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Sonic hedgehog promotes angiogenesis and osteogenesis in a coculture system consisting of primary osteoblasts and outgrowth endothelial cells.

ABSTRACT: 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.

SUBMITTER: Dohle E

PROVIDER: S-EPMC2875976 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The repair and regeneration of large bone defects, including the formation of functional vasculature, represents a highly challenging task for tissue engineering and regenerative medicine. Recent studies have shown that vascularization and ossification can be stimulated by mild heat stress (MHS), which would offer the option to enhance the bone regeneration process by relatively simple means. However, the mechanisms of MHS-enhanced angiogenesis and osteogenesis, as well as potential risks for the treated cells are unclear. We have investigated the direct effect of MHS on angiogenesis and osteogenesis in a co-culture system of human outgrowth endothelial cells (OECs) and primary osteoblasts (pOBs), and assessed cytotoxic effects, as well as the levels of various heat shock proteins (HSPs) synthesized under these conditions. Enhanced formation of microvessel-like structures was observed in co-cultures exposed to MHS (41°C, 1 h), twice per week, over a time period of 7-14 days. As shown by real-time polymerase chain reaction (PCR), the expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and tumor necrosis factor-alpha was up-regulated in MHS-treated co-cultures 24 h post-treatment. At the protein level, significantly elevated VEGF and Ang-1 concentrations were observed in MHS-treated co-cultures and pOB mono-cultures compared with controls, indicating paracrine effects associated with MHS-induced angiogenesis. MHS-stimulated co-cultures and OEC mono-cultures released higher levels of Ang-2 than untreated cultures. On the other hand MHS treatment of co-cultures did not result in a clear effect regarding osteogenesis. Nevertheless, real-time PCR demonstrated that MHS increased the expression of mitogen-activated protein kinase, interleukin-6, and bone morphogenetic protein 2, known as HSP-related molecules in angiogenic and osteogenic regulation pathways. In agreement with these observations, the expression of some selected HSPs also increased at both the mRNA and protein levels in MHS-treated co-cultures.

Project description:Sonic Hedgehog (Shh) signaling induces neovascularization and angiogenesis. It is not known whether the hedgehog signaling pathway in endothelial cells is essential to angiogenesis. Smoothened (Smo) transduces hedgehog signaling across the cell membrane. This study assessed whether endothelial Smoothened-dependent Shh signaling is required for Shh-mediated angiogenesis and ischemic tissue repair. Endothelial-specific smoothened knockout mice, eSmoNull were created using Cre-lox recombination system. eSmoNull mice had no observable phenotype at baseline and showed normal cardiac function. Smoothened in CD31+ cells isolated from eSmoNull hearts was significantly reduced compared to CD31+ cells from eSmoWT littermate control hearts. Fluorescence immunostaining of eSmoNull heart sections showed Smo expression in endothelial cells was abolished. The hind-limb ischemia (HLI) model was used to assess the response to ischemic injury. Perfusion ratio, limb motor function, and limb necrosis were not significantly different after HLI between eSmoNull mice and eSmoWT. Capillary densities in the ischemic limb in eSmoNull mice were also similar to eSmoWT at 4 weeks after HLI. Next, response to exogenous Shh was assessed in the corneal angiogenesis model. There was no significant difference in corneal angiogenesis induced by administration of Shh pellets between eSmoWT and eSmoNull mice. Furthermore, in vitro experiments demonstrated that direct Shh had limited effects on endothelial cell proliferation and migration. However, conditioned media from Shh-treated fibroblasts had a more potent effect on endothelial cell proliferation and migration than non-treated conditioned media. Furthermore, Shh treatment of fibroblasts dramatically stimulated angiogenic growth factor expression, including PDGF-B, VEGF-A, HGF and IGF. PDGF-B was the most upregulated and may contribute to the large neo-vessels associated with Shh-induced angiogenesis. Taken together, these data demonstrate that Shh signaling via Smoothened in endothelial cells is not required for angiogenesis and ischemic tissue repair. Shh signaling via stromal cells likely mediates its angiogenic effects.

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Sonic hedgehog promotes angiogenesis and osteogenesis in a coculture system consisting of primary osteoblasts and outgrowth endothelial cells.

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