A small interfering RNA targeting Lnk accelerates bone fracture healing with early neovascularization

Lab Invest. 2013 Sep;93(9):1036-53. doi: 10.1038/labinvest.2013.93. Epub 2013 Jul 29.

Abstract

Lnk, an intracellular adapter protein, is expressed in hematopoietic cell lineages, which has recently been proved as an essential inhibitory signaling molecule for stem cell self-renewal in the stem cell factor-c-Kit signaling pathway with enhanced hematopoietic and osteogenic reconstitution in Lnk-deficient mice. Moreover, the therapeutic potential of hematopoietic stem/endothelial progenitor cells (EPCs) for fracture healing has been demonstrated with mechanistic insight into vasculogenesis/angiogenesis and osteogenesis enhancement in the fracture sites. We report here, Lnk siRNA-transfected endothelial commitment of c-kit+/Sca-1+/lineage- subpopulations of bone marrow cells have high EPC colony-forming capacity exhibiting endothelial markers, VE-Cad, VEGF and Ang-1. Lnk siRNA-transfected osteoblasts also show highly osteoblastic capacity. In vivo, locally transfected Lnk siRNA could successfully downregulate the expression of Lnk at the fracture site up to 1 week, and radiological and histological examination showed extremely accelerated fracture healing in Lnk siRNA-transfected mice. Moreover, Lnk siRNA-transfected mice exhibited sufficient therapeutic outcomes with intrinstic enhancement of angiogenesis and osteogenesis, specifically, the mice demonstrated better blood flow recovery in the sites of fracture. In our series of experiments, we clarified that a negatively regulated Lnk system contributed to a favorable circumstance for fracture healing by enhancing vasculogenesis/angiogenesis and osteogenesis. These findings suggest that downregulation of Lnk system may have the clinical potential for faster fracture healing, which contributes to the reduction of delayed unions or non-unions.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Animals
  • Bone Marrow Cells / metabolism
  • Cell Proliferation
  • Chi-Square Distribution
  • Fractures, Bone / metabolism*
  • Hematopoietic Stem Cells / cytology
  • Hematopoietic Stem Cells / metabolism
  • Histocytochemistry
  • Intracellular Signaling Peptides and Proteins / analysis
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Laser-Doppler Flowmetry
  • Male
  • Membrane Proteins
  • Mice
  • Mice, Inbred C57BL
  • Neovascularization, Physiologic / genetics
  • Neovascularization, Physiologic / physiology*
  • Osteoblasts / cytology
  • Osteoblasts / metabolism
  • Osteogenesis / genetics
  • Osteogenesis / physiology
  • Phenotype
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism*
  • Regional Blood Flow
  • Statistics, Nonparametric
  • Transfection
  • Wound Healing / genetics
  • Wound Healing / physiology*
  • X-Ray Microtomography

Substances

  • Adaptor Proteins, Signal Transducing
  • Intracellular Signaling Peptides and Proteins
  • Lnk protein, mouse
  • Membrane Proteins
  • RNA, Small Interfering