Data from: Regeneration of glycocalyx by heparan sulfate and sphingosine 1-phosphate restores inter-endothelial communication

Vasculoprotective endothelium glycocalyx (GCX) shedding plays a critical role in vascular disease. Previous work demonstrated that GCX degradation disrupts endothelial cell (EC) gap junction connexin (Cx) proteins, likely blocking interendothelial molecular transport that maintains EC and vascular tissue homeostasis to resist disease. Here, we focused on GCX regeneration and tested the hypothesis that vasculoprotective EC function can be stimulated via replacement of GCX when it is shed. We used EC with [i] intact heparan sulfate (HS), the most abundant GCX component; [ii] degraded HS; or [iii] HS that was restored after enzyme degradation, by cellular self-recovery or artificially. Artificial HS restoration was achieved via treatment with exogenous HS, with or without the GCX regenerator and protector sphingosine 1- phosphate (S1P). In these cells we immunocytochemically examined expression of Cx isotype 43 (Cx43) at EC borders and characterized Cx-containing gap junction activity by measuring interendothelial spread of gap junction permeable Lucifer Yellow dye. With intact HS, 60% of EC borders expressed Cx43 and dye spread to 2.88 ± 0.09 neighboring cells. HS degradation decreased Cx43 expression to 30% and reduced dye spread to 1.87± 0.06 cells. Cellular self-recovery of HS restored baseline levels of Cx43 and dye transfer. Artificial HS recovery with exogenous HS partially restored Cx43 expression to 46% and yielded dye spread to only 1.03 ± 0.07 cells. Treatment with both HS and S1P, recovered HS and restored Cx43 to 56% with significant dye transfer to 3.96 ± 0.23 cells. This is the first evidence of GCX regeneration in a manner that effectively restores vasculoprotective EC communication.

血管保护性内皮糖萼（glycocalyx, GCX）脱落与血管疾病的发生发展密切相关。既往研究显示，GCX降解会破坏内皮细胞（endothelial cell, EC）间隙连接的连接蛋白（connexin, Cx），或可阻断维持内皮细胞与血管组织稳态以抵御疾病的内皮细胞间分子转运过程。本研究聚焦于GCX再生，验证了当GCX脱落后，通过补充GCX可刺激血管保护性内皮细胞功能这一假说。本研究采用三类内皮细胞模型：[i] 完整表达硫酸乙酰肝素（heparan sulfate, HS，GCX中含量最丰富的组分）的细胞；[ii] HS发生酶促降解的细胞；以及[iii] 经酶降解后通过细胞自主修复或人工手段恢复HS表达的细胞。人工HS恢复可通过外源性HS处理实现，可联合或不联合GCX的再生与保护因子鞘氨醇1-磷酸（sphingosine 1-phosphate, S1P）。针对上述细胞，我们采用免疫细胞化学法检测了内皮细胞边界处连接蛋白亚型43（connexin isotype 43, Cx43）的表达水平，并通过检测间隙连接可通透的荧光黄染料（Lucifer Yellow dye）在内皮细胞间的扩散程度，表征了含连接蛋白的间隙连接的功能活性。当HS完整表达时，60%的内皮细胞边界可检测到Cx43表达，荧光黄染料可扩散至2.88 ± 0.09个邻近细胞。HS降解使Cx43阳性细胞边界占比降至30%，染料扩散范围缩减至1.87 ± 0.06个细胞。通过细胞自主修复恢复HS表达后，Cx43表达与染料传递效率均可恢复至基线水平。仅采用外源性HS进行人工HS恢复时，仅能将Cx43阳性占比部分恢复至46%，染料扩散范围仅为1.03 ± 0.07个细胞。而联合使用HS与S1P进行处理时，可有效提升HS恢复效果，Cx43阳性占比恢复至56%，且染料扩散范围显著提升至3.96 ± 0.23个细胞。本研究首次证实，通过GCX再生可有效恢复血管保护性内皮细胞间的通讯功能。