Role of Angiogenic CXC Chemokines in Intramembranous Bone Repair

描述（由申请人提供）： 骨的创伤性破坏和对骨的血液供应的中断引发一系列事件，最终导致骨的完全修复，因为间充质干细胞（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。在CXC基序上游具有标志性glu-leu-arg（ELR）序列的CXC趋化因子（ELR+ CXC趋化因子）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。假设在炎性阶段期间加工的ELR+ CXC趋化因子CXCL 5和CXCL 8通过以下方式在膜内骨修复中起关键作用：1）刺激MSC的趋化性以在修复位点获得临界质量的MSC，2）在骨修复期间帮助MSC分化成OB，和3）在成骨愈合的炎症阶段启动血管生成以支持MSC凝聚和“肉芽组织”。有证据表明，经典的Wnt/2-catenin信号是新骨形成所必需的。因此，我们进一步假设经典Wnt/2-连环蛋白信号传导机制参与CXCL 5和CXCL 8的加工，随后旁分泌刺激血管生成，并且CXCL 5和CXCL 8还可以通过上调蛋白激酶D（PKD）和激活促分裂原活化蛋白激酶（MAPK）信号传导途径促进MSC迁移和成骨分化，以实现骨修复。具体目标是：1）证明在成骨分化起始期间CXCL 5和CXCL 8的加工通过Wnt/2-连环蛋白信号传导途径发生; 2）证明CXCL 5和CXCL 8通过CXCR 1和/或CXCR 2导致人和小鼠MSC的迁移和成骨分化（hMSC; mMSC）在体外模型中，导致迁移和成骨分化的细胞信号传导通过PKD和MAPK的下游活化发生路径; 3）确定是否由于缺乏小鼠CXC受体（mCXCR）（与人CXCR 2同源）而在迁移和分化中存在mMSC（来自骨髓）或OB特异性缺陷，和/或在mCXCR缺失小鼠中发现的骨表型和较少的骨愈合是否是由于ELR+ CXC趋化因子刺激的血管生成不足。将分别通过实时RT-PCR和ELISA分析ELR+ CXC和成骨标志物mRNA和蛋白。MSC迁移将通过Transwell测定进行。CXC受体和下游PKD和MAPK信号传导的过表达或抑制将分别使用转染的表达载体或siRNA技术进行。体内研究将使用组织形态学分析来评估mCXCR敲除小鼠颅骨缺损模型中的骨愈合和新血管形成。了解骨修复的早期事件可能有助于开发和提供加速骨愈合的治疗方法，以将骨恢复到疾病前的强度和健康水平。 公共卫生相关性： 与退伍军人健康的相关性目前的研究建议与在现役战区经历过创伤性损伤的退伍军人的护理有关。在持久自由行动（OEF）和伊拉克自由行动（OIF）中观察到的战斗损伤类型的特征表明，创伤性骨损伤是一个普遍的问题。事实上，在持久自由行动（OEF）和伊拉克自由行动（OIF）中观察到的战斗伤害类型表明，所有肢体创伤中有26%是骨折（Owens BD，Kragh，Jr JF，Macaitis J，Svoboda SJ，Wenke JC。伊拉克自由行动和持久自由行动中四肢伤口的特征。J Orthop Trauma 21：254-257，2007）。头部和面部创伤占战斗伤害的2% - 20%，这取决于中东冲突的战区。因此，创伤性骨损伤被发现与积极的军事活动。在那些创伤性骨损伤，要么愈合缓慢或不愈合，或在面部和颅骨损伤的情况下，也可能有伴随的视觉或听觉缺陷或创伤性脑损伤，发病率增加，长期康复，甚至长期残疾，这些退伍军人面临着对退伍军人本身，他们的家庭和国家的财政资源造成损失。重要的是要了解骨修复的早期事件。这些事件包括从来自骨髓的更原始的细胞（例如间充质干细胞）产生称为成骨细胞的新骨形成细胞，以及恢复新的血液供应到骨。通过称为血管生成（损伤后从现有血管中萌发新血管）的过程重建骨骼的血液供应对于骨修复的发生是必要的和关键的。了解骨修复中的这些关键因素可能有助于开发和提供加速骨愈合的疗法，以将骨恢复到疾病前的强度和健康水平。

DESCRIPTION (provided by applicant): Traumatic disruption of bone and the interruption of blood supply to bone initiate a cascade of events eventually leading to full repair of bone given the circumstances that mesenchymal stem cells (MSCs) can differentiate into osteoblasts (OBs) and that new blood supply can be re-established. A key step of bone repair occurs in the early inflammatory phase where there is an elaboration of CXC chemokines. CXC chemokines having a signature glu-leu-arg (ELR) sequence upstream to the CXC motif (ELR+ CXC chemokines) 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 chemokines, CXCL5 and CXCL8, elaborated during the inflammatory phase, play a key role in intramembranous bone repair by 1) stimulating chemotaxis of MSCs to achieve a critical mass of MSCs at the repair site, 2) aiding in the differentiation of MSCs into OBs during bone repair, and 3) initiating angiogenesis to support MSC condensation and "granulation tissue" in the inflammatory phase of osteogenic healing. There is evidence that canonical Wnt/2-catenin signaling is required for new bone formation. Thus we further hypothesize that canonical Wnt/2-catenin signaling is mechanistically involved in the elaboration of CXCL5 and CXCL8 with subsequent paracrine stimulation of angiogenesis, and