Neural Mechanisms in Heterotopic Ossification

描述(申请人提供)：我们最近证明了周围神经系统(PNS)中的感觉神经与异常的骨形成之间的联系，称为异位骨化(HO)。从这些研究来看，骨形态发生蛋白2(BMP2)似乎直接诱导神经炎症的表达，从而导致神经周祖细胞的扩张和迁移。然后，这些细胞经历棕色脂肪生成和抑制神经炎症，两者都消融了它们的扩张，并显著减弱了HO。同样值得注意的是，这些棕色脂肪细胞样细胞表达许多软骨细胞标记物，表明它们可能正在经历软骨形成。这些神经周祖细胞在HO诱导48小时内也表达VEGFA和D，这与新骨形成区域出现新的flk1+血管相一致。同时，神经内神经室的其他细胞也表达成骨细胞转录因子Osterix。这些细胞还表达存在于神经内皮细胞上的claudin 5。这些数据共同引导了我们进一步的假设：周围神经可能通过常驻祖细胞的迁移直接参与HO的形成，形成异位软骨和骨。此外，这些细胞的扩张和迁移可能是它们共同调节的产物，涉及到内神经细胞内的内皮。我们设计了三个具体目标来检验这一假设。(1)确定神经周围神经前体细胞是否正在进行软骨形成。星形细胞标记GLAST在神经内的神经周祖细胞上唯一表达，我们将使用他莫昔芬调节(ERT)GLAST-YFP小鼠来评估它们的血统命运。我们还将在UCP-DTA小鼠中诱导HO，在白喉毒素存在的情况下，UCP1+细胞被消融，并在没有白喉毒素的情况下表征软骨的形成。(2)确定周围神经内皮细胞样祖细胞在HO过程中是否发生成骨。我们将进一步鉴定这些细胞，寻找与神经前体细胞、内皮细胞和成骨细胞相关的标记。此外，我们将使用ERT-WNT1-YFP小鼠和/或ERT-PDGFR？-YFP小鼠来追踪这些细胞的初步神经谱系，并分析产生的YFP+细胞的异位骨。此外，我们使用ERT-WNT1-CRE x Floted Osterix选择性地去除这些细胞中Osterix的表达，并分析下游的骨形成。(3)研究HO过程中神经外膜扩张、血管萌发和神经内祖细胞扩张和迁移的规律。我们将测试特定的生长因子(如LIF、PDGF、VEGF、CSF)是否在HO的早期阶段受到调控，导致紧密连接的溶解以及神经周围细胞的扩张，随后是棕色脂肪生成以及轴突和新血管的萌发。

DESCRIPTION (provided by applicant): We have recently demonstrated a connection between sensory nerves in the peripheral nervous system (PNS) and the aberrant bone formation known as heterotopic ossification (HO). From these studies bone morphogenetic protein type 2 (BMP2) appears to directly induce expression of neural inflammation, that leads to the expansion and migration of perineurial progenitors. These cells then undergo brown adipogenesis and suppression of neural inflammation, both ablated their expansion and significantly attenuate HO. Also of note, these brown adipocyte-like cells expressed many chondrocyte markers suggesting that they may be undergoing chondrogenesis. These perineurial progenitors also express VEGF A and D within 48 hours of induction of HO, which coincides with the appearance of new flk1+ vessels in the region of new bone formation. Simultaneously, other cells from the endoneurial compartment of the nerve were found to express the osteoblast transcription factor osterix. These cells also express claudin 5, which is present on endoneurial endothelial cells. The data collectively has led us to further hypothesize; that peripheral nerves may directly contribute to HO through the migration of resident progenitors to form both the heterotopic cartilage and bone. Further, the expansion of these cells and their migration may be a product of their co-regulation involving the endothelium within the endoneurium. We have devised three specific aims to test this hypothesis. (1) To determine whether perineurial progenitors are undergoing chondrogenesis. The astrocytic marker GLAST is uniquely expressed on the perineurial progenitors within the nerve, and we will use tamoxifen-regulated (Ert) GLAST-YFP mice to evaluate their lineage fate. We will also induce HO in UCP-DTA mice, where UCP1+ cells are ablated in the presence of diptheria toxin and characterize the formation of cartilage in their absence. (2) To determine if endoneurial endothelial-like progenitors within peripheral nerves undergo osteogenesis during HO. We will further characterize the cells for markers associated with neural progenitors, endothelial cells, and osteoblasts. Additionally, we will use Ert-Wnt1-YFP mice and/or Ert-PDGFR?-YFP mice to trace the tentative neural lineage of these cells, and analyze the resultant heterotopic bone for YFP+ cells. Further, we selectively remove osterix expression in these cells, using an Ert-Wnt1-Cre x Floxed osterix and analyze downstream bone formation. (3) To characterize the regulation of perineurial expansion, vascular sprouting and endoneurial progenitor expansion and migration during HO. We will test whether specific growth factors (such as LIF, PDGF,VEGF, CSF) expression are regulated during the early stages of HO, leading to the dissolution of the tight junctions as well as expansion of perineurial cells followed by brown adipogenesis as well as sprouting of axons and new vessels.