Department of Life Sciences, Manchester Metropolitan University, Manchester M1 5GD, UK.
Cardiovascular Research Institute, University of Manchester, Manchester M13 9PL, UK.
Cells. 2024 Feb 8;13(4):312. doi: 10.3390/cells13040312.
Heparan sulphate (HS) can act as a co-receptor on the cell surface and alterations in this process underpin many pathological conditions. We have previously described the usefulness of mimics of HS (glycomimetics) in protection against β-glycerophosphate-induced vascular calcification and in the restoration of the functional capacity of diabetic endothelial colony-forming cells in vitro. This study aims to investigate whether our novel glycomimetic compounds can attenuate glycated low-density lipoprotein (g-LDL)-induced calcification by inhibiting RAGE signalling within the context of critical limb ischemia (CLI). We used an established osteogenic in vitro vascular smooth muscle cell (VSMC) model. Osteoprotegerin (OPG), sclerostin and glycation levels were all significantly increased in CLI serum compared to healthy controls, while the vascular calcification marker osteocalcin (OCN) was down-regulated in CLI patients vs. controls. Incubation with both CLI serum and g-LDL (10 µg/mL) significantly increased VSMC calcification vs. controls after 21 days, with CLI serum-induced calcification apparent after only 10 days. Glycomimetics (C2 and C3) significantly inhibited g-LDL and CLI serum-induced mineralisation, as shown by a reduction in alizarin red (AR) staining and alkaline phosphatase (ALP) activity. Furthermore, secretion of the osteogenic marker OCN was significantly reduced in VSMCs incubated with CLI serum in the presence of glycomimetics. Phosphorylation of cyclic AMP response element-binding protein (CREB) was significantly increased in g-LDL-treated cells vs. untreated controls, which was attenuated with glycomimetics. Blocking CREB activation with a pharmacological inhibitor 666-15 replicated the protective effects of glycomimetics, evidenced by elevated AR staining. In silico molecular docking simulations revealed the binding affinity of the glycomimetics C2 and C3 with the V domain of RAGE. In conclusion, these findings demonstrate that novel glycomimetics, C2 and C3 have potent anti-calcification properties in vitro, inhibiting both g-LDL and CLI serum-induced VSMC mineralisation via the inhibition of LDLR, RAGE, CREB and subsequent expression of the downstream osteogenic markers, ALP and OCN.
硫酸乙酰肝素 (HS) 可作为细胞表面的协同受体，其在这一过程中的改变是许多病理状况的基础。我们之前已经描述了 HS 类似物 (糖模拟物) 在预防β-甘油磷酸诱导的血管钙化和恢复糖尿病内皮祖细胞体外功能能力方面的有用性。本研究旨在探讨我们的新型糖模拟化合物是否可以通过抑制 RAGE 信号通路来减轻糖基化低密度脂蛋白 (g-LDL) 诱导的临界肢体缺血 (CLI) 中的血管钙化。我们使用了一种已建立的成骨性体外血管平滑肌细胞 (VSMC) 模型。与健康对照组相比，CLI 血清中的骨保护素 (OPG)、骨硬化蛋白和糖化水平均显著升高，而 CLI 患者的血管钙化标志物骨钙素 (OCN) 则降低。与对照组相比，孵育 CLI 血清和 g-LDL(10µg/mL)21 天后明显增加了 VSMC 钙化，而 CLI 血清诱导的钙化在 10 天后即可观察到。糖模拟物 (C2 和 C3) 显著抑制 g-LDL 和 CLI 血清诱导的矿化，如茜素红 (AR) 染色和碱性磷酸酶 (ALP) 活性降低所示。此外，在存在糖模拟物的情况下，用 CLI 血清孵育的 VSMCs 中骨生成标志物 OCN 的分泌显著减少。与未经处理的对照组相比，g-LDL 处理的细胞中环磷酸腺苷反应元件结合蛋白 (CREB) 的磷酸化显著增加，糖模拟物可减弱该作用。用药理学抑制剂 666-15 阻断 CREB 激活可复制糖模拟物的保护作用，这可通过 AR 染色升高来证明。计算机分子对接模拟显示，糖模拟物 C2 和 C3 与 RAGE 的 V 结构域具有结合亲和力。总之，这些发现表明，新型糖模拟物 C2 和 C3 在体外具有强大的抗钙化特性，通过抑制 LDLR、RAGE、CREB 及其下游成骨标志物 ALP 和 OCN 的表达，抑制 g-LDL 和 CLI 血清诱导的 VSMC 矿化。
