Fibroblast Growth Factor Signaling Mediates Pulmonary Endothelial Glycocalyx Reconstitution

Abstract

The endothelial glycocalyx is a heparan sulfate (HS)-rich endovascular structure critical to endothelial function. Accordingly, endothelial glycocalyx degradation during sepsis contributes to tissue edema and organ injury. We determined the endogenous mechanisms governing pulmonary endothelial glycocalyx reconstitution, and if these reparative mechanisms are impaired during sepsis. We performed intravital microscopy of wild-type and transgenic mice to determine the rapidity of pulmonary endothelial glycocalyx reconstitution after nonseptic (heparinase-III mediated) or septic (cecal ligation and puncture mediated) endothelial glycocalyx degradation. We used mass spectrometry, surface plasmon resonance, and in vitro studies of human and mouse samples to determine the structure of HS fragments released during glycocalyx degradation and their impact on fibroblast growth factor receptor (FGFR) 1 signaling, a mediator of endothelial repair. Homeostatic pulmonary endothelial glycocalyx reconstitution occurred rapidly after nonseptic degradation and was associated with induction of the HS biosynthetic enzyme, exostosin (EXT)-1. In contrast, sepsis was characterized by loss of pulmonary EXT1 expression and delayed glycocalyx reconstitution. Rapid glycocalyx recovery after nonseptic degradation was dependent upon induction of FGFR1 expression and was augmented by FGF-promoting effects of circulating HS fragments released during glycocalyx degradation. Although sepsis-released HS fragments maintained this ability to activate FGFR1, sepsis was associated with the downstream absence of reparative pulmonary endothelial FGFR1 induction. Sepsis may cause vascular injury not only via glycocalyx degradation, but also by impairing FGFR1/EXT1-mediated glycocalyx reconstitution.

Circulating Heparan Sulfate Fragments Attenuate Histone-Induced Lung Injury Independently of Histone Binding

“…In contrast, HS oligosaccharides prevented CTH-induced cytotoxicity in a size-independent (Fig 6c) fashion. These protective effects were similarly observed using primary human lung microvascular endothelial cells, supporting the previously-demonstrated (10) relevance of HPMEC-ST1.6R cells (Fig 6d). The HPMEC-ST1.6R cytoprotective effects of HS were sulfation-dependent (Fig 6e).…”

“…As circulating HS fragments in sepsis are highly-sulfated, we used heparin (a highly-sulfated HS) oligosaccharides to model septic circulating HS fragments, as previously described (10). As demonstrated by representative isotherms in Fig 1, CTH bound heparin fragments 10 saccharides in length (degree of polymerization 10, dp10) or longer.…”

“…HPMEC-ST1.6R cells, a human pulmonary microvascular endothelial cell line, were generously provided by Dr. Ronald Unger (University of Mainz), the originator of the cell line (11). We have previously demonstrated the relevance of this transformed cell line to primary lung endothelial cells (10). The TLR4 inhibitor TAK-242 was purchased from Calbiochem/EMD Millipore (Billerica, MA); the TLR2 inhibitor CU CPT 22 was purchased from Tocris (Bristol, UK).…”

“…To model highly-sulfated circulating HS fragments, we purchased heparin (a highly-sulfated HS) and selectively desulfated heparin oligosaccharides of various sizes from Iduron (Manchester, UK), as previously described (10). Calf thymus histones (CTH, a mixture of histones H1, H2A, H2B, H3, and H4), histone H1, and histone H3 were purchased from Sigma-Aldrich (St. Louis, MO).…”

“…We have previously identified that plasma HS fragments may persist in humans for up to 5 days after ICU admission for respiratory failure (8). These circulating fragments are highly sulfated (similar to heparin, a highly-sulfated HS (9)) and are typically larger than 6 saccharides in length (degree of polymerization 6, dp6), structural characteristics indicative of an ability to bind positively-charged residues of soluble growth factors (10). We hypothesized that circulating HS would similarly intercept circulating histones and neutralize their cytotoxic effect, thereby serving as an endogenous defense against histone-induced lung injury.…”