Endothelial glycocalyx injury is involved in heatstroke-associated coagulopathy and protected by N-acetylcysteine

Abstract

Introduction: Damage to endothelial glycocalyx (EGCX) can lead to coagulation disorders in sepsis. Heat stroke (HS) resembles sepsis in many aspects; however, it is unclear whether EGCX injury is involved in its pathophysiology. The purpose of this study was to examine the relationship between the damage of EGCX and the development of coagulation disorders during HS.

Methods: We retrospectively collected 159 HS patients and analyzed coagulation characteristics and prognosis of HS patients with or without disseminated intravascular coagulation (DIC). We also replicated a rat HS model and measured coagulation indexes, pulmonary capillary EGCX injury in HS rats. Finally, we evaluated the effect of the antioxidant N-acetylcysteine (NAC) on HS-initiated EGCX injury and coagulation disorders.

Results: Clinical data showed that HS patients complicated with DIC had a higher risk of death than HS patients without DIC. In a rat HS model, we found that rats subjected to heat stress developed hypercoagulability and platelet activation at the core body temperature of 43°C, just before the onset of HS. At 24 h of HS, the rats showed a consumptive hypo-coagulation state. The pulmonary capillary EGCX started to shed at 0 h of HS and became more severe at 24 h of HS. Importantly, pretreatment with NAC substantially alleviated EGCX damage and reversed the hypo-coagulation state in HS rats. Mechanically, HS initiated reactive oxidative species (ROS) generation, while ROS could directly cause EGCX damage. Critically, NAC protected against EGCX injury by attenuating ROS production in heat-stressed or hydrogen peroxide (H₂O₂)-stimulated endothelial cells.

Discussion: Our results indicate that the poor prognosis of HS patients correlates with severe coagulation disorders, coagulation abnormalities in HS rats are associated with the damage of EGCX, and NAC improves HS-induced coagulopathy, probably through its protection against EGCX injury by preventing ROS generation.

Keywords: N-acetylcysteine; coagulopathy; endothelial glycocalyx; heat stroke; hyaluronic acid; syndecan-1.

Figure 1

Flow of the enrollment process for HS patients. A total of 182 heat stroke (HS) patients from June 2009 to December 2020 were screened for eligibility. After the exclusion of 16 patients according to the predetermined criteria and 7 patients with data missing on primary outcome, 159 cases were included and divided into HS with DIC and HS without DIC groups.

Figure 2

Survival curves in HS patients and HS rats. (A) Kaplan–Meier curve shows a significant difference in 28-day survival between heat stroke (HS) patients with DIC (n=77) and without DIC (n=82) (p<0.0001). (B, C) Changes of core body temperature (Tc) and systolic blood pressure (SBP) in rats subjected to heat stress from 0 min to 200 min with an interval of 10 min in comparison with control rats. Arrows indicate the time point of Tc and SBP starting to fall and the onset of HS. (D) Kaplan–Meier curve shows a significant difference in 9-day survival between HS (n=48) and control (n=24) rats (p<0.0001), and the cumulative proportion of deaths in HS rats.

Figure 3

An evolution of abnormal coagulation function is the characteristic in HS rats. (A) The standard coagulation parameters PT, APTT, FIB, and PLT were assessed in rats at a Tc of 41 °C, 42 °C, 43 °C and 0, 2, 6, 24, 72, 216 h after HS (n=6 per group). (B) The trend of changes in viscoelastic coagulation parameters ACT, CR, and PF was measured in rats at a Tc of 41 °C, 42 °C, 43 °C and 0, 2, 6, 24, 72, 216 h after HS (n=6 per group). Due to the large differences in some time points, values represent as fold changes of the baseline (100%) of the control group to improve aesthetics of graphics. *p<0.05, **p<0.01 compared with the control group. PT, prothrombin time; APTT, activated partial thromboplastin time; FIB, fibrinogen; PLT, platelet count; ACT, activated clotting time; CR, clot rate; PF, platelet function.

Figure 4

Substantially elevated circulating coagulation-related biomarkers and proinflammatory cytokines in HS rats. (A) Plasma levels of coagulation-related biomarkers TAT, vWF, PAP, and ET-1 were assessed at 0, 2, 6, 24, 72, and 216 h post HS. (B) At the indicated time points, serum IL-6 and TNF-α concentrations were detected at 0, 2, 6, 24, 72 and 216 h post HS. Data are presented as mean ± SD from six rats per time point. *p<0.05, **p<0.01 compared with the control group. TAT, thrombin-antithrombin complex; vWF, von Willebrand factor; PAP, plasmin-antiplasmin complex; ET-1, endothelin-1.

