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Theranostics 2021; 11(16):7715-7734. doi:10.7150/thno.58410 This issue Cite

Research Paper

Exosomal lncRNA-H19 promotes osteogenesis and angiogenesis through mediating Angpt1/Tie2-NO signaling in CBS-heterozygous mice

Jyotirmaya Behera¹, Anil Kumar², Michael J. Voor³, Neetu Tyagi^1✉

1. Bone Biology Laboratory, Department of Physiology, School of Medicine, University of Louisville, Louisville, KY 40202, USA.
2. James Graham Brown Cancer Center, Department of Microbiology & Immunology, University of Louisville, KY 40202, USA.
3. Departments of Orthopaedic Surgery and Bioengineering, School of Medicine and Speed School of Engineering, University of Louisville, Louisville, KY 40202, USA.

✉ Corresponding author: Neetu Tyagi, Ph.D., FAPS. Associate Professor, Bone Biology Laboratory, Department of Physiology, Health Sciences Center, A-1201, University of Louisville, Louisville, KY 40202. Phone: 502-852-4145; Fax: 502-852-6239; E-mail: n0tyag01edu. More

Abstract

Rationale: Emerging evidence indicates that the growth of blood vessels and osteogenesis is tightly coordinated during bone development. However, the molecular regulators of intercellular communication in the bone microenvironment are not well studied. Therefore, we aim to investigate whether BMMSC-Exo promotes osteogenesis and angiogenesis via transporting lnc-H19 in the CBS- heterozygous mouse model.

Methods: Using RT2 lncRNA PCR array screening, we identify a bone-specific, long noncoding RNA-H19 (lncRNA-H19/lnc-H19) in exosomes derived from bone marrow mesenchymal stem cells (BMMSC-Exo) during osteogenesis. Using bioinformatics analysis, we further discovered the seed sequence of miR-106a that could bind to lnc-H19. A luciferase reporter assay was performed to demonstrate the direct binding of miR-106a to the target gene angiopoietin 1 (Angpt1). We employed an immunocompromised Nude mouse model, to evaluate the effects of BMMSC-Exo on angiogenesis in vivo. Using a micro-CT scan, we monitored microstructural changes of bone in the experimental mice.

Results: BMMSC-Exo possessed exosomal characteristics including exosome size, and typical markers including CD63, CD9, and TSD101. In vitro, BMMSC-Exo significantly promoted endothelial angiogenesis and osteogenesis. Mechanistic studies have shown that exosomal lnc-H19 acts as “sponges” to absorb miR-106 and regulate the expression of angiogenic factor, Angpt1 that activates lnc-H19/Tie2-NO signaling in mesenchymal and endothelial cells. Both of these effects on osteogenesis and angiogenesis are inhibited by antagonizing Tie2 signaling. Treatment of BMMSC-Exo also restored the bone formation and mechanical quality in vivo.

Conclusion: These findings provide a novel insight into how the extracellular role of exosomal lnc-H19 affects osteogenesis and angiogenesis through competing endogenous RNA networks.

Keywords: lncRNA-H19 regulation, miRNA sponge, Bone formation, Angiogenesis, Extracellular vesicles

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