Chronic Alcohol Reduces Bone Mass Through Inhibiting Proliferation and Promoting Aging of Endothelial Cells in Type-H Vessels.

Alcohol consumption is regarded as one of the leading risk factors for secondary osteopenia. Angiogenesis and osteogenesis coupled by type-H vessels coordinate the biological process of bone homeostasis to prevent osteopenia. This study aimed to determine whether chronic alcohol inhibits type-H vessel-dependent bone formation. Two-month-old mice were fed with 5% (v/v) alcohol liquid diet (28% of calories) or normal liquid diet every day for 2 months. The tibias were isolated and detected with X-ray and microcomputed tomography. Paraffin-embedded or frozen tibial sections were prepared and used for immunohistochemical or immunofluorescence staining, respectively. Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of alcohol, including 0 mM (0%), 8.7 mM (0.5%), 52 mM (3%), or 87 mM (5%) alcohol for 12 h. The conditioned medium of the above HUVEC cells was collected to culture human bone marrow-mesenchymal stem cells (BM-MSCs), which were induced to differentiate into osteoblasts in vitro. The alcoholic diet retarded the bone growth and led to osteoporosis, impaired bone formation of osteoblasts, and decreased CD31 hi EMCN hi type-H vessel formation through inhibiting proliferation and promoting aging of endothelial cells in mice. Alcohol treatment obviously increased the expression of p16, while significantly decreased the expression of Bmi-1, CDK6, Cyclin D, E2F1, and bone morphogenetic protein (BMP)2 compared with vehicle. Alcohol inhibited the differentiation of BM-MSCs into osteoblasts through reducing the BMP2 secretion of endothelial cells in type-H vessels. Alcoholic diet impaired CD31 hi EMCN hi type-H vessel formation through inhibiting proliferation and promoting aging of endothelial cells through Bmi-1/p16 signaling, and inhibited the differentiation of BM-MSCs into osteoblasts through reducing the BMP2 secretion of endothelial cells in type-H vessels. This study provides a basis for developing a new treatment strategy targeting aging endothelial cells of type-H vessel to prevent alcoholic osteopenia.