Laboratory of Periodontal Biology, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.
Faculty of Dentistry, Universidad Andres Bello, Santiago, Chile.
Int Endod J. 2023 Oct;56(10):1270-1283. doi: 10.1111/iej.13957. Epub 2023 Aug 6.
AIM: Angiogenesis contributes to the development of apical periodontitis, periodontitis, and other oral pathologies; however, it remains unclear how this process is triggered. The aim was to evaluate whether lipopolysaccharide (LPS) from Porphyromonas endodontalis and Porphyromonas gingivalis induced angiogenesis-related effects in vitro via TLR2 and TLR4. METHODOLOGY: Porphyromonas endodontalis LPS (ATCC 35406 and clinical isolate) was purified with TRIzol, whereas P. gingivalis LPS was obtained commercially. The effects of the different LPS (24 h) in endothelial cell migration were analysed by Transwell assays, following quantification in an optical microscope (40×). The effects of LPS on FAK Y397 phosphorylation were assessed by Western blotting. Angiogenesis in vitro was determined in an endothelial tube formation assay (14 h) in Matrigel in the absence or presence of either LPS. IL-6 and VEGF-A levels were determined in cell supernatants, following 24 h treatment with LPS, and measured in multiplex bead immunoassay. The involvement of TLR2 and TLR4 was assessed with blocking antibodies. The statistical analysis was performed using STATA 12® (StataCorp LP). RESULTS: The results revealed that P. endodontalis LPS, but not P. gingivalis LPS, stimulated endothelial cell migration. Pre-treatment with anti-TLR2 and anti-TLR4 antibodies prevented P. endodontalis LPS-induced cell migration. P. endodontalis LPS promoted FAK phosphorylation on Y397, as observed by an increased p-FAK/FAK ratio. Both P. gingivalis and P. endodontalis LPS (ATCC 35406) induced endothelial tube formation in a TLR-2 and -4-dependent manner, as shown by using blocking antibodies, however, only TLR2 blocking decreased tube formation induced by P. endodontalis (clinical isolate). Moreover, all LPS induced IL-6 and VEGF-A synthesis in endothelial cells. TLR2 and TLR4 were required for IL-6 induction by P. endodontalis LPS (ATCC 35406), while only TLR4 was involved in IL-6 secretion by the other LPS. Finally, VEGF-A synthesis did not require TLR signalling. CONCLUSION: Porphyromonas endodontalis and P. gingivalis LPS induced angiogenesis via TLR2 and TLR4. Collectively, these data contribute to understanding the role of LPS from Porphyromonas spp. in angiogenesis and TLR involvement.
目的：血管生成有助于根尖周炎、牙周炎和其他口腔病理学的发展；然而，目前尚不清楚这一过程是如何引发的。本研究旨在评估牙髓卟啉单胞菌和牙龈卟啉单胞菌的脂多糖（LPS）是否通过 TLR2 和 TLR4 在体外诱导血管生成相关作用。
方法：用 TRIzol 从牙髓卟啉单胞菌 LPS（ATCC 35406 和临床分离株）中纯化，而牙龈卟啉单胞菌 LPS 则从商业途径获得。通过 Transwell 分析检测不同 LPS（24 小时）对内皮细胞迁移的影响，然后在光学显微镜（40×）下进行定量分析。通过 Western blot 检测 LPS 对 FAK Y397 磷酸化的影响。在无 LPS 或存在 LPS 的情况下，在 Matrigel 中进行内皮管形成试验（14 小时），以确定体外血管生成情况。用 LPS 处理 24 小时后，用多重珠免疫分析测定细胞上清液中 IL-6 和 VEGF-A 的水平。用阻断抗体评估 TLR2 和 TLR4 的参与情况。统计分析使用 STATA 12®（StataCorp LP）进行。
结果：结果表明，牙髓卟啉单胞菌 LPS 但不是牙龈卟啉单胞菌 LPS 刺激内皮细胞迁移。用抗 TLR2 和抗 TLR4 抗体预处理可阻止牙髓卟啉单胞菌 LPS 诱导的细胞迁移。P. endodontalis LPS 促进 FAK 在 Y397 上的磷酸化，表现为 p-FAK/FAK 比值增加。用阻断抗体显示，牙龈卟啉单胞菌和牙髓卟啉单胞菌 LPS（ATCC 35406）均以 TLR-2 和 -4 依赖的方式诱导内皮管形成，但只有 TLR2 阻断可减少由牙髓卟啉单胞菌（临床分离株）诱导的管形成。此外，所有 LPS 均诱导内皮细胞中 IL-6 和 VEGF-A 的合成。牙髓卟啉单胞菌 LPS（ATCC 35406）诱导 IL-6 表达需要 TLR2 和 TLR4，而其他 LPS 分泌 IL-6 仅需要 TLR4。最后，VEGF-A 的合成不需要 TLR 信号。
结论：牙髓卟啉单胞菌和牙龈卟啉单胞菌 LPS 通过 TLR2 和 TLR4 诱导血管生成。总之，这些数据有助于了解牙髓卟啉单胞菌和牙龈卟啉单胞菌 LPS 在血管生成和 TLR 参与中的作用。
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