ld-MECs. By further blocking TGF-\u3b21 with anti-TGF-\u3b21 antibodies or with Ly-364947 (a specific inhibitor TGF-\u3b21-receptor) adhesion to Ld-MECs and NCAM expression respectively was partially restored. TGF-\u3b21 contributes to HCC-induced vascular alterations by affecting the interaction between HCC-StCs and Ld-MECs through a down-modulation of NCAM expression