Department of Microbiology, Escuela Nacional de Ciencias Biológicas of Instituto Politécnico Nacional, Carpio y Plan de Ayala S/N. Col. Santo Tomas. Deleg. Miguel Hidalgo, CP 11340, Mexico City, Mexico.
Graefes Arch Clin Exp Ophthalmol. 2013 Jan;251(1):53-62. doi: 10.1007/s00417-012-2130-5. Epub 2012 Aug 14.
Keratitis caused by Staphylococcus aureus often leads to Vascular Endothelial Growth Factor (VEGF)-dependent neovascularization, but contribution of peptidoglycan (PGN), muramyl dipeptide (MDP) and lipoteichoic acid (LTA) from S. aureus to VEGF-dependent neovascularization has not been well-studied. This work was focused on the analysis of S. aureus cell wall components in the production of VEGF family members (VEGF-A, VEGF-B, VEGF-C and VEGF-D) in ocular limbal fibroblasts.
Primary culture of human limbal fibroblasts (PCHLFs) were stimulated with PGN, MDP, and LTA, and VEGF family; toll-like receptor 2 (TLR2), nucleotide-binding oligomerization domain 1 (NOD1), and NOD2 expression were determined by RT-PCR. Anti-TLR2 antibody, epidermal growth factor receptor (EGFR) signaling inhibitors (AG1478 and PD98059), and NFκB activation were used to analyze VEGF-A by ELISA. TLR2 and NOD1 expression were analyzed by flow cytometry.
The stimulation of PCHLFs with PGN and MDP increased the levels of VEGF-A expression (mRNA and protein) in a time-dependent and dose-dependent manner. VEGF-B, VEGF-C and VEGF-D were expressed constitutively, and no further induction was observed in stimulated PCHLFs. LTA did not increase the expression levels of the VEGF family. TLR2 mRNA and protein were increased only when PCHLFs were stimulated with PGN. Treatment with an anti-TLR2 antibody blocked the interaction of PGN with the receptor, inhibiting VEGF-A over-expression; the presence of anti-TLR2 antibodies did not affect the over-production of VEGF-A after MDP treatment. PCHLFs stimulated with PGN and MDP, but not with LTA, activated NFκB. MDP stimulated the production of NOD1 and NOD2 mRNAs in a time-dependent and dose-dependent manner, and NOD2 protein was only increased by MDP. Treatment of PCHLFs with AG1478 and PD98059 inhibitors prior to stimulation with MDP resulted in the inhibition of VEGF-A over-production, compared with PCHLFs stimulated with MDP alone.
Taken together, these results suggest that limbal fibroblasts produce VEGF-A through PGN-TLR2-NFκB and MDP-NOD2-EGFR.
金黄色葡萄球菌引起的角膜炎常导致血管内皮生长因子(VEGF)依赖性新生血管形成，但金黄色葡萄球菌的肽聚糖(PGN)、胞壁酰二肽(MDP)和脂磷壁酸(LTA)对 VEGF 依赖性新生血管形成的贡献尚未得到很好的研究。本工作集中分析了眼角膜缘成纤维细胞中金黄色葡萄球菌细胞壁成分在 VEGF 家族成员(VEGF-A、VEGF-B、VEGF-C 和 VEGF-D)产生中的作用。
用 PGN、MDP 和 LTA 刺激原代培养的人角膜缘成纤维细胞(PCHLFs)，通过 RT-PCR 检测 VEGF 家族；Toll 样受体 2(TLR2)、核苷酸结合寡聚化结构域 1(NOD1)和 NOD2 的表达。用抗 TLR2 抗体、表皮生长因子受体(EGFR)信号抑制剂(AG1478 和 PD98059)和 NFκB 激活来分析 ELISA 中的 VEGF-A。用流式细胞术分析 TLR2 和 NOD1 的表达。
PGN 和 MDP 刺激 PCHLFs 可使 VEGF-A 的表达(mRNA 和蛋白)呈时间和剂量依赖性增加。VEGF-B、VEGF-C 和 VEGF-D 呈组成性表达，在受刺激的 PCHLFs 中没有进一步诱导。LTA 没有增加 VEGF 家族的表达水平。只有当 PCHLFs 受到 PGN 刺激时，TLR2 mRNA 和蛋白才会增加。用抗 TLR2 抗体阻断 PGN 与受体的相互作用，抑制 VEGF-A 的过度表达；用 MDP 处理后，用抗 TLR2 抗体处理不会影响 VEGF-A 的过度产生。PGN 和 MDP 刺激 PCHLFs 而非 LTA 激活 NFκB。MDP 以时间和剂量依赖性方式刺激 NOD1 和 NOD2 mRNA 的产生，而 NOD2 蛋白仅受 MDP 增加。在用 MDP 刺激 PCHLFs 之前，用 AG1478 和 PD98059 抑制剂处理可抑制 VEGF-A 的过度产生，与单独用 MDP 刺激 PCHLFs 相比。
综上所述，这些结果表明角膜缘成纤维细胞通过 PGN-TLR2-NFκB 和 MDP-NOD2-EGFR 产生 VEGF-A。
