Abstract
Heterotopic ossification (HO), or endochondral bone formation at nonskeletal sites, often results from traumatic injury and can lead to devastating consequences. Alternatively, the ability to harness this phenomenon would greatly enhance current orthopedic tools for treating segmental bone defects. Thus, understanding the earliest events in this process potentially would allow us to design more targeted therapies to either block or enhance this process. Using a murine model of HO induced by delivery of adenovirus-transduced cells expressing bone morphogenetic protein 2 (BMP-2), we show here that one of the earliest stages in this process is the establishment of new vessels prior to the appearance of cartilage. As early as 48 hours after induction of HO, we observed the appearance of brown adipocytes expressing vascular endothelial growth factors (VEGFs) simultaneous with endothelial progenitor replication. This was determined by using a murine model that possesses the VEGF receptor 2 (Flk1) promoter containing an endothelial cell enhancer driving the expression of nuclear-localized yellow fluorescent protein (YFP). Expression of this marker has been shown previously to correlate with the establishment of new vasculature, and the nuclear localization of YFP expression allowed us to quantify changes in endothelial cell numbers. We found a significant increase in Flk1-H2B::YFP cells in BMP-2-treated animals compared with controls. The increase in endothelial progenitors occurred 3 days prior to the appearance of early cartilage. The data collectively suggest that vascular remodeling and growth may be essential to modify the microenvironment and enable engraftment of the necessary progenitors to form endochondral bone. © 2010 American Society for Bone and Mineral Research.
Highlights
Endochondral bone formation is thought to proceed through an ordered series of events starting with the proliferation and ‘‘condensation’’ of presumptive mesenchymal cells to form avascular cartilage
Using a murine model that possesses the vascular endothelial growth factors (VEGFs) receptor 2 (Flk1) promoter containing an endothelial cell enhancer driving the expression of yellow fluorescent protein (YFP),(1) we confirmed recent data from our laboratory using a model of heterotopic ossification that suggested that vessels may play an essential role in the induction of chondrogenesis.[2]
The data cannot differentiate between expansion of cells expressing VEGF-A and -D and elevated transcription within cells residing in the area, the results suggest that these potent endothelial growth factors are rapidly and transiently increased within the site of new bone formation prior to the onset of cartilage
Summary
Endochondral bone formation is thought to proceed through an ordered series of events starting with the proliferation and ‘‘condensation’’ of presumptive mesenchymal cells to form avascular cartilage. Using a murine model that possesses the VEGF receptor 2 (Flk1) promoter containing an endothelial cell enhancer driving the expression of YFP,(1) we confirmed recent data from our laboratory using a model of heterotopic ossification that suggested that vessels may play an essential role in the induction of chondrogenesis.[2]. Vessels invade the perichondrium and hypertrophic zone and are required for the replacement of cartilage by bone.[3] The angiogenic factor vascular endothelial growth factor (VEGF) promotes vascular invasion via specific receptors, including Flk (VEGF receptor 2) expressed in endothelial cells, in the perichondrium or surrounding tissue.[4,5] These events of cartilage matrix remodeling and vascular invasion are necessary for the migration and differentiation of osteoblasts and osteoclasts, which remove mineralized cartilage matrix and replace it with bone. A series of cell signaling events is induced by the hypoxic state of the tissues, resulting in upregulation of hypoxia inducible factor (HIF1), which, in turn, upregulates a series of factors including several VEGFs
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