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

Abstract

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.

Localized endothelial‐based control of platelet aggregation and coagulation under flow: A proof‐of‐principle vessel‐on‐a‐chip study

“…The heparan sulphates appear to be the most active GAGs with anticoagulant activity, making up 50% to 90% of the endothelial glycocalyx . Degradation of the heparan sulphates by heparinase III in vitro has also been shown by others . With the textbook knowledge that negative charges in the glycocalyx serve to inactivate thrombin in an antithrombin‐dependent way, our results are the first to demonstrate experimentally that heparinase‐induced proteolysis of heparan sulphates can affect the anticoagulant potential of endothelial cells under flow conditions.…”

“…9 Degradation of the heparan sulphates by heparinase III in vitro has also been shown by others. 25,30 With the textbook knowledge that negative charges in the glycocalyx serve to inactivate thrombin in an antithrombin-dependent way, our results are the first to demonstrate experimentally that heparinase-induced proteolysis of heparan sulphates can affect the anticoagulant potential of endothelial cells under flow conditions. Transgenic, heparanase-deficient mice show an increased thrombosis tendency after vascular injury, 32 which is in agreement with the present data.…”

“…Subsequently, we investigated the possible anticoagulant role of glycosaminoglycans in the endothelial glycocalyx. The HUVEC on collagen/TF (coated 500 pmol/L) were pretreated with heparinase III before blood flow, as a manner to cleave off the negatively charged heparan sulphate side chains . This pretreatment did not affect the cell viability (Figure S3).…”

“…Microfluidic chambers with collagen/ TF coating and 40% to 60% coverage of HUVEC were subjected to perfusion with whole blood (DiOC 6 -labeled platelets), followed by recalcified plasma (AF546-fibrinogen), for 10 minutes as described for Figure 2 arinase III before blood flow, as a manner to cleave off the negatively charged heparan sulphate side chains. 25 This pretreatment did not affect the cell viability ( Figure S3). However, in the flow assay, it resulted in a shortening of the time to fibrin formation ( Figure 5A).…”

Metastatic cancer cell attachment to endothelium is promoted by endothelial glycocalyx sialic acid degradation

“…Analysis was performed using NIH ImageJ software. For detailed methods, see SI Materials and Methods and our previously published work …”