Role of Angiogenic CXC Chemokines in Intramembranous Bone Repair


Principal Investigator: Dean T Yamaguchi
Abstract: DESCRIPTION (provided by applicant): Traumatic injury of craniofacial bone and the interruption of bone blood supply initiate a cascade of events to repair disrupted bone given the circumstances that mesenchymal stem cells (MSCs) can differentiate into osteoblasts (OBs) and that new blood supply can be re-established. A critical step of bone repair occurs in the early inflammatory phase where there is an elaboration of CXC chemokines. CXC chemokines have a signature glu-leu-arg (ELR) sequence upstream to the CXC motif (ELR+ CXC chemokines) and are also uniquely angiogenic. These ELR+ CXC chemokines bind to either CXC receptor 1 (CXCR1) and/or CXC receptor 2 (CXCR2), to enable their chemotactic and angiogenic effects. It is hypothesized that the ELR+ CXC chemokine, CXCL5, elaborated during the inflammatory phase of healing is regulated by components of non-canonical Wnt signaling and surprisingly, secreted frizzled related protein-1 (sFRP-1). CXCL5 then plays a key role in intramembranous bone repair by 1) initiating angiogenesis to establish an infrastructure for granulation tissue formation in the inflammatory phase of osteogenic healing and by 2) stimulating the chemoattraction of MSCs, resulting in MSC condensation that leads to osteogenic differentiation of MSCs. The specific aims are: 1. Define the mechanism of the up-regulation of CXCL5 through non-canonical Wnt signaling and by sFRP-1;2. Explore the role of the CXCR2 in MSC migration and osteogenic differentiation in vitro;3. Establish that manipulation of CXCR2 levels or a short course of sFRP administration in vivo will enhance angiogenesis and bone repair in a mouse cranial defect model. Wnt5a stimulation of CXCL5 mRNA and protein will be tested by real time RT- PCR and ELISA, respectively, using both recombinant Wnt5a and conditioned medium from L-cells expressing Wnt5a. Experiments will be done in human MSCs (hMSCs) that have been transduced to express human telomerase reverse transcriptase (TERT) to enhance their longevity in vitro. Potential frizzled (Fzl) receptors that can bind Wnt5a and RoR2, a non-canonical Wnt co-receptor, will be assessed by FACS. In addition to sFRP-1, the other sFRP isoforms will be examined to see if CXCL5 can also be induced, and formal dose responses and time courses will be done. Downstream signaling mechanisms that may be responsible for CXCL5 expression such as NF-[unreadable]B, or the mitogen-activated protein kinase (MAPK) pathways will be explored using luciferase reporter assays, appropriate siRNAs and small molecule inhibitors, and assessment of activated phosphorylation states of these signaling molecules. The functionality of CXCL5 expression stimulated by Wnt5a or sFRPs will be tested to see if angiogenesis can be stimulated in an endothelial tube formation assay. Next, the role of the CXCR2 will be examined to see if CXCL5 and CXCL8, whose production is stimulated by canonical Wnt signaling, can stimulate chemotaxis and osteogenic differentiation of hMSC TERT cells that have been transduced with various CXCR2 constructs (wild type, constitutively active, and inactive). Chemotaxis will be assessed by Transwell assay and osteogenesis determined by mRNA expression of various osteogenic markers. Downstream CXCR2 signaling involving signal transducer and activator of transcription-3 (STAT-3), phosphoinositide 3-kinase (PI3K), and MAPK will be assessed as above in addition to utilizing small molecule inhibitors of CXCR2. Parallel chemotaxis and osteogenic differentiation studies will be done in mouse MSCs derived from mice with intact and globally knocked out mouse CXC receptor (mCXCR, a homolog of CXCR2). Finally, in vivo studies will be done using the hMSC TERT CXCR2 constructs to see if calvarial defect healing is improved in both wild type and mCXCR knockout mice and if short term sFRP-1 administration along with administering hMSC TERT CXCR2 will increase angiogenesis and subsequent calvarial defect healing in wild type mice. Understanding how Wnt signaling can regulate ELR+ CXC chemokines, a foundation for angiogenesis during the initial phase of intramembranous bone healing, should lead to novel interventions to restore damaged bone to pre-morbid levels of strength and soundness.
Funding Period: 2009-04-01 - 2017-03-31
more information: NIH RePORT

