Department of Bioengineering, The Pennsylvania State University, University Park, PA 16802, USA.
Microvasc Res. 2010 Dec;80(3):394-401. doi: 10.1016/j.mvr.2010.06.005. Epub 2010 Jun 21.
The endothelial glycocalyx is well endowed with the glycosaminoglycans (GAGs) heparan sulfate, chondroitin sulfate and hyaluronan. The current studies aimed to assess the relative contributions of each of these GAGs to the thickness and permeability of the glycocalyx layer by direct enzymatic removal of each using micropipettes to infuse heparinase, chondroitinase and hyaluronidase into post-capillary venules of the intestinal mesentery of the rat. The relative losses of GAGs due to enzymatic removal were compared with stimulated shedding of glycans induced by superfusing the mesentery with 10(-)(7)M fMLP. Thickness of the glycocalyx was assessed by infiltration of the glycocalyx with circulating FITC labeled 70kDa dextran (Dx70) and measuring the distance from the dye front to the surface of the endothelium (EC), which averaged 463nm under control conditions. Reductions in thickness were 43.3%, 34.1% and 26.1% following heparinase, chondroitinase and hyaluronidase, respectively, and 89.7% with a mixture of all three enzymes. Diffusion coefficients of FITC in the glycocalyx were determined using a 1-D diffusion model. By comparison of measured transients in radial intensity of a bolus of FITC with that of a computational model a diffusion coefficient D was obtained. Values of D were obtained corresponding to the thickness of the layer demarcated by Dx70 (D(Dx70)), and a smaller sublayer 173nm above the EC surface (D(173)), prior to and following enzyme infusion and superfusion with fMLP. The magnitude of D(Dx70) was twice that of D(173) suggesting that the glycocalyx is more compact near the EC surface. Chondroitinase and hyaluronidase significantly increased both D(Dx70) and D(173). However, heparinase decreased D(Dx70), and did not induce any significant change for the D(173). These observations suggest that the three GAGs are not evenly distributed throughout the glycocalyx and that they each contribute to permeability of the glycocalyx to a differing extent. The fMLP-induced shedding caused a reduction in glycocalyx thickness (which may increase permeability) and as with heparinase, decreased the diffusion coefficient of solutes (which may decrease permeability). This behavior suggests that the removal of heparan sulfate may cause a collapse of the glycocalyx which counters decreases in thickness by compacting the layer to maintain a constant resistance to filtration.
内皮糖萼富含糖胺聚糖 (GAGs) 肝素、硫酸软骨素和透明质酸。目前的研究旨在通过使用微吸管直接酶去除每种 GAG，评估它们各自对糖萼层厚度和通透性的相对贡献，将肝素酶、软骨素酶和透明质酸酶注入大鼠肠肠系膜的毛细血管后静脉。将由于酶去除而导致的 GAG 相对损失与用 10(-)(7)M fMLP 超滤液刺激糖萼聚糖脱落进行比较。糖萼的厚度通过循环 FITC 标记的 70kDa 葡聚糖 (Dx70) 渗透到糖萼中来评估，并测量染料前沿到内皮 (EC) 表面的距离，在对照条件下平均为 463nm。用肝素酶、软骨素酶和透明质酸酶处理后，厚度分别减少了 43.3%、34.1%和 26.1%，而用三种酶的混合物处理则减少了 89.7%。使用一维扩散模型确定 FITC 在糖萼中的扩散系数。通过比较 FITC bolus 的径向强度测量瞬变与计算模型，获得扩散系数 D。在酶输注和 fMLP 超滤液之前和之后，获得与 Dx70 标记的层厚度相对应的 D 值 (D(Dx70))，以及 EC 表面上方 173nm 的较小亚层 (D(173))。D(Dx70)的值是 D(173)的两倍，这表明糖萼在靠近 EC 表面的地方更加紧凑。软骨素酶和透明质酸酶显著增加了 D(Dx70)和 D(173)。然而，肝素酶降低了 D(Dx70)，并且没有引起 D(173)的任何显著变化。这些观察结果表明，这三种 GAG 并非均匀分布在糖萼中，它们各自对糖萼的通透性的贡献程度不同。fMLP 诱导的脱落导致糖萼厚度减小（这可能增加通透性），并且与肝素酶一样，降低了溶质的扩散系数（这可能降低通透性）。这种行为表明，肝素硫酸盐的去除可能导致糖萼塌陷，通过使层致密化来抵消厚度的减小，从而保持对过滤的恒定阻力。
