Abstract
Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v) in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC) Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis.
Highlights
Bone morphogenetic proteins (BMPs) belong to the larger TGF-β family of extracellular ligands and are involved in a multitude of developmental and homeostatic processes [1]
An in vitro co-culture system containing a feeder layer of ADSCs, which are similar to mesenchymal stem cells, and Endothelial cord forming cells (ECFCs), a subtype of umbilical cord blood-derived endothelial cells which can form vascular networks, was used to analyze BMP effects on cord formation [17,18]
When HUVECs were pretreated with BMP7 variant (BMP7v) and cultured in Matrigel, there was a large reduction in the number of endothelial tubes formed
Summary
Bone morphogenetic proteins (BMPs) belong to the larger TGF-β family of extracellular ligands and are involved in a multitude of developmental and homeostatic processes [1]. BMP9 and BMP10 are high affinity ligands for the type I receptor ALK1, expressed on endothelial cells and possibly playing a role in endothelial cell dynamics [12]. BMP4 induced human umbilical vein endothelial cell sprouting, migration and tube formation [13,14] along with paracrine effects on tumor vasculature in malignant melanoma [15]. These observations as well our previous results showing that a BMP7 variant (BMP7v) was able to reduce glioblastoma brain invasion and associated angiogenesis [16] led us to further investigate the mechanism of this angiogenic inhibition
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