"Introduction: Neo-angiogenesis describes the development of new blood vessels and takes place during bone healing in the fracture gap. Angiogenesis and the establishment of a functional vascular network is closely associated with bone formation and critical for scar-free healing. Type H vascular endothelial cells (ECs), characterized by high expression of CD31 and endomucin (Emcn) have been identified as key regulators for angiogenesis-osteogenesis coupling. However, the underlying mechanisms that drive revascularization, especially in the early phases from hematoma formation to cartilage development largely remain unknown. Here, we especially want to draw attention to the role of mechanosensors YAP/TAZ in ECs and how they affect angiogenesis and osteogenesis. By modulating YAP and TAZ in ECs we further want to unravel the different phases of angiogenesis during bone healing.
Methodology: We analyzed vessel ingrowth and organization under different fixation stabilities during bone regeneration. Female, 12 weeks aged mice with endothelial YAP/TAZ dKO were sacrificed 7 days or 14 days post-osteotomy, respectively. Soft callus formation, cell organization and ECM deposition within the fracture gap was analyzed by immunohistological protocols and second harmonic imaging (SHI). To characterize H type vessel formation, CD31 and endomucin antibodies were used. Confocal microscopy was employed to analyze the target proteins during the onset of neo-vascularization in bone regeneration. Endochondral ossification was analyzed in areas of bone healing and compared against areas in the growth plate.
Results: We could show that H type vessels (CD31hiEmcnhi) are present in the osteotomy gap, suggesting that they play a key role in de novo angiogenesis during pathological and regenerative processes. Further, we could show that H type vessels are surrounded by osterix expressing pre-osteoblasts, supporting their important role in angiogenesis-osteogenesis coupling during bone healing. Conditionally knocking out YAP/TAZ in ECs leads to an increase in bone vasculature in the hypoxic microenvironment of the osteotomy gap. Further, EC YAP/TAZ dKO induces more vessel crosses, suggesting that vessels fail to stabilize and build a functional basement membrane. Whereas in the pre-cartilage phase complete vascular invasion can be observed, vessels regress in areas of cartilage development in later stages. This suggests that two different mechanisms of angiogenesis exist during bone healing. Angiogenesis via endochondral ossification in the osteotomy gap shows similarities to the bone formation process at the growth plate, however, the latter being more organized.
Conclusion: Next to a downregulation of inflammation, successful bone regeneration requires angiogenesis. We explored here the role of EC mechanosensors YAP/TAZ and their ability to regulate the build-up of a capillary network. We could further show, that revascularization during bone healing occurs via two distinct pathways. Neo-angiogenesis at the pre-cartilage phase follows different mechanisms than angiogenesis via a cartilage template in later stages of bone healing. Understanding the mechanisms of angiogenesis and the effect of EC YAP/TAZ is essential to develop new therapies how to best accelerate bone healing, which would particularly important for the treatment of bone fractures in the elderly.
Neto, F. et al., eLife; 7:e31037 (2018)
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