Circulating Cell-free Hemoglobin Upregulates Active Heparanase to Drive Endothelial Glycocalyx Destruction and Inflammation in Sepsis

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Abstract Rationale Endothelial injury is a hallmark of sepsis pathophysiology. Endothelial barrier integrity is partially regulated by the endothelial glycocalyx, a matrix of proteoglycans and glycosaminoglycans (GAGs) lining the vascular lumen. However, in sepsis, this glycocalyx is degraded by heparanase; glycocalyx breakdown is linked to worse sepsis outcomes, but the mechanisms inducing heparanase activity in sepsis are unknown. We previously found that plasma levels of hemoglobin released from red blood cells (cell-free hemoglobin, CFH) are elevated in clinical sepsis and associate with worse outcomes. We also showed that CFH is associated with increased glycocalyx degradation in septic humans and mice (Bogart, ATS 2024). We therefore hypothesized that CFH upregulates heparanase production to drive glycocalyx shedding in sepsis. Methods To test the effect of CFH on heparanase expression in mice after 24 hours of intraabdominal polymicrobial sepsis with elevated CFH (CS [cecal slurry, 2.0mg/g] + intravenous [IV] CFH, 0.15mg/g), we quantified total plasma heparanase (ELISA). We also measured heparanase transcript abundance (RT-qPCR) and active enzyme levels (western blot, WB) in pulmonary endothelial cells isolated from these animals. Additionally, primary human lung microvascular endothelial cells (HLMVECs) treated with tumor necrosis factor alpha (TNFα, 50ng/mL) to simulate sepsis were exposed to CFH (1.0mg/mL) with or without the hemoprotein reductant acetaminophen (APAP, 24mcg/mL) for 24 hours to investigate the mechanism by which CFH may induce heparanase synthesis. Results Total plasma heparanase levels were higher in the CS+CFH treated animals compared to CS alone (430.5±98.9 vs. 61.8±9.6 pg/mL, p=0.001, Figure A), consistent with our prior data that circulating glycocalyx components are elevated in these animals. Heparanase transcript abundance (Relative expression [RE]: 3.57±0.20 vs. 1.00±0.02, p=0.022) and active enzyme levels (RE: 1.55±0.07 vs. 0.73±0.09, p=0.024, Figure B) were also increased in mice receiving CS+CFH. HLMVECs treated with TNFα+CFH had elevated heparanase mRNA (RE vs. TNFα control: 3.26±0.47 vs. 0.40±0.10, p=0.046) and active protein production (RE vs. TNFα control: 2.70±0.43 vs. 0.42±0.15, p=0.033, Figure C). The effect of CFH on heparanase transcription (RE: 0.57±0.16, p=0.047) and enzyme activation (RE: 0.23±0.07, p=0.046, Figure C) was blocked by APAP. Conclusions In sepsis, CFH induces heparanase transcription in lung microvascular endothelial cells, leading to active enzyme synthesis and subsequent endothelial glycocalyx degradation, which may disrupt the endothelial barrier and cause downstream organ injury. Heparanase induction can be blocked by APAP, a hemoprotein reductant, indicating that the effects of CFH on heparanase induction are driven by the oxidant effects of CFH.

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