Human subjects. Human samples and accompanying clinical data were collected from ICU patients enrolled in the NIRFS study, a substudy of the MESSI cohort, which is a single-center, prospective cohort of patients admitted to the ICU at the Hospital of the University of Pennsylvania. This study was approved by the IRB of the University of Pennsylvania. Subjects or their available surrogates provided written informed consent.

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.

Sepsis induces heparanase-mediated degradation of the endothelial glycocalyx, a heparan sulfate-enriched endovascular layer critical to vascular homeostasis, releasing highly sulfated domains of heparan sulfate into the circulation. These domains are oligosaccharides rich in heparin-like trisulfated disaccharide repeating units. Using a chemoenzymatic approach, an undecasaccharide containing a uniformly 13C-labeled internal 2-sulfoiduronic acid residue was synthesized on a p-nitrophenylglucuronide acceptor. Selective periodate cleavage afforded a heparin nonasaccharide having a natural structure. This 13C-labeled nonasaccharide was intravenously administered to septic (induced by cecal ligation and puncture, a model of polymicrobial peritonitis-induced sepsis) and nonseptic (sham) mice. Selected tissues and biological fluids from the mice were harvested at various time points over 4 hours, and the 13C-labeled nonasaccharide was recovered and digested with heparin lyases. The resulting 13C-labeled trisulfated disaccharide was quantified, without interference from endogenous mouse heparan sulfate/heparin, using liquid chromatography–mass spectrometry with sensitive and selective multiple reaction monitoring. The 13C-labeled heparin nonasaccharide appeared immediately in the blood and was rapidly cleared through the urine. Plasma nonasaccharide clearance was only slightly prolonged in septic mice (t1/2 ∼ 90 minutes). In septic mice, the nonasaccharide penetrated into the hippocampus but not the cortex of the brain; no hippocampal or cortical brain penetration occurred in sham mice. The results of this study suggest that circulating heparan sulfates are rapidly cleared from the plasma during sepsis and selectively penetrate the hippocampus, where they may have functional consequences.

Extracellular histones are cationic damage-associated molecular pattern molecules capable of directly inducing cellular injury via charge-mediated interactions with plasma membranes. Accordingly, histones released into the plasma during critical illness are known to contribute to the onset and propagation of lung injury. Vascular injury (with consequent degradation of the endothelial glycocalyx) simultaneously releases anionic heparan sulfate fragments (hexa- to octasaccharides in size) into the plasma. It is unknown whether this endogenous release of heparan sulfate fragments modulates charge-dependent histone cytotoxicity, or if exogenous heparan sulfate fragments could therapeutically attenuate histone-induced lung injury. Using isothermic calorimetry, we found that extracellular histones only bind to heparan sulfate fragments ≥ 10 saccharides in size, suggesting that glycocalyx-derived heparan sulfate hexa/octasaccharides are incapable of intercepting/neutralizing circulating histones. However, we found that even heparan sulfate fragments incapable of histone binding (e.g. tetrasaccharides) attenuated histone-induced lung injury in vivo, suggesting a direct, size-independent protective effect of heparan sulfate. We found that histones had no effect on human neutrophils ex vivo but exerted toll-like receptor-independent cytotoxicity on human pulmonary microvascular endothelial cells in vitro. This cytotoxicity could be prevented by either the addition of negatively-charged (i.e. highly-sulfated) heparan sulfate tetrasaccharides (incapable of binding histones) or decasaccharides (capable of binding histones). Taken together, our findings suggest that heparan sulfate oligosaccharides may directly exert pulmonary endothelial-protective effects that attenuate histone-mediated lung injury.

Sepsis outcomes are heavily dependent on the development of septic organ injury, but no interventions exist to interrupt or reverse this process. microRNA-223 (miR-223) is known to be involved in both inflammatory gene regulation and host-pathogen interactions key to the pathogenesis of sepsis. The goal of this study was to determine the role of miR-223 as a mediator of septic kidney injury. Using miR-223 knockout mice and multiple models of experimental sepsis, we found that miR-223 differentially influences acute kidney injury (AKI) based on the model used. In the absence of miR-223, mice demonstrated exaggerated AKI in sterile models of sepsis (LPS injection) and attenuated AKI in a live-infection model of sepsis (cecal ligation and puncture). We demonstrated that miR-223 expression is induced in kidney homogenate after cecal ligation and puncture, but not after LPS or fecal slurry injection. We investigated additional potential mechanistic explanations including differences in peritoneal bacterial clearance and host stool virulence. Our findings highlight the complex role of miR-223 in the pathogenesis of septic kidney injury, as well as the importance of differences in experimental sepsis models and their consequent translational applicability.