that CXCL5 and CXCL8 can also foster MSC migration and osteogenic differentiation via the upregulation of protein kinase D (PKD) and activation of the mitogen-activated protein kinase (MAPK) signaling pathways to enable bone repair. The specific aims are to: 1) demonstrate that the elaboration of CXCL5 and CXCL8 during the initiation of osteogenic differentiation occurs through the Wnt/2-catenin signaling pathway; 2) show that CXCL5 and CXCL8 through CXCR1 and/or CXCR2 leads to migration and osteogenic differentiation of human and mouse MSCs (hMSC; mMSC) models in vitro and that cellular signaling leading to migration and osteogenic differentiation occurs through PKD and downstream activation of the MAPK pathways; 3) establish if there is a mMSC (from bone marrow) or OB- specific defect in migration and differentiation due to the lack of the mouse CXC receptor (mCXCR) (homologous to human CXCR2), and/or if the bone phenotype and less bone healing found in mCXCR null mice is due to insufficient ELR+ CXC chemokine-stimulated angiogenesis. ELR+ CXC and osteogenic markers mRNA and protein will be analyzed by real-time RT-PCR and ELISA, respectively. MSC migration will be done by Transwell assay. Overexpression or inhibition of CXC receptors and downstream PKD and MAPK signaling will be done using transfected expression vectors or siRNA technology, respectively. In vivo studies will use histomorphometric analyses to assess bone healing and new blood vessel formation in a calvarial defect model in mCXCR knockout mice. Understanding the early events 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. PUBLIC HEALTH RELEVANCE: RELEVANCE TO VETERANS HEALTH The current research proposal is every relevant to the care of veterans who have experienced traumatic injury in active combat theaters. Characterization of the types of combat injuries observed in Operation Enduring Freedom (OEF) and Operation Iraqi Freedom (OIF) has shown that traumatic bone injury is a prevalent problem. In fact the types of combat injuries observed in Operation Enduring Freedom (OEF) and Operation Iraqi Freedom (OIF) has shown that 26% of all extremity wounds are fractures (Owens BD, Kragh, Jr JF, Macaitis J, Svoboda SJ, Wenke JC. Characterization of extremity wounds in Operation Iraqi Freedom and Operation Enduring Freedom. J Orthop Trauma 21:254-257, 2007). Head and face trauma accounts for 2% - 20% of combat injuries depending on the theater of Middle East conflict. Thus traumatic bone injury is found to be associated with active military campaigns. In those traumatic bone injuries that either heal slowly or do not heal, or in the case of facial and skull bone injuries that could also have attendant visual or hearing deficits or 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 bone repair. These events include the generation of new bone forming cells called osteoblasts from more primitive cells from bone marrow (e.g. mesenchymal 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.