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-?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.

Public Health Relevance

Head, jaw, and face trauma accounts for 26% of combat injuries in OEF/OIF. In those traumatic bone injuries that either heal slowly or do not heal, or in the case of facial and skull bone injures with attendant traumatic brain injury, increased morbidity, long-term rehabilitation, and even long-term disabilities are faced by such Veterans that take a toll on the Veterans themselves, their families, and the financial resources of the country. It is important to understand the early events in craniofacial bone repair to effect optimal results. These events include the generation of new bone forming cells called osteoblasts from more primitive stem cells, and the restoration of new blood supply to bone. Re-establishing blood supply to bone by a process called angiogenesis (the sprouting of new blood vessels from existing blood vessels after injury) is necessary and critical for bone repair to occur. Knowledge of these key elements in bone repair may help to develop and deliver therapies that would hasten bone healing to restore bone to pre-morbid levels of strength and soundness.

National Institute of Health (NIH)
Veterans Affairs (VA)
Non-HHS Research Projects (I01)
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Endocrinology B (ENDB)
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VA Greater Los Angels Healthcare System
Los Angeles
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