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

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 siRNAtransfected endothelial commitment of c-kit þ /Sca-1 þ /lineage À 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.

Clinical Impact of Circulating CD34-Positive Cells on Bone Regeneration and Healing

“…37 In the second series of studies, we clarified that downregulation of the Lnk system through transfection of a Lnk siRNA in mice contributed to favorable conditions for fracture healing by enhancing vasculogenesis/angiogenesis and osteogenesis. 38 In this study, we also demonstrated that Lnk was also expressed in KSL cells and osteoblasts, and that Lnk siRNAtransfected KSL cells and osteoblasts showed increased proliferation activity and high vasculogenic and osteogenic activity. These findings suggest that inhibition of Lnk may have therapeutic potential by promoting an environment conducive to vasculogenesis/angiogenesis and osteogenesis and by facilitating terminal differentiation of osteoblasts leading to enhanced fracture healing.…”

“…27 Therefore, we conducted several studies to prove the efficacy of circulating CD34-positive cells, which have potential for osteogenesis [28][29][30] and angiogenesis/ vasculogenesis, for bone fracture healing. [31][32][33][34][35][36][37][38] In this review, the current concepts and strategies in circulating CD34 + cellbased therapy and its potential applications for bone repair will be highlighted.…”

Small Players Ruling the Hard Game: siRNA in Bone Regeneration

“…Atelocollagen is a decomposition product of type I collagen that is positively charged and thus can complex with siRNA . Kawakami and colleagues used Atelocollagen to complex the siRNA against Lnk (an inhibitory signaling molecule for stem cell renewal) in order to accelerate fracture healing. Primary mouse bone marrow cells and osteoblasts were transfected with siRNA against Lnk using lipofection.…”

The Use of Endothelial Progenitor Cells for the Regeneration of Musculoskeletal and Neural Tissues

“…The bone fracture model of Lnk-deficient mice shows accelerated angiogenesis, fracture healing, and remodeling through the enhancement of mobilization and the recruitment of bone marrow EPCs [37]. In addition, Lnk siRNA transfection enhances the function of EPCs for vascularization and improves fracture healing [38]. Therefore, the inhibition of Lnk may have therapeutic potential to enhance fracture healing.…”