Plasmin is Essential to Prevent and Resolve Heterotopic Ossification

描述（由申请人提供）：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。骨骼肌损伤通常发生在骨骼肌损伤后，由于疼痛、肌肉损失和肌肉/关节运动受限，在最初的创伤性损伤后很长一段时间内都会造成严重的发病率，原因尚不清楚。目前，还没有安全的药物或手术手段来预防或选择性去除HO。因此，有必要阐明HO的潜在原因，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，我们探索了一种临床观察提示的机制，即HO最常见于损害凝血系统纤溶臂功能的损伤，如烧伤、爆炸伤和头部创伤。为了支持我们的假设，我们发现，仅部分缺乏纤溶酶原（Plg-/+）（纤溶酶的前体和纤溶系统的初级蛋白酶）的小鼠，在损伤后骨骼肌中发展出强大的HO。此外，我们确定在Plg-/+小鼠骨骼肌损伤之前靶向纤溶蛋白的主要抑制剂α2-抗纤溶蛋白完全预防HO。目的：我们假设1)需要一个阈值水平的纤溶酶活性来阻止骨骼肌损伤后HO的发生，2)增强的纤溶酶活性可以阻止HO的发生和/或在成熟之前解决未成熟的HO。方法：两种目的均采用我们建立的心毒素诱导HO模型。目的1将利用反义寡核苷酸（ASO）设计，在HO诱导前逐渐降低野生型小鼠的纤溶酶原表达，并通过x射线和微ct监测疾病进展。通过基因表达、血浆抗原和纤溶酶活性水平来测定纤溶酶原。在本研究的目标2中，我们将给Plg-/+小鼠注射靶向α2-抗纤溶酶的ASO，以在损伤后立即或损伤后两周（HO发生后）增强纤溶酶的活性。与Aim 1一样监测纤溶酶原表达、纤溶酶活性和HO的发展。影响：本文提出的实验有可能阐明急性HO的机制，从而直接影响HO患者的临床治疗。临床：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设计量化纤溶活性的研究提供了必要的信息，因为它与人类HO有关。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：这项工作可能为一种新的药理学方法提供原则性证据，包括靶向给药策略，通过增强纤维蛋白溶解来预防、阻止和去除HO。

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.