Pathological angiogenesis is essential for tumor growth and metastasis. Endothelial cells (EC) and pericytes form blood vessels under pro-angiogenic conditions. Although pericytes are found on the microvasculature of various tumors, including neuroblastoma, the mechanism underlying their recruitment to the endothelium and their role in angiogenesis remain unclear. Our laboratory has previously demonstrated that matrix metalloproteinase-9 (MMP-9) expressed in more aggressive neuroblastoma is critical for pericyte recruitment along EC (Jodele et al. Cancer Res 65:3200-3208, 2005). MMPs are involved in multiple stages of tumor progression including angiogenesis. They can degrade extracellular matrix (ECM) proteins, as well as cleave ECM- or cell-associated growth factors (sheddase activity), thereby enhancing their bioavailability. Our current work examines the mechanism by which MMP-9 promotes pericyte recruitment and tests the possibility that MMP-9 may affect the release and activation of platelet-derived growth factor β (PDGFβ) and heparin-binding EGF-like growth factor (HB-EGF), two potent pericyte attractants and stimulatory factors. Human brain pericytes expressing PDGFβ receptor (PDGFR-β) and ErbB2 (HB-EGF) receptor, and human brain microvascular EC (HBMEC) expressing PDGFβ and HB-EGF were co-cultured in transwell assays and examined for migration. We observed a 45±5% increase in pericyte migration towards EC stimulated with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). In contrast, pericytes failed to attract HBMEC. In addition, we found that treating HBMEC with VEGF and bFGF increased the production of soluble MMP-9 in the culture medium. Consistent with our hypothesis, we observed that pericyte migration towards HBMEC was inhibited by 80±4% in the presence of the MMP inhibitor AG3340, whereas it was increased by 49±10% with activated recombinant human MMP-9. We obtained similar inhibitory effects on pericyte recruitment with AG3340 using Matrigel endothelial tube formation assays. Consistent with a role for PDGFβ and HB-EGF in pericyte recruitment, pericyte migration towards stimulated HBMEC was inhibited by 60±8% in the presence of a PDGF function-blocking antibody, and by 50±3% in the presence of Imatinib, which disrupts PDGFβ/PDGFR-β signaling. Migration was also inhibited by 39±15% with a PDGFR-β tyrosine kinase inhibitor, and by 39±6% in the presence of an HB-EGF function-blocking antibody. Taken together, this data suggests that MMP-9 plays an integral role in recruiting pericytes to EC, possibly through its sheddase activity on PDGFβ and HB-EGF. Ultimately, understanding the mechanism behind pericyte recruitment could lead to new therapeutic interventions in the treatment of neuroblastoma.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA