Karine Bonjean

Histone deacetylases inhibitors as anti-angiogenic agents altering vascular endothelial growth factor signaling

Angiogenesis is a complex biological process involving the coordinated modulation of many genes. Histone deacetylases (HDAC) are a growing family of enzymes that mediate the availability of chromatin to the transcriptional machinery. Trichostatin-A (TSA) and suberoylanilide hydroxamic acid (SAHA), two HDAC inhibitors known to relieve gene silencing, were evaluated as potential antiangiogenic agents. TSA and SAHA were shown to prevent vascular endothelial growth factor (VEGF)-stimulated human umbilical cord endothelial cells (HUVEC) from invading a type I collagen gel and forming capillary-like structures. SAHA and TSA inhibited the VEGF-induced formation of a CD31-positive capillary-like network in embryoid bodies and inhibited the VEGF-induced angiogenesis in the CAM assay. TSA also prevented, in a dose-response relationship, the sprouting of capillaries from rat aortic rings. TSA inhibited in a dose-dependent and reversible fashion the VEGF-induced expression of VEGF receptors, VEGFR1, VEGFR2, and neuropilin-1. TSA and SAHA upregulated the expression by HUVEC of semaphorin III, a recently described VEGF competitor, at both mRNA and protein levels. This effect was specific to endothelial cells and was not observed in human fibroblasts neither in vascular smooth muscle cells. These observations provide a conspicuous demonstration that HDAC inhibitors are potent anti-angiogenic factors altering VEGF signaling.

Bone Sialoprotein Mediates Human Endothelial Cell Attachment and Migration and Promotes Angiogenesis

Bone sialoprotein (BSP) is a secreted glycoprotein primarily found in sites of biomineralization. Recently, we demonstrated that BSP is strongly upregulated in osteotropic cancers and particularly those that exhibit microcalcifications. BSP contains an Arg-Gly-Asp (RGD) motif found in other adhesive molecules that interact with cellular integrins. In bone, BSP has been shown to mediate the attachment of osteoblasts and osteoclasts via alpha(v)beta(3) integrin receptors. Ligands for alpha(v)beta(3) integrin are considered to play a central role during angiogenesis. Therefore, we used human umbilical vein endothelial cells (HUVECs) to study the potential role of BSP in angiogenesis. We found that purified eukaryotic recombinant human BSP (rhBSP) is able to promote both adhesion and chemotactic migration of HUVECs in a dose-dependent manner. These interactions involve HUVEC alpha(v)beta(3) integrin receptors and the RGD domain of BSP. Indeed, HUVECs attach to a recombinant BSP fragment containing the RGD domain, whereas this response is not observed with the same fragment in which RGD has been mutated to Lys-Ala-Glu (KAE). A cyclic RGD BSP peptide inhibits both adhesion and migration of HUVECs to rhBSP. Moreover, anti-alpha(v)beta(3) but not anti-alpha(v)beta(5) monoclonal antibodies also prevent BSP-mediated adhesion and migration of HUVECs. We observed that both rhBSP and the RGD BSP recombinant fragment stimulated ongoing angiogenesis on the chorioallantoic chick membrane assay. BSP angiogenic activity was inhibited by anti-alpha(v)beta(3) antibody, and the KAE BSP fragment was inactive. Our findings represent the first report implicating BSP in angiogenesis. BSP could play a critical role in angiogenesis associated with bone formation and with tumor growth and metastatic dissemination.

Zoledronate inhibits alpha v beta 3 and alpha v beta 5 integrin cell surface expression in endothelial cells

Zoledronate exhibits antiangiogenic properties in vitro and in vivo. Integrins alphavbeta3 and alphavbeta5 are involved in angiogenesis. Because zoledronate inhibits endothelial cell adhesion, the authors explored the hypothesis that it could alter these integrins recruitment to focal adhesion sites. Human umbilical vein endothelial cells (HUVECs) were treated with zoledronate or with mevalonate pathway intermediates geranylgeraniol (GGOH) and farnesol (FOH). Zoledronate generated a significant decrease in alphavbeta3 and alphavbeta5 expression at HUVEC cell surface using flow cytometry and immunofluorescence. This inhibition was reversed by GGOH but not by FOH. Cells cotreated with zoledronate and GGOH were able to attach to vitronectin through alphavbeta3 and alphavbeta5, as confirmed by the use of specific function-blocking antibodies. The authors showed that zoledronate alters endothelial cell integrin-mediated adhesion. This effect is likely to contribute to the previously demonstrated antiangiogenic effect of zoledronate. Whether this mechanism of action also applies to metastatic tumor cells is under investigation.