Purine metabolism enhances neovascularization of type H vessels in the induced membrane technique
Yung-Heng Hsu, Guan-Lin Lee, Yu-Chih Lin, Mei-Feng Chen, Yuhan Chang, Ying-Yu Wu, Chih-Chien Hu
Yung-Heng Hsu, Guan-Lin Lee, Yu-Chih Lin, Mei-Feng Chen, Yuhan Chang, Ying-Yu Wu, Chih-Chien Hu
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Research Article Metabolism Vascular biology

Purine metabolism enhances neovascularization of type H vessels in the induced membrane technique

Abstract

The induced membrane technique (IMT) is a 2-stage surgical intervention for critical-sized bone defects (CSBD), yet the metabolic mechanisms driving neovascularization within the induced membrane remain unclear. Here, we combined a rat IMT model, metabolomic profiling, and endothelial assays to delineate the role of purine metabolism in neovascularization of type H vessels. Using a rat IMT model and metabolomic profiling, we identified purine metabolism as the most substantial pathway during the formation of induced membranes, with consistent trends of adenosine, inosine, hypoxanthine, and xanthosine found in both serum and induced membranes. Histological analysis revealed abundant CD31hiEMCNhi type H vessels, critical for osteogenesis, within the induced membrane. Inhibition of purine metabolism suppressed the CD31hiEMCNhi type H phenotype in human umbilical vein endothelial cells, whereas treatment with inosine, hypoxanthine, or xanthosine promoted endothelial activation and the type H phenotype. Notably, inosine and hypoxanthine displayed parallel changes across consistent systemic (serum) and local alterations (induced membranes), highlighting their potential as serum indicators of induced membrane formation. Collectively, these findings uncover a previously unrecognized metabolic mechanism driving neovascularization of type H vessels in induced membranes and suggest purine metabolites as promising indicators and therapeutic targets for improving IMT outcomes as well as CBSD treatment.

Authors

Yung-Heng Hsu, Guan-Lin Lee, Yu-Chih Lin, Mei-Feng Chen, Yuhan Chang, Ying-Yu Wu, Chih-Chien Hu

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Figure 7

Inhibition of purine metabolism suppresses angiogenic capacity and type H phenotype in HUVECs.

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Inhibition of purine metabolism suppresses angiogenic capacity and type ...
HUVECs were seeded into chamber slides with 5% FBS medium, then serum starved in fresh 0.5% FBS medium for 24 hours and treated with purine metabolism inhibitors: mycophenolic acid (MA, 20 μM), mycophenolate mofetil (MM, 20 μM), and L-alanosine (L-Ala, 20 μM). DMSO was used as solvent control. (A) Immunofluorescence images of HUVECs after 3-day treatment, stained for DAPI (blue), CD31 (green), and EMCN (red). (B and C) In vitro data are based on 3 independent experiments performed in triplicate. Fluorescence intensity was quantified using ImageJ (green, CD31; red, EMCN). (D) Representative images of tube formation after 24-hour treatment. (E–I) Quantification of angiogenesis parameters — including total number of nodes, junctions, segments, meshes, and length — was analyzed by ImageJ. In vitro data are based on 3 independent experiments with triplicate measurements. All data are presented as box plots. Box plots represent the median (line), interquartile range (box), and minimum-to-maximum values (whiskers). P < 0.05 was considered statistically significant. Scale bars: 50 μm (A); 250 μm (D). EMCN, endomucin.