ABSTRACT
Bone regeneration requires a well-orchestrated cellular and molecular response including robust vascularization and recruitment of mesenchymal and osteogenic cells. In femoral fractures, angiogenesis and osteogenesis are closely coupled during the complex healing process. Here, we show with advanced longitudinal intravital multiphoton microscopy that early vascular sprouting is not directly coupled to osteoprogenitor invasion during calvarial bone regeneration. Early osteoprogenitors emerging from the periosteum give rise to bone-forming osteoblasts at the injured calvarial bone edge. Microvessels growing inside the lesions are not associated with osteoprogenitors. Subsequently, osteogenic cells collectively invade the vascularized and perfused lesion as a multicellular layer, thereby advancing regenerative ossification. Vascular sprouting and remodeling result in dynamic blood flow alterations to accommodate the growing bone. Single cell profiling of injured calvarial bones demonstrates mesenchymal stromal cell heterogeneity comparable to femoral fractures with increase in cell types promoting bone regeneration. Expression of angiogenesis and hypoxia-related genes are slightly elevated reflecting ossification of a vascularized lesion site. Endothelial Notch and VEGF signaling alter vascular growth in calvarial bone repair without affecting the ossification progress. Our findings may have clinical implications for bone regeneration and bioengineering approaches.
Subject(s)
Bone Regeneration , Mesenchymal Stem Cells , Neovascularization, Physiologic , Osteogenesis , Skull , Animals , Bone Regeneration/physiology , Mice , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Vascular Endothelial Growth Factor A/metabolism , Osteoblasts/cytology , Osteoblasts/metabolism , Male , Receptors, Notch/metabolism , Receptors, Notch/genetics , Mice, Inbred C57BL , Signal Transduction , Female , AngiogenesisABSTRACT
Blood vessels are integrated into different organ environments with distinct properties and physiology (Augustin and Koh, 2017). A striking example of organ-specific specialization is the bone vasculature where certain molecular signals yield the opposite effect as in other tissues (Glomski et al., 2011; Kusumbe et al., 2014; Ramasamy et al., 2014). Here, we show that the transcriptional coregulators Yap1 and Taz, components of the Hippo pathway, suppress vascular growth in the hypoxic microenvironment of bone, in contrast to their pro-angiogenic role in other organs. Likewise, the kinase Lats2, which limits Yap1/Taz activity, is essential for bone angiogenesis but dispensable in organs with lower levels of hypoxia. With mouse genetics, RNA sequencing, biochemistry, and cell culture experiments, we show that Yap1/Taz constrain hypoxia-inducible factor 1α (HIF1α) target gene expression in vivo and in vitro. We propose that crosstalk between Yap1/Taz and HIF1α controls angiogenesis depending on the level of tissue hypoxia, resulting in organ-specific biological responses.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/metabolism , Endothelial Cells/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Neovascularization, Physiologic/genetics , Trans-Activators/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Cycle Proteins/genetics , Cell Hypoxia/genetics , Hippo Signaling Pathway , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Male , Mice , Mice, Inbred C57BL , Osteogenesis/genetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Trans-Activators/genetics , YAP-Signaling ProteinsABSTRACT
Parathyroid hormone (PTH) acts on osteoblasts and functions as an essential regulator of calcium homeostasis and as a mediator of bone remodeling. We previously reported that PTH stimulates the expression of matrix metalloproteinase-13 (MMP-13) in rat osteoblasts and that MMP-13 plays a key role in bone remodeling, endochondral bone formation, and bone repair. Recent evidence indicated that microRNAs (miRNAs) have regulatory functions in bone metabolism. In this study, we hypothesized that the down-regulation of miRNAs that target MMP-13 by PTH leads to the stimulation of MMP-13 expression in osteoblasts. We used various bioinformatic tools to identify miRNAs that putatively target rat MMP-13. Among these miRNAs, the expression of miR-532-5p in rat osteoblasts decreased at 4 h of PTH-treatment, whereas MMP-13 mRNA expression was maximal at the same time point. When an miR-532-5p mimic was transiently transfected into UMR-106-01 cells, MMP-13 mRNA and protein expression decreased. Using a luciferase reporter assay system, we also identified that miR-532-5p directly targeted the 3' UTRs of MMP-13 gene. Based on these results, we suggest that PTH-induced down-regulation of miR-532-5p resulted in the stimulation of MMP-13 expression in rat osteoblasts. This study identified a significant role of miRNA in controlling bone remodeling via PTH-stimulated MMP-13 expression. This finding enhances our understanding of bone metabolism and bone-related diseases and it could provide information regarding the usage of miRNAs as therapeutic agents or biomarkers.
