Despite intensive efforts, the lineages of progenitor cells that respond to BMP signaling and directly contribute to the formation of ectopic bone, as originally described by Urist5, have remained elusive. We used Cre/loxP lineage tracing methods in the mouse to identify the origin of cells that are directly responsible for ectopic skeletogenesis. In the present study, we show that soft-tissue injury and associated inflammatory changes induce and synchronize endochondral heterotopic ossification in transgenic mice that misexpress BMP4 at the neuromuscular junction13. Intramuscular injection of BMP2 or BMP4 protein is also sufficient to elicit ectopic skeletogenesis, and both models of heterotopic ossification closely resemble the progression of histopathological events observed in patients with fibrodysplasia ossificans progressiva10. In both models, we demonstrated that progenitor cells that express Tie2 in their developmental history contribute substantially to every stage of the endochondral anlagen, including the fibroproliferative, chondrogenic, and osteogenic stages.
In addition to Tie2 expression in endothelial cells, in which it regulates growth, remodeling, and maintenance of the vasculature, Tie2 is also expressed by hematopoietic stem cells29,30, raising the possibility that Tie2+ progenitors that participate in heterotopic ossification arise from the hematopoietic system and not from endothelium of the local vasculature. Recent studies on bone marrow transplantation, however, while pointing to the importance of hematopoietic cells in triggering heterotopic ossification, have clearly shown that the preosseous skeletal anlagen is derived from cells of nonhematopoietic origin9. These data strongly suggest that the labeled cells in heterotopic lesions of Tie2-Cre;R26R mice arise from the endothelium of the local vasculature, in response to injury and BMP signaling.
BMP receptors are highly expressed on endothelial cells in vivo, and the BMP-Smad pathway potently activates the endothelium32. In addition, BMPs have the ability to redirect the differentiation of connective tissue progenitor cells33-35 to orchestrate an endothelial-to-mesenchymal transition in these cells, often through inflammatory cell intermediates36-40. Interestingly, misexpression of constitutively active ACVR1/ALK2, a BMP type-I receptor and the gene mutated in fibrodysplasia ossificans progressiva, is sufficient to stimulate an endothelial-to-mesenchymal transformation in endothelial cells of the heart41. Notably, BMP4, as well as hypoxia and inflammatory cytokines—conditions and factors that are present in the earliest preosseous lesions of heterotopic ossification—upregulate Tie2 in endothelial cells, which contributes to the angiogenic response39,42. The ongoing expression of Tie2 and other endothelial markers at all stages of BMP-induced ossification is likely a response to these local environmental cues and is entirely consistent with previous reports in two animal models of bone regeneration and fracture callus formation43. Thus, the heterotopic skeleton is not only supplied by a robust vasculature, it is actually derived from and formed in part by vascular cells. Of note, normal developing cartilage and bone of the embryonic skeleton are not derived from Tie2+ progenitors (unpublished observations), suggesting key differences in the etiology of normotopic and heterotopic bone.
Clinical studies have identified inflammatory cells in the earliest fibrodysplasia ossificans progressiva lesions7,11, and long-term quiescence of fibrodysplasia ossificans progressiva following chronic immune suppression has been noted8,9. Cunningham et al.44 showed that BMPs have profound effects on the recruitment of monocytes, known precursors of tissue macrophages. Subsequent studies have shown that BMP receptors are robustly expressed on monocytes and tissue macrophages45. Importantly, previous studies have identified macrophage recruitment to the early injury site following muscle trauma in the Nse-BMP4 mice13. These data strongly suggest that macrophages are involved in the induction of injury-induced heterotopic ossification in Nse-BMP4 transgenic mice. Our working model of BMP4-induced heterotopic ossification, which integrates the inflammatory reaction to muscle injury, the secretion of BMPs, and the cross-talk between cells of the innate and adaptive immune system, is shown in Figure 6. Importantly, our study shows that muscle injury and associated inflammatory changes are sufficient to trigger fibrodysplasia ossificans progressiva-like heterotopic ossification in a setting of chronically stimulated BMP activity.
In both models of heterotopic ossification used in the present study, approximately 50% of the heterotopic chondrogenic and osteogenic cells were derived from Tie2+ progenitor cells. Since nearly 100% of endothelium is labeled in Tie2-Cre mice (unpublished observations), incomplete labeling of the BMP-induced skeletal anlagen is not a consequence of inefficient labeling of the Tie2 lineage. Rather, these data indicate that at least one additional progenitor population (Tie2-negative) can respond to BMP2/4 and injury signals and can participate in ectopic skeletogenesis. Among possible progenitors, satellite cells—muscle stem cells responsible for muscle repair—were particularly attractive candidates. Satellite cells in culture downregulate the myogenic program and express osteogenic markers in response to BMP signaling12,46 (unpublished observations). Further, fibrodysplasia ossificans progressiva lesions and BMP-induced heterotopic ossification are restricted to skeletal muscle and associated soft tissues, raising the possibility that ectopic skeletogenesis is mediated by stem and/or progenitor cells specific to muscle tissue. Surprisingly, however, satellite cells contributed minimally to BMP-induced heterotopic lesions, even when the satellite cell pool was activated by cardiotoxin-induced muscle injury prior to administration of BMP2. At the time of writing, we were evaluating other stem cell sources in muscle tissue47 for their osteogenic capacity.
Previous studies have shown that early fibroproliferative lesional stromal cells of fibrodysplasia ossificans progressiva and BMP-induced lesions express multiple smooth muscle lineage markers11. Smooth muscle marker expression could reflect a vascular smooth muscle origin or could result from de novo activation of smooth muscle markers in lesional cells originating from other sources. Using SMMHC-Cre transgenic mice15, we did not observe a contribution of vascular smooth muscle cells to any stage of BMP-induced heterotopic ossification. The origin of smooth muscle-like cells in heterotopic lesions, therefore, remains to be determined. One candidate is the pericyte, a smooth muscle-like mural cell of the microvasculature that exhibits multilineage differentiation capacity47. Importantly, pericytes exhibit chondrogenic and osteogenic differentiation potential in some settings47, making them an excellent candidate progenitor of heterotopic ossification. Pericytes are difficult to evaluate at present, however, because pericyte-specific reagents for lineage tracing have not yet been developed.
The discovery that Tie2-expressing vascular cells contribute to the heterotopic endochondral anlagen will aid in the development of cell-specific therapeutic strategies to treat fibrodysplasia ossificans progressiva and more common conditions of BMP-associated heterotopic endochondral ossification. Given the prevalence of BMP signaling in diverse cellular processes, the ability to target specific cell populations is of primary importance in order to minimize collateral effects. Mouse models of fibrodysplasia ossificans progressiva should prove invaluable for testing treatment modalities and drug discovery. In this regard, while the two mouse models of heterotopic ossification used herein recapitulate important clinical features of fibrodysplasia ossificans progressiva, they are not perfect models for the condition. We are currently developing gene-targeted mice that harbor the same activating mutation in the ACVR1/ALK2 BMP receptor that causes classic fibrodysplasia ossificans progressiva in all affected individuals3. Studies in those mice will enable a more comprehensive understanding of the cellular triggers and the repertoire of responsive progenitor cells in fibrodysplasia ossificans progressiva and will provide a systematic background for the development of the most appropriate medications and treatment for the condition.
NOTE: The authors thank Bob Caron for his invaluable technical assistance. The study was received in original form August 8, 2008 and accepted September 25, 2008.
Investigation performed at the University of Pennsylvania, Philadelphia, Pennsylvania, and the University of Connecticut, Storrs, Connecticut
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