Department of Biomedical Engineering, The City College of New York, New York, New York; and.
Am J Physiol Heart Circ Physiol. 2014 Feb;306(3):H363-72. doi: 10.1152/ajpheart.00687.2013. Epub 2013 Nov 27.
Endothelial cells (ECs) are covered by a surface glycocalyx layer that forms part of the barrier and mechanosensing functions of the blood-tissue interface. Removal of albumin in bathing media induces collapse or shedding of the glycocalyx. The electrostatic interaction between arginine residues on albumin, and negatively charged glycosaminoglycans (GAGs) in the glycocalyx have been hypothesized to stabilize the glycocalyx structure. Because albumin is one of the primary carriers of the phospholipid sphingosine-1-phosphate (S1P), we evaluated the alternate hypothesis that S1P, acting via S1P1 receptors, plays the primary role in stabilizing the endothelial glycocalyx. Using confocal microscopy on rat fat-pad ECs, we demonstrated that heparan sulfate (HS), chondroitin sulfate (CS), and ectodomain of syndecan-1 were shed from the endothelial cell surface after removal of plasma protein but were retained in the presence of S1P at concentrations of >100 nM. S1P1 receptor antagonism abolished the protection of the glycocalyx by S1P and plasma proteins. S1P reduced GAGs released after removal of plasma protein. The mechanism of protection from loss of glycocalyx components by S1P-dependent pathways was shown to be suppression of metalloproteinase (MMP) activity. General inhibition of MMPs protected against loss of CS and syndecan-1. Specific inhibition of MMP-9 and MMP-13 protected against CS loss. We conclude that S1P plays a critical role in protecting the glycocalyx via S1P1 and inhibits the protease activity-dependent shedding of CS, HS, and the syndecan-1 ectodomain. Our results provide new insight into the role for S1P in protecting the glycocalyx and maintaining vascular homeostasis.
内皮细胞 (ECs) 被表面糖萼层覆盖，该层构成了血液组织界面屏障和机械感应功能的一部分。在孵育介质中去除白蛋白会导致糖萼层的塌陷或脱落。白蛋白上的精氨酸残基与糖萼层中带负电荷的糖胺聚糖 (GAG) 之间的静电相互作用被假设为稳定糖萼层结构。由于白蛋白是磷脂鞘氨醇-1-磷酸 (S1P) 的主要载体之一，我们评估了替代假设，即 S1P 通过 S1P1 受体发挥主要作用稳定内皮糖萼。通过在大鼠脂肪垫 ECs 上进行共聚焦显微镜检查，我们证明在去除血浆蛋白后，肝素硫酸盐 (HS)、软骨素硫酸盐 (CS) 和 syndecan-1 的外显子从内皮细胞表面脱落，但在浓度>100 nM 的 S1P 存在下保留。S1P1 受体拮抗作用消除了 S1P 和血浆蛋白对糖萼的保护作用。S1P 降低了去除血浆蛋白后释放的 GAG。S1P 通过依赖于 S1P 的途径保护糖萼成分免于丢失的机制被证明是抑制金属蛋白酶 (MMP) 活性。MMP 的一般抑制可防止 CS 和 syndecan-1 的丢失。特异性抑制 MMP-9 和 MMP-13 可防止 CS 丢失。我们得出结论，S1P 通过 S1P1 在保护糖萼中发挥关键作用，并抑制 CS、HS 和 syndecan-1 外显子的蛋白酶活性依赖性脱落。我们的研究结果为 S1P 在保护糖萼和维持血管内稳态中的作用提供了新的见解。