Detail Information

Research Grants30

  1. BBP (Bone Morphogenetic Protein Binding Peptide) and Bone Healing
    SAMUEL SCOTT MURRAY; Fiscal Year: 2013
    ..We will also determine how the native bone protein spp24 (secreted phosphoprotein-24 kDa) inhibits BMP/TGF-b-dependent tumor cell growth. ..
    John V Fahy; Fiscal Year: 2013
    ..Including studies in human biospecimens in a PPG that promises to advance understanding of airway TH2 inflammation in ways that are highly relevant to patients with asthma. ..
  3. Role of Angiogenesis in Distraction Osteogenesis
    ..The completion of Aims 1 and 2 will determine if ECs are a primary cell source of BMP2 in regulating skeletal healing after surgery or injury and if BMP2 has a functional role in vasculogenesis. ..
  4. Economics of Health, Wealth, and Well-Being
    DAVID A WISE; Fiscal Year: 2013
    ..Subproject 8 explores ways of Improving Health and Health Care for Minority and Aging Populations. ..
  5. Alcohol Effects on SDF1-Mediated Stem Cell Homing Following Bone Fracture Injury
    John J Callaci; Fiscal Year: 2013
  6. Wnt and PPARy Signaling in Nell-1 and BMP2 Mediated Bone Regeneration
    Chia Soo; Fiscal Year: 2013
    ..Moreover, increased induction of osteogenesis and suppression of adipogenesis among mesenchymal stem cells will lead to marked improvements in clinical bone repair. ..
  7. Functional roles of muscle in bone repair
    Theodore Miclau; Fiscal Year: 2013
  8. Regional differences in neural crest and mesodermal derived calvarial bone healin
    Michael T Longaker; Fiscal Year: 2013
    ..Identification of skull bones with superior healing potential provides insight into novel strategies that can be employed to regenerate, rather than reconstruct, skull bones. ..
  9. Inhibited Intramembranous Bone Healing in Diabetes
    Philip C Trackman; Fiscal Year: 2013
  10. PTHrP/IHH Microenvironment Control for Growth Plate Tissue Engineering
    JUAN MANUEL TABOAS; Fiscal Year: 2013
    ..We will implant these constructs in dorsal subcutaneous pockets of immunodeficient mice and use histological and micro-CT assays to evaluate construct growth and GP-like structure maintenance. ..
    Paul Greengard; Fiscal Year: 2013
    ..Results from the three Projects will complement each other. In addition, there will be a significant level of collaboration between the three Projects, as well as close interaction of the three Projects with the Scientific Core. ..
  12. Signaling in Inflammation, Stress, and Tumorigenesis
    GEORGE ROBERT STARK; Fiscal Year: 2013
    ..abstract_text> ..
  13. Translational Optimization of Bone Regeneration in the Irradiated Mandible
    Steven R Buchman; Fiscal Year: 2013
    ..The current proposal entails developing those synergies and innovative solutions in order to translate our findings from the bench to the operative suite to improve the treatment for this severely compromised patient population. ..
  14. Bone Tissue Engineering Using Mineralized Collagen-GAG Scaffolds
    Timothy A Miller; Fiscal Year: 2013
    ..We anticipate that changes in integrin signaling are responsible for enhanced differentiation and reduced contraction of rBMSCs in MC-GAG scaffolds. ..
  15. Rapid mobilization of endogenous progenitor cells for bone healing
    CLARE E YELLOWLEY-GENETOS; Fiscal Year: 2013
    ..It is expected that efficient mobilization of stem cells shortly after injury, could circumvent the need for bone marrow aspiration, and complications associated with in vitro cell manipulation. ..
  16. Osteocyte Regulation of Bone/Muscle with Age
    Lynda F Bonewald; Fiscal Year: 2013
    ..The results of these experiments should lead to novel therapeutics for the prevention and treatment of both osteoporosis and sarcopenia. ..
  17. PTH Effects of Craniofacial Allografts
    Edward M Schwarz; Fiscal Year: 2013
    ..abstract_text> ..
  18. Glycan Modulation of Inflammatory Responses
    Ajit P Varki; Fiscal Year: 2013
    ..abstract_text> ..
  19. The Role of BMP2 in the Regenerative Effects of MSC in Fracture Repair
    TIMOTHY JOSEPH MYERS; Fiscal Year: 2013