Plasmin is Essential to Prevent and Resolve Heterotopic Ossification

描述（由申请方提供）：异位骨化（HO）是一种导致软组织结构矿化和潜在骨化的病理过程。由于许多未知的原因，HO经常发生在骨骼肌损伤之后，由于疼痛、肌肉损失和限制的肌肉/关节运动，在初始创伤性损伤之后很长时间内造成显著的发病率。目前，没有安全的药理学或外科手术手段来预防或选择性去除HO。因此，有必要阐明HO的根本原因，以指导旨在预防和治疗这种毁灭性疾病的新疗法的战略发展。因此，我们探索了临床观察所提示的机制，即HO最常发生在损害凝血系统纤溶臂功能的损伤后，例如：烧伤、爆炸伤和头部创伤。为了支持我们的假设，我们已经发现，仅部分缺乏纤溶酶原（Plg-/+）（纤溶酶的前体和纤维蛋白溶解系统的主要蛋白酶）的小鼠在损伤后在骨骼肌中发展出稳健的HO。此外，我们确定在Plg-/+小鼠骨骼肌损伤之前靶向纤溶酶的主要抑制剂α2-抗纤溶酶完全防止HO。目的：我们推测：1）需要纤溶酶活性的阈值水平来防止骨骼肌损伤后HO的启动，2）增强的纤溶酶活性可以防止HO的启动和/或在成熟之前解决未成熟的HO。方法：两个目的均采用我们建立的肉毒毒素诱导的HO模型。目的1将利用反义寡核苷酸（阿索），旨在逐步敲低野生型小鼠的纤溶酶原表达之前HO诱导，疾病的进展将监测与X射线和microCT。将通过基因表达、血浆抗原和纤溶酶活性水平测量纤溶酶原。在本提案的目的2中，我们将在损伤后立即或损伤后两周、HO发生后，向Plg-/+小鼠给予靶向α2-抗纤溶酶的阿索，以增强纤溶酶的活性。如目的1所述，监测纤溶酶原表达、纤溶酶活性和HO的发展。影响力：本文提出的实验有可能阐明急性HO的潜在机制，从而直接影响HO患者的临床治疗。临床上：我们的实验可以帮助识别有HO风险的患者并改善临床结果，因为这项工作为未来旨在量化与人类HO相关的纤溶活性的研究提供了必要的信息。此外，由于软组织钙化在多种疾病中起着重要作用，这些发现有可能揭示预防和治疗其他病理过程（如动脉粥样硬化和瓣膜钙化）的新方法。治疗方面：这项工作可以提供一种新的药理学方法，包括有针对性的给药策略，以防止，逮捕和消除HO通过增强纤维蛋白溶解的原则性证据。

DESCRIPTION (provided by applicant): Heterotopic ossification (HO) is a pathologic process resulting in mineralization and potential ossification of soft tissue structures. For reasons largey unknown, HO often occurs following skeletal muscle injury, imposing significant morbidity long after the initial traumatic insult due to pain, muscle loss and restricted muscle/joint motion. Currently, there are no safe pharmacological or surgical means of preventing or selectively removing HO. Thus, there is a need for elucidation of the underlying cause of HO to guide strategic development of novel therapies designed to prevent and treat this devastating disease. Hence, we explored a mechanism suggested by the clinical observation that HO most commonly occurs following injuries that impair the function of the fibrinolytic arm of the coagulation system such as: burns, blast wounds and head trauma. In support of our hypothesis we have discovered that mice with only a partial deficiency of plasminogen (Plg-/+), the precursor of plasmin and primary protease of the fibrinolytic system, develop robust HO in skeletal muscle after injury. In addition, we determined that targeting the primary inhibitor of plasmin, α2-antiplasmin, prior to skeletal muscle injury in Plg-/+ mice completely prevents HO. Aims: We postulate that 1) a threshold level of plasmin activity is required to prevent initiation f HO after skeletal muscle injury and 2) enhanced plasmin activity can prevent initiation of HO and/or resolve immature HO prior to maturation. Methods: Both aims employ our established Cardiotoxin-induced HO model. Aim 1 will make use of antisense oligonucleotides (ASO) designed to incrementally knock down plasminogen expression in wild type mice prior to HO induction, and disease progression will be monitored with X-ray and microCT. Plasminogen will be measured by gene expression, plasma antigen and plasmin activity levels. In Aim 2 of this proposal we will dose Plg-/+ mice with an ASO targeting α2-antiplasmin to enhance plasmin's activity either immediately following injury or two weeks after injury, after the development of HO. Plasminogen expression, plasmin activity and development of HO will be monitored as in Aim 1. Impact: The experiments proposed here have the potential to elucidate a mechanism underlying acute HO thereby directly impact clinical treatment of patients with HO. Clinically: Our experiments could help identify patients at risk for HO and improve clinical outcomes, as this work provides necessary information for future studies designed to quantify fibrinolytic activity as it relates to HO in humans. In addition, as soft tissue calcification plays a significat role in multiple diseases, these findings have the potential to reveal novel means of preventing and treating other pathologic processes such as atherosclerosis and valvular calcification. Therapeutically: This work may provide proof-of-principle evidence for a novel pharmacological approach, including a targeted dosing strategy, to prevent, arrest and remove HO through enhanced fibrinolysis.