Supplementary Material for: Porphyromonas gingivalis Gingipains-Mediated Degradation of Plasminogen Activator Inhibitor-1 Leads to Delayed Wound Healing Responses in Human Endothelial Cells

Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is constitutively produced by endothelial cells and plays a vital role in maintaining vascular homeostasis. Chronic periodontitis is an inflammatory disease characterized by bleeding of periodontal tissues that support the tooth. In this study, we aimed to determine the role of PAI-1 produced by endothelial cells in response to infections caused by the primary periodontal pathogen Porphyromonas gingivalis. We demonstrated that P. gingivalis infection resulted in significantly reduced PAI-1 levels in human endothelial cells. This reduction in PAI-1 levels could be attributed to the proteolysis of PAI-1 by P. gingivalis proteinases, especially lysine-specific gingipain-K (Kgp). We demonstrated the roles of these degradative enzymes in the endothelial cells using a Kgp-specific inhibitor and P. gingivalis gingipain-null mutants, in which the lack of the proteinases resulted in the absence of PAI-1 degradation. The degradation of PAI-1 by P. gingivalis induced a delayed wound healing response in endothelial cell layers via the low-density lipoprotein receptor-related protein. Our results collectively suggested that the proteolysis of PAI-1 in endothelial cells by gingipains of P. gingivalis might lead to the deregulation of endothelial homeostasis, thereby contributing to the permeabilization and dysfunction of the vascular endothelial barrier.纤溶酶原激活物抑制剂-1（Plasminogen activator inhibitor-1, PAI-1）是一种丝氨酸蛋白酶抑制剂，由内皮细胞组成性表达，在维持血管稳态中发挥关键作用。慢性牙周炎是一种以支撑牙齿的牙周组织出血为特征的炎症性疾病。本研究旨在明确内皮细胞产生的PAI-1在应对主要牙周致病菌牙龈卟啉单胞菌（Porphyromonas gingivalis）感染时的作用。研究证实，牙龈卟啉单胞菌感染可显著降低人内皮细胞中的PAI-1水平。PAI-1水平的这一降低可归因于牙龈卟啉单胞菌蛋白酶，尤其是赖氨酸特异性牙龈素-K（lysine-specific gingipain-K, Kgp）对PAI-1的蛋白水解作用。通过使用Kgp特异性抑制剂以及牙龈素缺失突变株（gingipain-null mutants）——该突变株因缺乏相关蛋白酶而无法介导PAI-1的降解，我们验证了这些降解酶在内皮细胞中的作用。牙龈卟啉单胞菌对PAI-1的降解可通过低密度脂蛋白受体相关蛋白（low-density lipoprotein receptor-related protein）延迟内皮细胞层的伤口愈合反应。综上，本研究结果表明，牙龈卟啉单胞菌的牙龈素对内皮细胞中PAI-1的蛋白水解作用可能导致内皮稳态失衡，进而促成血管内皮屏障的通透性增加与功能异常。