Role of Angiogenic CXC Chemokines in Intramembranous Bone Repair

描述(由申请人提供)： 在骨髓间充质干细胞(MSCs)可以分化为成骨细胞(OB)并且可以重新建立新的血液供应的情况下，创伤性骨破坏和骨血供中断会引发一系列事件，最终导致骨的完全修复。骨修复的一个关键步骤发生在炎症的早期阶段，在这个阶段有CXC趋化因子的详细阐述。在CXC基序上游有一个标志性的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。这些ELR+CXC趋化因子与CXC受体1(CXCR1)和/或CXC受体2(CXCR2)结合，以实现其趋化和血管生成作用。据推测，在炎症阶段形成的ELR+CXC趋化因子CXCL5和CXCL8在膜内骨修复中发挥关键作用：1)刺激MSCs的趋化，使修复部位的MSCs达到临界质量；2)在骨修复过程中帮助MSCs分化为OB；3)在成骨愈合的炎症阶段启动血管生成，支持MSC凝聚和肉芽组织。有证据表明，规范的Wnt/2-catenin信号是新骨形成所必需的。因此，我们进一步假设，规范的Wnt/2-catenin信号机制参与了CXCL5和CXCL8的形成以及随后的旁分泌刺激血管生成，并且CXCL5和CXCL8还可以通过上调蛋白激酶D(PKD)和激活丝裂原活化蛋白激酶(MAPK)信号通路来促进MSC迁移和成骨分化，从而使骨修复成为可能。其具体目的是：1)证明CXCL5和CXCL8在成骨分化起始过程中的表达是通过Wnt/2-catenin信号通路发生的；2)证明CXCL5和CXCL8通过CXCR1和/或CXCR2在体外诱导人和小鼠MSCs(hMSC；MMSC)迁移和成骨分化，以及细胞信号导致迁移和成骨分化是通过PKD和MAPK通路下游激活而发生的；3)确定是否由于缺乏小鼠CXC受体(MCXCR)(与人CXCR2同源)，在迁移和分化过程中存在MMSC或OB特异性缺陷，和/或mCXCR缺失小鼠的骨表型和骨愈合较差是否由于ELR+CXC趋化因子刺激的血管生成不足所致。ELR+CXC和成骨标记物的mRNA和蛋白将分别用实时定量RT-PCR和ELISA法进行分析。MSC的迁移将通过Transwell实验进行。CXC受体的过度表达或抑制以及下游的PKD和MAPK信号转导将分别使用转基因表达载体或siRNA技术完成。体内研究将使用组织形态计量学分析来评估mCXCR基因敲除小鼠颅骨缺损模型中的骨愈合和新血管形成。了解骨骼修复的早期事件可能有助于开发和提供加速骨骼愈合的治疗方法，以使骨骼恢复到患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提案与在现役作战战区经历过创伤的退伍军人的护理完全相关。对持久自由行动(OEF)和伊拉克自由行动(OIF)中观察到的战斗伤害类型的描述表明，创伤性骨损伤是一个普遍存在的问题。事实上，在持久自由行动(OEF)和伊拉克自由行动(OIF)中观察到的战伤类型表明，所有四肢创伤中有26%是骨折(Owens BD、Kragh、JRJF、Macaitis J、Svoboda SJ、Wenke JC)。《伊拉克自由行动》和《持久自由行动》中四肢创伤的特征。创伤骨科杂志21：254-257,2007)。根据中东冲突战区的不同，头部和面部创伤占战斗伤害的2%-20%。因此，创伤性骨损伤被发现与积极的军事行动有关。在那些愈合缓慢或不愈合的创伤性骨骼损伤中，或者在面部和颅骨损伤的情况下，还可能伴随着视力或听力障碍或创伤性脑损伤，这些退伍军人面临着发病率增加、长期康复甚至长期残疾的问题，这对退伍军人本身、他们的家庭和国家的财政资源造成了损害。了解骨修复的早期事件是很重要的。这些事件包括从更原始的骨髓细胞(例如，间充质干细胞)生成称为成骨细胞的新的骨形成细胞，以及恢复对骨的新的血液供应。通过一种称为血管生成(损伤后从现有血管长出新血管)的过程重建骨骼的血液供应是骨修复所必需的，也是至关重要的。了解骨骼修复中的这些关键因素可能有助于开发和提供加速骨骼愈合的治疗方法，以使骨骼恢复到患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.