heparitin-sulfate and sphingosine-1-phosphate

The damage of the endothelial glycocalyx as a consequence of ischemia and/or reperfusion injury (IRI) following kidney transplantation has come at the spotlight of research due to potential associations with delayed graft function, acute rejection as well as long-term allograft dysfunction. The disintegration of the endothelial glycocalyx induced by IRI is the crucial event which exposes the denuded endothelial cells to further inflammatory and oxidative damage. The aim of our review is to present the currently available data regarding complex links between shedding of the glycocalyx components, like syndecan-1, hyaluronan, heparan sulphate, and CD44 with the activation of intricate immune system responses, including toll-like receptors, cytokines and pro-inflammatory transcription factors. Evidence on modes of protection of the endothelial glycocalyx and subsequently maintenance of endothelial permeability as well as novel nephroprotective molecules such as sphingosine-1 phosphate (S1P), are also depicted. Although advances in technology are making the visualization and the analysis of the endothelial glycocalyx possible, currently available evidence is mostly experimental. Ongoing progress in understanding the complex impact of IRI on the endothelial glycocalyx, opens up a new era of research in the field of organ transplantation and clinical studies are of utmost importance for the future.

Topics: Endothelium; Glycocalyx; Heparitin Sulfate; Humans; Hyaluronic Acid; Ischemia; Kidney; Kidney Transplantation; Lysophospholipids; Reperfusion Injury; Sphingosine

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.

Topics: Animals; Cell Communication; Cells, Cultured; Connexin 43; Endothelial Cells; Gap Junctions; Gene Expression Regulation; Glycocalyx; Heparitin Sulfate; Humans; Lysophospholipids; Rats; Sphingosine

Sphingosine 1-phosphate (S1P) protects glycocalyx against shedding, playing important roles in endothelial functions. We previously found that glycocalyx on endothelial cells (ECs) was shed after plasma protein depletion. In the present study, we investigated the role of S1P on the recovery of glycocalyx, and tested whether it is mediated by phosphoinositide 3-kinase (PI3K) pathway. After depletion of plasma protein, ECs were treated with S1P for another 6h. And then, the major components of glycocalyx including syndecan-1 with attached heparan sulfate (HS) and chondroitin sulfate (CS) on endothelial cells were detected using confocal fluorescence microscopy. Role of PI3K in the S1P-induced synthesis of glycocalyx was confirmed by using the PI3K inhibitor (LY294002). Syndecan-1 with attached HS and CS were degraded with duration of plasma protein depletion. S1P induced recovery of syndecan-1 with attached HS and CS. The PI3K inhibitor LY294002 abolished the effect of S1P on recovery of glycocalyx. Thus, S1P induced synthesis of glycocalyx on endothelial cells and it is mediated by PI3K pathway.

Topics: Animals; Cells, Cultured; Chondroitin Sulfates; Endothelium, Vascular; Fluorescent Antibody Technique; Glycocalyx; Heparitin Sulfate; Lysophospholipids; Microscopy, Confocal; Phosphatidylinositol 3-Kinases; Rats; Sphingosine; Syndecan-1

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.

Topics: Adipose Tissue; Animals; Cells, Cultured; Chondroitin Sulfates; Endothelial Cells; Glycocalyx; Heparitin Sulfate; Lysophospholipids; Matrix Metalloproteinase 13; Matrix Metalloproteinase 9; Rats; Sphingosine; Syndecan-1

Sphingosine 1-phosphate (S1P), a lysophospholipid, plays an important chemotactic role in the migration of lymphocytes and germ cells, and is known to regulate aspects of central nervous system development such as neurogenesis and neuronal migration. Its role in axon guidance, however, has not been examined. We show that sphingosine kinase 1, an enzyme that generates S1P, is expressed in areas surrounding the Xenopus retinal axon pathway, and that gain or loss of S1P function in vivo causes errors in axon navigation. Chemotropic assays reveal that S1P elicits fast repulsive responses in retinal growth cones. These responses require heparan sulfate, are sensitive to inhibitors of proteasomal degradation, and involve RhoA and LIM kinase activation. Together, the data identify downstream components that mediate S1P-induced growth cone responses and implicate S1P signalling in axon guidance.

Topics: Animals; Axons; Embryo, Nonmammalian; Enzyme Activation; Gene Expression Regulation, Developmental; Growth Cones; Heparitin Sulfate; Humans; Lim Kinases; Lysophospholipids; Mice; Phosphotransferases (Alcohol Group Acceptor); Proteasome Inhibitors; Receptors, Lysosphingolipid; Retina; rhoA GTP-Binding Protein; Signal Transduction; Sphingosine; Visual Pathways; Xenopus laevis