Role of Angiogenesis in Distraction Osteogenesis

描述（由申请人提供）：骨骼损伤的愈合受损与发病率增加和功能状态下降相关，每年对超过800，000例愈合受损的骨折进行外科矫形治疗。血液供应不足是所有类型骨科治疗中愈合受损的主要原因。骨形态发生蛋白2（BMP 2）在促进间充质干细胞（MSC）向成骨细胞分化中起重要作用;而血管内皮生长因子（VEGF）在血管内皮细胞（EC）的募集和分化中起关键作用。目前，普遍认为BMP 2是由MSC和成骨细胞产生的，或者是从细胞外基质中的隔离储存中蛋白水解释放的。我们对转基因小鼠的初步研究表明，在骨折和截骨部位附近以及骨修复组织内的血管内皮细胞是修复部位BMP 2的主要来源。VEGF也已被证明在骨愈合中起作用，然而，它们是否通过成骨细胞中受体介导的信号传导以直接方式起作用仍有很大争议。我们实验室的其他初步工作表明，虽然分化的成骨细胞大量表达VEGF，但外源性加入的VEGF既不促进MSC成骨分化，也不增强分化的成骨细胞功能。总的来说，这些数据使我们得出以下假设：纳塔尔后骨修复和再生过程中血管和骨组织的发育是通过正旁分泌反馈回路调节的，该反馈回路是通过EC表达BMP 2和成骨细胞表达VEGF建立的。这一假说是从先前关于骨修复过程中BMP 2和VEGF的细胞来源和功能的一般信念的范式转变。将使用牵张成骨（DO）的鼠模型来解决这一假设。DO对于所提出的研究具有三个优点：a）新骨形成主要通过缺乏广泛软骨的膜内过程发生; B）它是能够诱导大量高度血管化骨组织的极其稳健的再生模型; c）它在临床环境中成功地用于在不存在BMP 2治疗的情况下治疗某些形式的骨不连。目的1通过阻断血管内皮生长因子受体1和2的活性改变血管形态发生，明确血管形态发生与内皮细胞中BMP 2诱导的功能关系。使用含有在内皮细胞中选择性表达的条件诱导型Cre基因的转基因小鼠，将在新血管内跟踪新生内皮细胞的发育，并将其共定位于表达BMP 2的细胞群。目的2将使用这些相同的转基因小鼠，通过在DO的不同阶段在EC中进行BMP 2基因消融来评估内皮细胞表达BMP 2在骨和血管组织形成中的功能作用。目标1和2的完成将确定EC是否是BMP 2在手术或损伤后调节骨骼愈合中的主要细胞来源，以及BMP 2是否在血管发生中具有功能性作用。

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.