Inosine from purine metabolism enhances fracture healing by coupling fibrinolysis and angiogenesis of type H vessels

Fibrinolysis-angiogenesis coupling is crucial for successful fracture healing, in which type H vessels play an indispensable role. However, the metabolic control of fibrinolysis-type H angiogenesis coupling in fracture healing remains unclear. We used a metabolomics approach to gain metabolic insights from mouse fracture models (0.3 mm and 1.0 mm femur defects). Furthermore, human umbilical vein endothelial cells (HUVECs) and MC3T3-E1 cells were employed as in vitro models to examine the effects of identified metabolites on endothelial events and osteogenesis, respectively. CD31 and endomucin (Emcn) were used for detecting type H markers, and tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) for fibrinolysis. A femur defect of 1.5 mm in mice was included to validate the therapeutic potential of identified metabolites by bone imaging and micro-CT. Purine metabolism is the most significant pathway in fracture healing; three purinergic metabolites, adenosine, adenine, and inosine, are regulated in mouse serum. According to the in vivo results, CD31^HiEmcn^Hi type H vessels are upregulated near the defect site in mouse femur and are associated with tPA expression, but not PAI-1. Additionally, in vitro experiments examining the endothelial functions of HUVECs demonstrated that these three metabolites promote cell migration and tube formation rather than proliferation. Fibrinolytic activity and type H phenotype in HUVECs were induced only by inosine through activation of the adenosine A2A receptor. Inosine, regulated during fracture healing, has the capacity to synchronously induce fibrinolysis and type H phenotype in line with osteogenesis, indicating its role in enhancing fracture healing.