FOXO1-mTOR pathway in vascular pericyte regulates the formation of type H vessels to control bone metabolism

Background

As the population aging progresses, age-related osteoporosis has become one of the most common and severe chronic degenerative diseases. Due to insufficient understanding of its complex pathomechanisms, current clinical treatments often suffer from many negative effects. Type H vessels play critical role in bone remodeling owing to their specialized function in coupling angiogenesis and osteogenesis. Increasing evidences have shown a close association between the age-related decline of type H vessels and bone loss. However, the underlying mechanisms whereby the regression of type H vessels with aging remain largely unknown.

Methods

Col2-Cre^ERT/Foxo1^flox/flox mice and FOXO1 inhibitor (AS1842856) treated adult (6 months) and middle aged (10 months) mice were utilized for evaluating the variations in bone volume, bone microarchitecture and type H vessels through micro-CT scanning analysis, histological staining and immunofluorescence staining. In vitro tube-forming and scratch assays were applied to evaluate the angiogenic capacity of human umbilical vein endothelial cells (HUVECs) exposed to AS1842856 or conditioned culture milieu of Human Brain Vascular Pericytes (HBVPs). The expression of pericyte marker proteins, myofibroblast-related proteins and genes in inhibitors-stimulated HBVPs were detected via western blot analysis and Reverse transcription-quantitative PCR (RT-qPCR). Furthermore, perivascular myofibroblastic-like transformation was confirmed in AS1842856-treated animal models through immunofluorescence staining. We also constructed Adipoq-Cre/Foxo1^flox/flox conditional knockout mice and measured their bone mass and type H vessels by micro-CT and immunofluorescence staining. Mechanistic experiments in vitro were conducted via detection of mTOR signalling expression in HBVPs with pharmacological intervention (AS1842856 and rapamycin), genetic knockdown of Foxo1, or FOXO1-overexpression plasmid treatment, verified by RT-qPCR, western blot analysis and cellular immunofluorescence staining. In vivo validation was conducted on Adipoq-Cre/Foxo1^flox/flox mice using immunofluorescence staining. Finally, alterations in osteo-morphology and type H vessels were verified in AS1842856-treated and rapamycin-treated aged mouse models.

Results

This study identified FOXO1 in pericytes as key components for the formation of type H vessels. We found that FOXO1 expression in pericytes decreases with aging, and pharmacological blocking with AS1842856 promoted type H vessels degeneration and increased bone loss in adult and middle-aged mice, while rapamycin prevented the above pathology in middle-aged mice. We further showed that the loss of FOXO1 in Adipoq⁺ pericytes led to degeneration of type H vessels and bone loss in mice. Mechanistically, the inhibition of FOXO1 by AS1842856 or knockdown of Foxo1 by siRNAs activated mTOR signaling, thereby resulting in the myofibroblastic transformation of pericytes. Furthermore, blocking mTOR signaling by rapamycin rescued the above effects in vitro and in vivo.

Conclusion

Our findings uncover a hitherto unknown role of FOXO1 in maintaining the phenotype and function of pericytes, thereby promoting formation of type H vessels. This suggests that targeting the FOXO1-mTOR pathway in pericytes could be a potential therapeutic approach to overcome the regression of type H vessels and bone degeneration with aging.

The translational potential of this article

Our research uncovers a previously unidentified role of FOXO1 in preserving pericyte characteristics and promoting the development of type H vessels. Future translational research targeting the FOXO1-mTOR pathway in pericytes may provide new strategies for the prevention and treatment of age-related osteoporosis in the clinic.