l-Mimosine and Dimethyloxaloylglycine Decrease Plasminogen Activation in Periodontal Fibroblasts

• Localización: Journal of periodontology, ISSN 0022-3492, Nº. 4, 2014, págs. 627-635
• Idioma: inglés
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• Resumen

  • Background: The use of prolyl hydroxylase inhibitors such as l-mimosine (L-MIM) and dimethyloxaloylglycine (DMOG) to improve angiogenesis is a new approach for periodontal regeneration. In addition to exhibiting pro-angiogenic effects, prolyl hydroxylase inhibitors can modulate the plasminogen activator system in cells from non-oral tissues. This study assesses the effect of prolyl hydroxylase inhibitors on plasminogen activation by fibroblasts from the periodontium.

    Methods: Gingival and periodontal ligament fibroblasts were incubated with L-MIM and DMOG. To investigate whether prolyl hydroxylase inhibitors modulate the net plasminogen activation, kinetic assays were performed with and without interleukin (IL)-1. Moreover, plasminogen activators and the respective inhibitors were analyzed by casein zymography, immune assays, and quantitative polymerase chain reaction.

    Results: The kinetic assay showed that L-MIM and DMOG reduced plasminogen activation under basal and IL-1�stimulated conditions. Casein zymography revealed that the effect of L-MIM involves a decrease in urokinase-type plasminogen activator activity. In agreement with these findings, reduced levels of urokinase-type plasminogen activator and elevated levels of plasminogen activator inhibitor 1 were observed.

    Conclusion: L-MIM and DMOG can reduce plasminogen activation by fibroblasts from the gingiva and the periodontal ligament under basal conditions and in the presence of an inflammatory cytokine.

    Periodontitis is a serious inflammatory disease that, if left untreated, leads to breakdown of the periodontal tissue and tooth loss. Consequently, treatment strategies that induce an anabolic response in the periodontium without stimulation of catabolic processes leading to periodontal tissue breakdown are required. Targeting oxygen sensors to improve tissue regeneration is a new therapeutic approach for treatment of inflammatory diseases.1-4 This strategy aims to enhance the local production of pro-angiogenic factors by pharmacologic inhibition of the oxygen sensors. This increase has been shown to support bone regeneration and wound healing.5,6 Cellular oxygen sensors include prolyl hydroxylases.7 Prolyl hydroxylases are active under normoxia, causing the degradation of the labile transcription factor hypoxia-inducible factor (HIF)1.2,3,7 Prolyl hydroxylases are inactive under hypoxia; thus HIF-1 is maintained and can induce the expression of pro-angiogenic molecules.2,3,8 Thus, inhibition of prolyl hydroxylases causes a cellular response that occurs under hypoxia.2 Typical inhibitors of prolyl hydroxylases are l-mimosine (L-MIM) and dimethyloxaloylglycine (DMOG).9 L-MIM was shown to effectively increase the pro-angiogenic response in fibroblasts from the gingiva, the periodontal ligament, and the pulp.10,11 However, it is still unknown whether L-MIM affects the catabolic processes underlying periodontitis.

    The plasminogen activator system is a key player in the catabolic processes. It involves the cleavage of plasminogen by plasminogen activators, which generates plasmin. Plasmin is an extracellular protease that degrades several components of the extracellular matrix such as collagen and proteoglycans. Furthermore, plasmin is capable of activating a number of other proteases that can break down tissue.12-14 Urokinase-type plasminogen activator (uPA) is a plasminogen activator that is associated with pericellular plasminogen activation. The activation of plasminogen is tightly controlled by plasminogen activator inhibitors (PAIs) such as PAI-1. Patients suffering from inflammatory diseases of the periodontium have significantly higher levels of plasminogen activators,15 which indicates an unbalanced plasminogen activation system. This results in the excessive production of plasmin.13,16 The plasminogen activator system is also involved in tissue regeneration. In the early phases of tissue regeneration, the fibrin-rich blood clot is organized as granulation tissue by the plasminogen activator system.13 Studies from uPA knockout mice show compromised healing and increased scar formation.17 Moreover, plasminogen is required to maintain a healthy periodontium and combat periodontitis as shown in preclinical models.18 Thus, the plasminogen activator system is a key player in both tissue breakdown and regeneration. Therefore, it is important to understand the impact of therapeutic strategies on plasminogen activation. It is not known whether HIF-1 stabilization by prolyl hydroxylase inhibitors modulates this important system. Several studies showed that stabilization of HIF-1 inhibits plasminogen activation in cells from non-oral tissues.19,20 However, whether prolyl hydroxylase inhibitors such as L-MIM and DMOG modulate the plasminogen activator system in oral fibroblasts is unknown.

    In this study, the authors assess the impact of L-MIM and DMOG on the plasminogen activator system of fibroblasts from the gingiva (GFs) and the periodontal ligament (PDLFs). Plasminogen activation is measured in response to L-MIM and DMOG by kinetic assays. To determine the mechanisms involved in this effect, the authors assess the effects of L-MIM and DMOG on plasminogen activators and plasminogen activator inhibitors by zymography, immunoassays, and quantitative polymerase chain reaction (PCR). The authors also assess the effect of prolyl hydroxylase inhibitors on interleukin (IL)-1�induced plasminogen activation to study their effects in response to inflammatory cytokines. This proof-of-concept study provides first insight into the impact of prolyl hydroxylase inhibitors on plasminogen activation in the periodontium.