Plasminogen in periodontitis and wound repair

Abstract: The plasminogen activator (PA) system plays a critical role in many physiological and pathological processes, such as fibrinolysis, extracellular matrix (ECM) degradation, wound healing, inflammation, and cancer. The key component of the PA system is plasmin, a broad-spectrum serine protease that is derived from its inactive form, plasminogen. The first aim of this thesis research was to determine the role of plasminogen in periodontitis, an inflammatory oral disease. The second aim was to explore the molecular mechanism by which plasminogen contributes to wound healing in the skin. Finally, the third aim was to investigate the possibility of using plasminogen as a treatment for skin wounds, especially for chronic wounds, such as diabetic wounds.Periodontitis is an oral disease that involves a bacterial infection, the inflammation of the periodontium, and the degradation of gum tissue and alveolar bone. This disease is irreversible and, in severe cases, can lead to loss of teeth due to the degradation of the periodontal ligament and alveolar bone. To study the effects of the PA system on oral health, we monitored the development of periodontitis in plasminogen-deficient mice and plasminogen activator-deficient mice. In control wild-type mice, periodontitis did not occur. However, in plasminogen-deficient mice, periodontitis developed rapidly within 20 weeks after birth. The morphological studies of plasminogen-deficient mice showed the detachment of gingival tissues, resorption of the cementum layer, formation of necrotic tissue, and severe alveolar bone degradation. Immunohistochemical staining showed the massive infiltration of neutrophils into the periodontal tissues. Interestingly, doubly deficient mice lacking both tissue-type plasminogen activator (tPA) and urokinase-type PA (uPA) developed periodontitis at a similar rate as the plasminogen-deficient mice, but mice lacking only tPA or uPA remained healthy. The intravenous injection of human plasminogen for 10 days into plasminogen-deficient mice led to the absorption of necrotic tissue, the diminution of inflammation, and the full regeneration of gum tissues. Notably, there was also partial re-growth of degraded alveolar bone.The wound healing process consists of three overlapping phases: inflammatory, proliferative, and remodeling. It has been postulated that the PA system plays an integral role in this process, and a lack of plasminogen leads to delayed wound healing in mice. To study the role of the PA system in wound healing, we monitored the responses of wild-type, plasminogen-deficient and diabetic mice to incision and burn wounds. We found that in addition to being delayed, the wound healing process in plasminogen-deficient mice was only superficial in nature. The plasminogen-deficient mice were unable to clear the provisional matrix after the formation of granulation tissue, and an extensive fibrin deposition. In addition, persistent inflammation was still present subcutaneously in these mice 60 days after introduction of the wound.The essential role of plasminogen in burn and incision wounds healing was further confirmed by reconstitution experiments. Both intravenous and subcutaneous administrations of human plasminogen to plasminogen-deficient mice led to a restored healing rate and wound maturation that was comparable to those of wild-type mice. We also demonstrated that plasminogen supplementation of plasminogen to wild-type and diabetic mice significantly improved the healing of cutaneous wounds. Plasminogen levels were not only temporally increased during the inflammation phase but also spatially concentrated at the site of the wound. The wound-specific accumulation of plasminogen after systemic supplementation is mainly due to the transportation of plasminogen by neutrophils and macrophages. Furthermore, the increased expression of interleukin 6 and the enhanced phosphorylation of STAT3 were observed in the wound after plasminogen treatment. These data indicate that plasminogen acts as a key pro-inflammatory regulator. It enhances pro-inflammatory cytokines and activates intracellular signaling events during wound healing.Taken together, the data obtained during the course of this project indicate that plasminogen is crucial for oral health in mice. We also demonstrate that supplementation of plasminogen to mice with periodontitis results in healing of gum tissues and significant re-growth of alveolar bone. Therefore, plasminogen may be a new drug that will be competitive to currently used oral health-related procedures, such as implantations and surgeries. Furthermore, we demonstrate for the first time that, in addition to its role in extracellular matrix degradation, plasminogen is a key pro-inflammatory factor that accumulates at the wound and potentiates the early inflammatory response during wound healing. Based on our findings, we propose the administration of plasminogen as a novel therapeutic strategy for the treatment of different types of wounds, including chronic diabetic wounds.