Kolanczyk Mateusz, Kühnisch Jirko, Kossler Nadine, Osswald Monika, Stumpp Sabine, Thurisch Boris, Kornak Uwe, Mundlos Stefan
Max Planck Institute for Molecular Genetics, FG Development & Disease, Berlin, Germany.
BMC Med. 2008 Jul 31;6:21. doi: 10.1186/1741-7015-6-21.
Bowing and/or pseudarthrosis of the tibia is a known severe complication of neurofibromatosis type 1 (NF1). Mice with conditionally inactivated neurofibromin (Nf1) in the developing limbs and cranium (Nf1Prx1) show bowing of the tibia caused by decreased bone mineralisation and increased bone vascularisation. However, in contrast to NF1 patients, spontaneous fractures do not occur in Nf1Prx1 mice probably due to the relatively low mechanical load. We studied bone healing in a cortical bone injury model in Nf1Prx1 mice as a model for NF1-associated bone disease. Taking advantage of this experimental model we explore effects of systemically applied lovastatin, a cholesterol-lowering drug, on the Nf1 deficient bone repair.
Cortical injury was induced bilaterally in the tuberositas tibiae in Nf1Prx1 mutant mice and littermate controls according to a method described previously. Paraffin as well as methacrylate sections were analysed from each animal. We divided 24 sex-matched mutant mice into a lovastatin-treated and an untreated group. The lovastatin-treated mice received 0.15 mg activated lovastatin by daily gavage. The bone repair process was analysed at three consecutive time points post injury, using histological methods, micro computed tomography measurements and in situ hybridisation. At each experimental time point, three lovastatin-treated mutant mice, three untreated mutant mice and three untreated control mice were analysed. The animal group humanely killed on day 14 post injury was expanded to six treated and six untreated mutant mice as well as six control mice.
Bone injury repair is a complex process, which requires the concerted effort of numerous cell types. It is initiated by an inflammatory response, which stimulates fibroblasts from the surrounding connective tissue to proliferate and fill in the injury site with a provisional extracellular matrix. In parallel, mesenchymal progenitor cells from the periost are recruited into the injury site to become osteoblasts. In Nf1Prx1 mice bone repair is delayed and characterised by the excessive formation and the persistence of fibro-cartilaginous tissue and impaired extracellular matrix mineralisation. Correspondingly, expression of Runx2 is downregulated. High-dose systemic lovastatin treatment restores Runx2 expression and accelerates new bone formation, thus improving cortical bone repair in Nf1Prx1 tibia. The bone anabolic effects correlate with a reduction of the mitogen activated protein kinase pathway hyper-activation in Nf1-deficient cells.
Our data suggest the potential usefulness of lovastatin, a drug approved by the US Food and Drug Administration in 1987 for the treatment of hypercholesteraemia, in the treatment of Nf1-related fracture healing abnormalities. The experimental model presented here constitutes a valuable tool for the pre-clinical stage testing of candidate drugs, targeting Nf1-associated bone dysplasia.
胫骨弯曲和/或假关节是1型神经纤维瘤病（NF1）已知的严重并发症。在发育中的四肢和颅骨中条件性失活神经纤维瘤蛋白（Nf1）的小鼠（Nf1Prx1）表现出胫骨弯曲，这是由于骨矿化减少和骨血管生成增加所致。然而，与NF1患者不同，Nf1Prx1小鼠不会发生自发性骨折，这可能是由于机械负荷相对较低。我们研究了Nf1Prx1小鼠皮质骨损伤模型中的骨愈合情况，以此作为NF1相关骨病的模型。利用这个实验模型，我们探讨了全身应用洛伐他汀（一种降胆固醇药物）对Nf1缺陷型骨修复的影响。
根据先前描述的方法，在Nf1Prx1突变小鼠和同窝对照小鼠的胫骨粗隆双侧诱导皮质损伤。对每只动物的石蜡切片和甲基丙烯酸甲酯切片进行分析。我们将24只性别匹配的突变小鼠分为洛伐他汀治疗组和未治疗组。洛伐他汀治疗组小鼠每天通过灌胃给予0.15 mg活化洛伐他汀。在损伤后的三个连续时间点，使用组织学方法、微型计算机断层扫描测量和原位杂交分析骨修复过程。在每个实验时间点，分析三只洛伐他汀治疗的突变小鼠、三只未治疗的突变小鼠和三只未治疗的对照小鼠。在损伤后第14天人道处死的动物组扩大到六只治疗的和六只未治疗的突变小鼠以及六只对照小鼠。
骨损伤修复是一个复杂的过程，需要多种细胞类型的协同努力。它由炎症反应启动，炎症反应刺激周围结缔组织中的成纤维细胞增殖，并用临时细胞外基质填充损伤部位。同时，骨膜中的间充质祖细胞被募集到损伤部位成为成骨细胞。在Nf1Prx1小鼠中，骨修复延迟，其特征是纤维软骨组织过度形成和持续存在以及细胞外基质矿化受损。相应地，Runx2的表达下调。高剂量全身洛伐他汀治疗可恢复Runx2表达并加速新骨形成，从而改善Nf1Prx1胫骨的皮质骨修复。骨合成代谢作用与Nf1缺陷细胞中有丝分裂原活化蛋白激酶途径的过度激活减少相关。
我们的数据表明，1987年被美国食品药品监督管理局批准用于治疗高胆固醇血症的药物洛伐他汀，在治疗Nf1相关骨折愈合异常方面具有潜在的应用价值。这里介绍的实验模型是用于针对Nf1相关骨发育异常的候选药物临床前阶段测试的有价值工具。
