Fig. 1. Markers expressed by cultured HSVECs. (A) Immunocytochemical visualization of the Von Willebrand factor. In the inset unspecific staining (obtained by omitting the primary antibody) is shown; weakly counterstaining with hematoxylin allows an easier identification of the cells. (B) Ethidium bromide-stained 1.5% agarose gel showing cDNA amplified with human AM specific primer from RNA of three control HSVEC cultures (left panel). The immunocytochemical demonstration of the AM peptide in the cells is shown in the right panel. (C) The melting curve (left panel) with a specific single peak demonstrating the presence of RAMP2 mRNA in HSVECs, together with the immunocytochemical demonstration of the RAMP2 and CRLR proteins in the cells (right panels). (D–F) Melting curves (left panels) indicating the expression of VEGF, VEGFR-2 and VEGFR-1 mRNA, respectively, together with the immunocytochemical visualization of the corresponding proteins (right panels). Bars are 50 μm.

Fig. 2. Effect of AM, FGF-2 and VEGF on HSVEC proliferation. (A) Effect on HSVEC proliferation of increasing concentrations of administered AM. A significant stimulatory effect was observed from the dose of 10⁻⁸ M. (B) In normal cells 10⁻⁸ M AM treatment induces an increase of the proliferation rate by about 100%, comparable to that obtained with FGF-2 or VEGF. (C) When the expression of RAMP2 in the cells was almost suppressed by gene silencing (see Section 2) the effect of 10⁻⁸ M AM on cell proliferation was no longer detectable, indicating that it was mediated by a functional AM1 receptor. Conversely, the proliferative action of FGF-2 or VEGF was not altered in this experimental condition. *p < 0.05 vs. the control group.

Fig. 3. Phase contrast micrographs illustrating the arrangement of HSVECs into a meshwork of capillary-like tubular structures when cultured on Matrigel for 18 h (A). 50 ng/mL FGF-2 (B) as well as 10⁻⁸ M AM (C) increased the density and complexity of the meshwork. Bar is 150 μm.

Fig. 4. Morphometric analysis of the effect of AM, FGF-2 and VEGF on the structure of the capillary meshwork formed by HSVECs when cultured on Matrigel. (A) Effect on HSVEC self-organization of increasing concentrations of administered AM. A significant increase of the cell pattern complexity was observed from the dose of 10⁻⁸ M. (B) In normal cells, 10⁻⁸ M AM (as well as FGF-2 or VEGF) significantly increased both dimensional (percent area covered by HSVECs and total length of the network per field) and topological (number of meshes and branching points per field) parameters of the capillary-like network. (C) When the expression of RAMP2 in the cells was almost suppressed by gene silencing the effect of 10⁻⁸ M AM on the capillary-like structures formation was no longer detectable, indicating that it was mediated by a functional AM1 receptor. Conversely, the morphogenetic action of FGF-2 or VEGF was not altered in this experimental condition. *p < 0.05 vs. the control group.

Fig. 5. (A) Western blot assessment of the VEGFR-2 protein (upper panel) and of the protein from the housekeeping gene GAPDH (lower panel) in control HSVEC and in HSVEC treated with 10⁻⁸ M AM or 0.4 μM SU5416. (B) The bands were quantitated by densitometry and data are expressed as the ratio of VEGFR-2 to GAPDH. No changes in the expression of VEGFR-2 were detected following the applied treatments.

Fig. 6. Effects of 10⁻⁸ M AM on the proliferation (left panels) and on the self-organization (right panels) of HSVECs following different protocols of VEGF signaling inhibition (see Section 2). (A) In HSVECs where the expression of VEGF was downregulated by gene silencing a significant proliferative and morphogenetic effect of AM on cultured HSVECs was still recognizable. (B) No changes in the effect of AM can be observed when cell proliferation and Matrigel assays were performed in the presence of the VEGFR-2 inhibitor V1, which acts as an antagonist of VEGFR-2, blocking the interaction of VEGF with that receptor. Conversely, in this experimental condition the effect of VEGF resulted inhibited as expected. (C) In the presence of the VEGFR-2 inhibitor SU5416, which blocks the tyrosine kinase activity of VEGFR-2 (i.e. the downstream signaling of the receptor without affecting its affinity for VEGF) not only was the effect of VEGF inhibited, but also the action of AM on both cell proliferation and self-organization resulted no longer detectable. As shown, FGF-2 was always active in all the above-mentioned experimental conditions. *p < 0.05 vs. the control group.

Fig. 7. Schematic representation of VEGFR-2 transactivation by AM. In addition to its function as a receptor for VEGF, Flk-1/KDR is used by some signaling pathways triggered by the AM1 receptor as the signal transducer for the activation of downstream targets leading to an angiogenic response in the vascular endothelial cell.

Real-time PCR primers

Semiquantitative RT-PCR assay of the effect of AM (10⁻⁸ M) on the relative mRNA expression.

Values are expressed as regulation factors with respect to the untreated control samples