Role of Angiogenesis in Distraction Osteogenesis

描述(由申请人提供)：骨骼损伤的愈合不良与发病率增加和功能状态下降有关，每年有超过800,000例骨折愈合受损的外科矫形治疗。在所有类型的骨科治疗中，血液供应不足是影响愈合的主要原因。骨形态发生蛋白2(BMP2)在促进间充质干细胞(MSC)成骨分化中起重要作用，而血管内皮生长因子(VEGFs)在血管内皮细胞(ECs)的募集和分化中起关键作用。目前，人们普遍认为BMP2是由骨髓间充质干细胞和成骨细胞产生的，或者是从细胞外基质中的隔离存储中蛋白分解释放出来的。我们对转基因小鼠的初步研究表明，在骨折和截骨部位以及骨修复组织内排列血管的内皮细胞是修复部位BMP2的主要来源。血管内皮生长因子也被证明在骨愈合中发挥作用，然而，它们是否通过成骨细胞中的受体介导的信号直接发挥作用仍存在很大争议。本实验室的其他前期工作表明，虽然分化的成骨细胞大量表达血管内皮生长因子(S)，但外源性添加的血管内皮生长因子(S)既不能促进MSC的成骨分化，也不能增强分化的成骨细胞功能。综上所述，我们得出以下假设：生后骨修复和再生过程中血管和骨组织的发育是通过一个正的旁分泌反馈环来调节的，该反馈环是通过内皮细胞表达BMP2和成骨细胞表达血管内皮生长因子而建立的。这一假说是对以前关于骨修复过程中BMP2和VEGF的细胞来源和功能的普遍看法的范式转变。一个牵张成骨(DO)的小鼠模型将被用来解决这一假说。对于拟议的研究，DO有三个优点：a)新骨形成主要通过膜内突起发生，缺乏广泛的软骨；b)它是一个非常健壮的再生模型，能够诱导大量高度血管化的骨组织；c)它成功地用于临床，在没有BMP2治疗的情况下治疗某些形式的骨不连。目的1通过阻断VEGFR 1和2的活性改变血管形态发生，明确血管形态发生与BMP2在内皮细胞中的诱导作用之间的功能关系。使用含有有条件诱导的Cre基因的转基因小鼠，这种基因在内皮细胞中选择性地表达，新生内皮细胞的发育将在新的血管内跟踪，并共同定位于表达BMP2的细胞群。目的2利用相同的转基因小鼠，研究内皮细胞BMP2表达在DO不同阶段内皮细胞BMP2基因去除在骨和血管组织形成中的功能作用。AIMS 1和2的完成将确定内皮细胞是否是BMP2的主要细胞来源，以调节手术或损伤后的骨骼愈合，以及BMP2是否在血管生成中具有功能作用。

DESCRIPTION (provided by applicant): Compromised healing of skeletal injuries is associated with increased morbidity and decreased functional status, and each year surgical orthopedic treatments are performed on over 800,000 fractures with impaired healing. Inadequate blood supply is a major cause of impaired healing in all types of orthopedic treatments. Bone morphogenetic protein 2 (BMP2) plays an essential role in promoting mesenchymal stem cell (MSC) osteogenic differentiation; while vascular endothelia growth factors (VEGFs) play crucial roles in the recruitment and differentiation of vascular endothelia cells (ECs). Currently, it is commonly accepted that BMP2 is either produced by MSCs and osteogenic cells or is proteolytically released from sequestered stores in the extracellular matrix. Our preliminary studies with transgenic mice have shown that ECs that line vessels adjacent to both fracture and osteotomy sites, and within bone repair tissues, are the main source of BMP2 at the repair site. VEGFs have also been shown to function in bone healing however, whether they act in a direct manner via receptor-mediated signaling in osteogenic cells is highly debated. Other preliminary work from our laboratory has shown that while differentiated osteoblasts abundantly express VEGF(s), exogenously added VEGF(s) neither promote MSC osteogenic differentiation nor enhance differentiated osteoblast function. Collectively, these data lead us to the following hypothesis: The development of vascular and bone tissues during post natal bone repair and regeneration is regulated through a positive paracrine feedback loop, which is established through the expression of BMP2 by ECs and VEGF expression by osteogenic cells. This hypothesis is a paradigm shift from prior general beliefs about the cell sources and functions of BMP2 and VEGF during bone repair. A murine model of distraction osteogenesis (DO) will be used to address this hypothesis. DO has three advantages for the proposed studies: a) new bone formation occurs primarily through an intramembranous process devoid of extensive cartilage; b) it is an extremely robust regenerative model capable of inducing immense amounts of highly vascularized bone tissues; c) it is successfully used in the clinical setting to treat some forms of nonunions in the absence of BMP2 treatment. Aim 1 will define the functional relationship between vessel morphogenesis and the induction of BMP2 in endothelial cells by altering vascular morphogenesis through blocking VEGFR 1 and 2 activities. Using transgenic mice containing a conditionally inducible Cre gene that is selectively expressed in endothelial cells, the development of nascent endothelial cells will be tracked within new vessels and co-localized to the cell populations that are expressing BMP2. Aim 2 will use these same transgenic mice to assess the functional role of BMP2 expression by endothelial cells in the formation of bone and vascular tissues by BMP2 gene ablation in ECs during the different phases of DO. 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.