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A number of previous studies documented the angiogenic potential of outgrowth endothelial cells in vitro and in vivo and provided evidence that therapeutic success could depend on coculture or coimplantation strategies. Thus, deeper insight into the molecular mechanisms underlying this pro-angiogenic effect of cocultures might provide new translational options for tissue engineering and regenerative medicine. One promising signaling pathway in bone repair involved in neoangiogenesis and bone formation is the sonic hedgehog (Shh) pathway. In this article, we focus on the effect of Shh on the formation of microvessel-like structures and osteoblastic differentiation in cocultures of primary osteoblasts and outgrowth endothelial cells. Already after 24 h of treatment, Shh leads to a massive increase in microvessel-like structures compared with untreated cocultures. Increased formation of angiogenic structures seems to correlate with the upregulation of vascular endothelial growth factor or angiopoietins (Ang-1 and Ang-2) studied at both the mRNA and protein levels. In addition, treatment with cyclopamine, an inhibitor of hedgehog signaling, blocked the formation of microvessel-like structures in the cocultures. However, exogenous Shh also resulted in the upregulation of several osteogenic differentiation markers in real-time polymerase chain reaction, as well as in an increased mineralization and alkaline phosphatase activity. The present data highlight the central role of the Shh pathway in bone regeneration and vascularization. Further, Shh might have the potential to improve both angiogenesis and osteogenesis in clinical applications in the future.

Sonic Hedgehog Promotes Angiogenesis and Osteogenesis in a Coculture System Consisting of Primary Osteoblasts and Outgrowth Endothelial Cells