Ascorbic acid attenuates endothelial permeability triggered by cell-free hemoglobin

Abstract

Background: Increased endothelial permeability is central to shock and organ dysfunction in sepsis but therapeutics targeted to known mediators of increased endothelial permeability have been unsuccessful in patient studies. We previously reported that cell-free hemoglobin (CFH) is elevated in the majority of patients with sepsis and is associated with organ dysfunction, poor clinical outcomes and elevated markers of oxidant injury. Others have shown that Vitamin C (ascorbate) may have endothelial protective effects in sepsis. In this study, we tested the hypothesis that high levels of CFH, as seen in the circulation of patients with sepsis, disrupt endothelial barrier integrity.

Methods: Human umbilical vein endothelial cells (HUVEC) were grown to confluence and treated with CFH with or without ascorbate. Monolayer permeability was measured by Electric Cell-substrate Impedance Sensing (ECIS) or transfer of ¹⁴C-inulin. Viability was measured by trypan blue exclusion. Intracellular ascorbate was measured by HPLC.

Results: CFH increased permeability in a dose- and time-dependent manner with 1 mg/ml of CFH increasing inulin transfer by 50% without affecting cell viability. CFH (1 mg/ml) also caused a dramatic reduction in intracellular ascorbate in the same time frame (1.4 mM without CFH, 0.23 mM 18 h after 1 mg/ml CFH, p < 0.05). Pre-treatment of HUVECs with ascorbate attenuated CFH induced permeability.

Conclusions: CFH increases endothelial permeability in part through depletion of intracellular ascorbate. Supplementation of ascorbate can attenuate increases in permeability mediated by CFH suggesting a possible therapeutic approach in sepsis.

Keywords: Ascorbic acid; Cell-free hemoglobin; Endothelial permeability; Oxidative stress.

Figure 1. Cell-free hemoglobin decreases electrical resistance across an endothelial cell monolayer

HUVECs were cultured to confluence on ECIS plates and electrical resistance was measured at 4,000 hz over time in response to 1 mg/mL CFH (A). In addition, CFH decreased electrical resistance at 18 hours in a dose-dependent manner (B). **p<0.05 by Mann Whitney U at 18 hours, *p < 0.05 by ANOVA.

Figure 2. Cell-free hemoglobin increases endothelial macromolecular permeability without altering viability

HUVECs were cultured to confluence on transwell plates and treated with cell-free hemoglobin (CFH, 0–1 mg/ml) for 18 h. CFH increased transfer of ¹⁴C-inulin (A) in a dose-dependent manner. Cell viability as measured by Trypan blue exclusion was not affected by CFH treatment for 18 hours (B). *p < 0.05 by ANOVA.

Figure 3. Cell-free hemoglobin depletes intracellular ascorbate

Confluent HUVECs were challenged with CFH (0–1 mg/ml) in antioxidant-free medium for 18 h, then intracellular ascorbate was measured by HPLC. *p < 0.05 by ANOVA.

Figure 4. Ascorbate prevents increases in endothelial permeability caused by CFH

HUVECs were cultured to confluence in antioxidant-free medium on transwell plates and loaded with ascorbate (0–60 uM) for 15 min prior to challenge with cell-free hemoglobin (CFH, 0.3 mg/ml) for 18 h. Ascorbate significantly blocked CFH mediated decrease in electrical resistance (A) and the increased transfer of ¹⁴C-inulin (B). *p < 0.05 by ANOVA.