Tie2

Tie2 is upregulated during distraction osteogenesis.

Identification of progenitor cells that contribute to heterotopic skeletogenesis.

"Individuals who have fibrodysplasia ossificans progressiva develop an ectopic skeleton because of genetic dysregulation of bone morphogenetic protein (BMP) signaling in the presence of inflammatory triggers.
We used Cre/loxP lineage tracing methods in the mouse to identify cell lineages that contribute to all stages of heterotopic ossification. Specific cell populations were permanently labeled by crossing lineage-specific Cre mice with the Cre-dependent reporter mice R26R and R26R-EYFP. Two mouse models were used to induce heterotopic ossification: (1) intramuscular injection of BMP2/Matrigel and (2) cardiotoxin-induced skeletal muscle injury in transgenic mice that misexpress BMP4 at the neuromuscular junction. The contribution of labeled cells to fibroproliferative lesions, cartilage, and bone was evaluated histologically by light and fluorescence microscopy. The cell types evaluated as possible progenitors included skeletal muscle stem cells (MyoD-Cre), endothelium and endothelial precursors (Tie2-Cre), and vascular smooth muscle (Smooth Muscle Myosin Heavy Chain-Cre [SMMHC-Cre]).
Vascular smooth muscle cells did not contribute to any stage of heterotopic ossification in either mouse model. Despite the osteogenic response of cultured skeletal myoblasts to BMPs, skeletal muscle precursors in vivo contributed minimally to heterotopic ossification, and this contribution was not increased by cardiotoxin injection, which induces muscle regeneration and mobilizes muscle stem cells. cells that expressed the vascular endothelial marker Tie2/Tek at some time in their developmental history contributed robustly to the fibroproliferative, chondrogenic, and osteogenic stages of the evolving heterotopic endochondral anlagen. endothelial markers were expressed by cells at all stages of heterotopic ossification. muscle injury and associated inflammation were sufficient to trigger fibrodysplasia ossificans progressiva-like heterotopic ossification in a setting of chronically stimulated BMP activity.
Tie2-expressing progenitor cells, which are endothelial precursors, respond to an inflammatory trigger, differentiate through an endochondral pathway, contribute to every stage of the heterotopic endochondral anlagen, and form heterotopic bone in response to overactive BMP signaling in animal models of fibrodysplasia ossificans progressiva. Thus, the ectopic skeleton is not only supplied by a rich vasculature, but appears to be constructed in part by cells of vascular origin. dysregulation of the BMP signaling pathway and an inflammatory microenvironment are both required for the formation of fibrodysplasia ossificans progressiva-like lesions."

"a recurrent heterozygous missense mutation in the BMP type-I receptor, activin receptor IA/activin-like kinase-2 (ACVR1/ALK2) [can cause heterotopic ossification]"

"a mutation in ACVR1 results in dysregulation of BMP signaling"

"Tie2, a receptor tyrosine kinase for angiopoietins, plays a critical role in the development of the embryonic vasculature and is ubiquitously expressed in early endothelial precursors during development and postnatal tissue repair"<-LSJL upregulates ANGPTL1(which interacts with Tie2) and ANGPTL2(which is related to inflammation which this study claims is needed for heterotopic ossification).

"While hematopoietic stem cells express Tie2, recent bone marrow transplantation studies have shown that cells of the hematopoietic system do not contribute to fibroproliferative, chondrogenic, or osteogenic stages of BMP-induced heterotopic ossification"

" Intramuscular injection of BMP2 or BMP4 protein is also sufficient to elicit ectopic skeletogenesis"

"the preosseous skeletal anlagen is derived from cells of nonhematopoietic origin"

"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."<-We need to study the endothelium and local vasculature of adult bone marrow.

"BMP receptors are highly expressed on endothelial cells in vivo, and the BMP-Smad pathway potently activates the endothelium"

"BMPs have the ability to redirect the differentiation of connective tissue progenitor cells to orchestrate an endothelial-to-mesenchymal transition in these cells, often through inflammatory cell intermediates"

"isexpression 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 heart"

"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 response"

Tie2 ligands angiopoietin-1 and angiopoietin-2 are coexpressed with vascular endothelial cell growth factor in growing human bone.

"we investigated the expression of the angiopoietins (Ang-1 and Ang-2) in human neonatal ribs. Ang-1 and Ang-2 exhibited similar patterns of staining in the growing rib. In the cartilage, expression of Ang-1 and Ang-2 increased with chondrocyte maturation. Ang-1, Ang-2, and VEGF were not detected in the resting zone except adjacent to vascular canals, and maximum expression was detected at the cartilage bone interface {Ang1, Ang2, and VEGF may play critical roles in cartilage canal formation}. In the cartilage, Ang-2 was more highly expressed than Ang-1 or VEGF, with staining observed in the proliferating, hypertrophic, and mineralized zones. In the bone, Ang-1, Ang-2, and VEGF were detected in modeling and remodeling sites. Ang-1 was detected in the majority of osteoblasts, osteoclasts, and in some marrow space cells. Ang-2 was expressed at variable levels by osteoblasts and osteoclasts in modeling and remodeling bone. VEGF was detected in cells at bone surfaces and in the marrow spaces. Strong staining for VEGF was observed in osteoblasts and osteoclasts in modeling and remodeling bone. In the perichondrium, Ang-1, Ang-2, and VEGF were most highly expressed adjacent to the hypertrophic zone and at sites of bone collar formation. In the periosteum, Ang-1, Ang-2, and VEGF expression colocalized with alkaline phosphatase expression. The distribution of Ang-1, Ang-2, and VEGF indicate these factors may play key roles in the regulation of angiogenesis at sites of endochondral ossification, intramembranous ossification, and bone turnover in the growing human skeleton."

"transgenic overexpression of Ang-2 resulted in failure of angiogenesis and normal vascular development due to disruption of endothelial-pericyte interactions"

"vascular invasion of the cartilage from the perichondrium was only observed in the resting zone, with focal expression of Ang-1, Ang-2, and VEGF in chondrocytes adjacent to the invading vascular canals."

"In the bone, all three factors were detected at sites of bone modeling and remodeling"<-So maybe we don't need to do anything special to induce these factors and just LSJL will be enough.

COMP-Ang1 promotes chondrogenic and osteogenic differentiation of multipotent mesenchymal stem cells through the Ang1/Tie2 signaling pathway.

"cartilage oligomeric matrix protein angiopoietin1 (COMP-Ang1), an Ang1 variant which is more potent than native Ang1 in phosphorylating Tie2 receptor was developed. The Ang1/Tie2 signaling system not only plays a pivotal role in vessel growth, remodeling, and maturation, but also protective and recruit effect on MSCs. Thus, the aim of the present study was to investigate the differentiate effect of Ang1/Tie2 signaling on MSCs in the presence of chondrogenic, osteogenic and adipogenic induction medium, and to determine the possible mechanisms.  MSCs cultured in each induction medium with COMP-Ang1 revealed strongly chondrogenic and osteogenic morphological change (3.5- and 2-fold, respectively){COMP-Ang1 is more chondrogenic than osteogenic} as well as up-regulate each gene, except for adipogenic differentiation. Phosphorylation of Tie2 expression lead to phosphorylation of p38 and AKT and then accelerating each differentiation of MSCs to chondrocytes and osteoblasts."

Comp-Ang1 increased cell proliferation maximally at 300 nanograms.  It also increased cell viability.  It also increased phosphorylation of Tie2 and this was downstream of the p38 and Akt pathway.