Department of Biomolecular Sciences, Section of Clinical Biochemistry and Molecular Genetics, University Carlo Bo Urbino, Italy.
Cardiovascular Biochemistry, Biomedical Research Institute Sant Pau (IIB-Sant Pau), Barcelona, Spain, C/Sant Antoni M. Claret 167, 08025 Barcelona, Spain; Molecular Biology and Biochemistry Department, Universitat Autònoma de Barcelona (UAB). Cerdanyola del Vallès, Spain.
Biochim Biophys Acta Mol Basis Dis. 2018 Dec;1864(12):3559-3567. doi: 10.1016/j.bbadis.2018.09.022. Epub 2018 Sep 19.
Electronegative LDL (LDL(-)) is involved in atherosclerosis through the activation of the TLR4/CD14 inflammatory pathway in monocytes. Matrix metalloproteinases (MMP) and their inhibitors (tissue inhibitors of metalloproteinase [TIMP]) are also crucially involved in atherosclerosis, but their modulation by LDL(-) has never been investigated. The aim of this study was to examine the ability of LDL(-) to release MMPs and TIMPs in human monocytes and to determine whether sulodexide (SDX), a glycosaminoglycan-based drug, was able to affect their secretion.
Native LDL (LDL(+)) and LDL(-) separated by anion-exchange chromatography were added to THP1-CD14 monocytes in the presence or absence of SDX for 24 h. A panel of 9 MMPs and 4 TIMPs was analyzed in cell supernatants with multiplex immunoassays. The gelatinolytic activity of MMP-9 was assessed by gelatin zymography. LDL(-) stimulated the release of MMP-9 (13-fold) and TIMP-1 (4-fold) in THP1-CD14 monocytes, as well as the gelatinolytic activity of MMP-9. Co-incubation of monocytes with LDL(-) and SDX for 24 h significantly reduced both the release of MMP-9 and TIMP-1 and gelatinase activity. In THP1 cells not expressing CD14, no effect of LDL(-) on MMP-9 or TIMP-1 release was observed. The uptake of DiI-labeled LDL(-) was higher than that of DiI-LDL(+) in THP1-CD14 but not in THP1 cells. This increase was inhibited by SDX. Experiments in microtiter wells coated with SDX demonstrated a specific interaction of LDL(-) with SDX.
LDL(-) induced the release of MMP-9 and TIMP-1 in monocytes through CD14. SDX affects the ability of LDL(-) to promote TIMP-1 and MMP-9 release by its interaction with LDL(-).
通过激活单核细胞中的 TLR4/CD14 炎症途径，负电低密度脂蛋白（LDL(-)）参与动脉粥样硬化。基质金属蛋白酶（MMP）及其抑制剂（金属蛋白酶组织抑制剂[TIMP]）也在动脉粥样硬化中起着至关重要的作用，但它们是否受 LDL(-)调节尚未得到研究。本研究旨在检测 LDL(-)是否能够释放人单核细胞中的 MMP 和 TIMP，并确定基于糖胺聚糖的药物 sulodexide（SDX）是否能够影响其分泌。
用阴离子交换色谱法分离的天然 LDL(+)和 LDL(-)，在存在或不存在 SDX 的情况下，加入 THP1-CD14 单核细胞中孵育 24 小时。用多重免疫分析检测细胞上清液中的 MMP-9 谱。通过明胶酶谱法评估 MMP-9 的明胶酶活性。LDL(-)刺激 THP1-CD14 单核细胞中 MMP-9（13 倍）和 TIMP-1（4 倍）的释放，以及 MMP-9 的明胶酶活性。24 小时内共孵育单核细胞与 LDL(-)和 SDX，可显著降低 MMP-9 和 TIMP-1 的释放和明胶酶活性。在不表达 CD14 的 THP1 细胞中，LDL(-)对 MMP-9 或 TIMP-1 的释放没有影响。在 THP1-CD14 细胞中，DiI 标记的 LDL(-)的摄取量高于 DiI-LDL(+)，但在 THP1 细胞中则不然。这种增加被 SDX 抑制。在 SDX 包被的微量滴定孔中的实验表明，LDL(-)与 SDX 之间存在特异性相互作用。
LDL(-)通过 CD14 诱导单核细胞中 MMP-9 和 TIMP-1 的释放。SDX 通过与 LDL(-)相互作用影响 LDL(-)促进 TIMP-1 和 MMP-9 释放的能力。
