Role of Angiogenic CXC Chemokines in Intramembranous Bone Repair

描述（由申请人提供）： 颅面骨的创伤性损伤和骨血液供应的中断引发了一系列事件来修复破坏的骨，因为在这种情况下，间充质干细胞（MSC）可以分化成成骨细胞（OB），并且可以重新建立新的血液供应。骨修复的关键步骤发生在早期炎症阶段，其中存在CXC趋化因子的加工。CXC趋化因子在CXC基序上游具有标志性glu-leu-arg（ELR）序列（ELR+ CXC趋化因子），并且也是独特的血管生成性的。这些ELR+ CXC趋化因子与CXC受体1（CXCR 1）和/或CXC受体2（CXCR 2）结合，以实现其趋化和血管生成作用。据推测，ELR+ CXC趋化因子，CXCL 5，在愈合的炎症阶段，阐述了由非经典Wnt信号的成分，令人惊讶的是，分泌卷曲相关蛋白-1（sFRP-1）。CXCL 5然后通过以下方式在膜内骨修复中起关键作用：1）启动血管生成以在成骨愈合的炎性阶段建立肉芽组织形成的基础结构，以及2）刺激MSC的化学吸引，导致MSC凝聚，从而导致MSC的成骨分化。具体目标是：1.明确CXCL 5通过非经典Wnt信号和sFRP-1上调的机制; 2.探讨CXCR 2在体外MSC迁移和成骨分化中的作用; 3.确定CXCR 2水平的操纵或体内sFRP给药的短期过程将增强小鼠颅骨缺损模型中的血管生成和骨修复。分别使用重组Wnt 5a和来自表达Wnt 5a的L细胞的条件培养基，通过真实的时间RT-PCR和ELISA测试Wnt 5a对CXCL 5 mRNA和蛋白质的刺激。实验将在已被转导以表达人端粒酶逆转录酶（TERT）以增强其体外寿命的人MSC（hMSC）中进行。将通过FACS评估可结合Wnt 5a和RoR 2（非典型Wnt共受体）的潜在卷曲（Fzl）受体。除sFRP-1外，还将检查其他sFRP亚型，以确定是否也可诱导CXCL 5，并将进行正式的剂量反应和时间过程。将使用荧光素酶报告基因测定、适当的siRNA和小分子抑制剂以及评估这些信号分子的活化磷酸化状态，探索可能负责CXCL 5表达的下游信号传导机制，如NF-B或促分裂原活化蛋白激酶（MAPK）途径。将测试由Wnt 5a或sFRP刺激的CXCL 5表达的功能性，以观察血管生成是否可以在内皮管形成测定中刺激。接下来，将检查CXCR 2的作用，以观察CXCL 5和CXCL 8（其产生由经典Wnt信号传导刺激）是否可以刺激已经用各种CXCR 2构建体（野生型、组成型活性和非活性）转导的hMSC TERT细胞的趋化性和成骨分化。将通过Transwell试验评估趋化性，并通过各种成骨标志物的mRNA表达确定成骨作用。除了使用CXCR 2的小分子抑制剂外，还将如上评估涉及信号转导和转录激活因子-3（STAT-3）、磷酸肌醇3-激酶（PI 3 K）和MAPK的下游CXCR 2信号传导。平行的趋化性和成骨分化研究将在源自具有完整和整体敲除的小鼠CXC受体（mCXCR，CXCR 2的同源物）的小鼠的小鼠MSC中进行。最后，使用hMSC TERT CXCR 2构建体进行体内研究，以观察野生型和mCXCR敲除小鼠中的颅骨缺损愈合是否得到改善，以及短期sFRP-1施用沿着施用hMSC TERT CXCR 2是否会增加野生型小鼠中的血管生成和随后的颅骨缺损愈合。了解Wnt信号传导如何调节ELR+ CXC趋化因子，这是膜内骨愈合初始阶段血管生成的基础，应该会导致新的干预措施，将受损骨恢复到发病前的强度和健康水平。

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-¿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.