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Syndecan ectodomain fragments decrease endothelial resistance via rho kinase.

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posted on 2019-05-15, 17:25 authored by Melanie Jannaway, Xiaoyuan Yang, Jamie E. Meegan, Danielle C. Coleman, Sarah Y. Yuan

The ability of syndecan ectodomains or their fragments generated following thrombin cleavage to modulate changes in endothelial barrier resistance was assessed using an electric cell-substrate impedance sensing assay, which measures changes in endothelial barrier resistance. (A) Intact rhS3ED (n = 4, p = 0.95) and rhS4ED (n = 4, p = 0.55) had no effect on transendothelial electrical resistance (TER). (B) rhS3ED fragments induced a highly significant drop in TER (n = 6, p<0.0001). (C) rhS4ED fragments significantly decreased TER (n = 8, p = 0.0002). (D) Fragments of non-glycosylated form of rhS4ED also generated significant decreases in TER (n = 5, p = 0.0011). (E) Pretreatment with Y27632 (10 μM, 1hr) significantly inhibits S3ED fragment-induced decreases in peak TER change (n = 4, p = 0.0045). (F) Pretreatment with Y27632 (10 μM, 1hr) significantly inhibits S4ED fragment-induced decreases in peak TER change (n = 4, p = 0.01). (G) Because thrombin is still present in the syndecan ectodomain fragment mixture, albeit at the low concentration of 0.5 U/ml, the ECIS assays were carried out following pre-treatment with the PAR1 receptor antagonist, RWJ56110. In the presence of RWJ56110, rhS3ED fragments mediated a significant decrease in TER response (n = 3, p<0.0001). (H) Following pre-treatment with RWJ56110, rhS4ED fragments induced a significant decrease in TER (n = 3, p<0.01). (I) S3ED (n = 4, p<0.0001) and S4ED (n = 4, p<0.0001) fragments significantly decrease TER response in human lung microvascular endothelial cells. Furthermore, S3ED fragments have a significantly bigger effect on TER than S4ED fragments (n = 4, p<0.0001). (J) TLR4 inhibitor, TAK-242, had no effect of the barrier disrupting effects of the S3ED (p = ns, n = 3) and (K) S4ED fragments (p = ns, n = 3).

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