Figure 5

HS rats display severe EGCX damage and increased release of syndecan-1 and HA in the circulation. The lung tissue section collected from control rats and HS rats at 0 h and 24 h post HS was assessed for pulmonary capillary EGCX under transmission electron microscope (A) and syndecan-1 expression by immunofluorescent staining with anti-syndecan-1 Ab and Alexa Flour 488-conjugated secondary Ab (B). Cell nuclei were stained with Hoche 33258. Results shown represent one experiment from a total of three separate experiments. Bars represent 1 μm in (A) and 100 µm in (B). (C, D) Plasma levels of syndecan-1 and HA were measured at the indicated time points post HS. Data are mean ± SD from six rats per time point. **p<0.01 compared with the control group. EGCX, endothelial glycocalyx; HA, hyaluronan acid.

Figure 6

Pretreatment with NAC ameliorates EGCX injury in HS rats. The lung tissue samples collected from the control, HS, and NAC+HS groups were assessed for pulmonary capillary EGCX under transmission electron microscope (A) and syndecan-1 expression by immunofluorescent staining with anti-syndecan-1 Ab and Alexa Flour 488-conjugated secondary Ab (B). Cell nuclei were stained with Hoche 33258. Results shown represent one experiment from a total of three separate experiments. Bars represent 200 nm in (A) and 25 µm in (B). (C) The thickness of syndecan-1 expression was quantitatively analyzed using Image J software. (D, E) Plasma levels of syndecan-1 and HA were measured at 2 h post HS between the control, HS, and NAC+HS groups. Data are mean ± SD from six rats per group. *p<0.05, **p<0.01 compared with the HS group. NAC, N-acetylcysteine; EGCX, endothelial glycocalyx; HA, hyaluronan acid.

Figure 7

Pretreatment with NAC attenuates proinflammatory cytokine release and improves the abnormal coagulation function in HS rats. Serum IL-6 and TNF-α (A), plasma standard coagulation parameters including PT, APTT, FIB, and PLT (B), and viscoelastic coagulation parameters including ACT, CR, and PF (C) were assessed at 2 h post HS. Data are mean ± SD from six rats per group. *p<0.05, **p<0.01 compared with the HS group. PT, prothrombin time; APTT, activated partial thromboplastin time; FIB, fibrinogen; PLT, platelet count; ACT, activated clotting time; CR, clot rate; PF, platelet function.

Figure 8

NAC prevents the release of vWF from the heat-stressed vascular endothelial cells by alleviating EGCX damage. (A) HUVECs collected from the control, HS, and NAC+HS groups at 2 h, 6 h, and 24 h after HS were assessed for EGCX under transmission electron microscope. Bars represent 200 nm. (B) vWF expression was detected by immunofluorescent staining with anti-vWF Ab and Alexa Flour 488-conjugated secondary Ab. Cell nuclei were stained with DAPI. Results shown represent one experiment from a total of three separate experiments. vWF multimers are normally compactly stored in the W-P bodies as graininess (arrowheads), and vWF ribbons or bands (arrows) are formed when VWF is released from the W-P bodies upon HS. Bars represent 50 μm. (C) vWF levels in culture supernatants collected from the control, HS, and NAC+HS groups were measured at the indicated time points post HS. Data are mean ± SD from six separate experiments per time point. **p<0.01 compared with the control group, #p<0.05, ##p<0.01 compared with the HS group. NAC, N-acetylcysteine; vWF, von Willebrand factor; EGCX, endothelial glycocalyx; HUVECs, human umbilical vein endothelial cells; W-P bodies, Weibel-Palade bodies.

Figure 9

NAC attenuates ROS production in the heat-stressed vascular endothelial cells and protects against EGCX damage in H₂O₂-treated HUVECs. HUVECs pretreated with or without NAC were subjected to HS for 0, 2, 6, and 24 h, and ROS production in the different groups was detected by FACScan analysis (A, B). HUVECs pretreated with or without NAC were incubated with H₂O₂ (100 μM) for 0, 2, 6, and 24 h, and cells collected at the indicated time points were assessed for EGCX under transmission electron microscope (C) and syndecan-1 expression by immunofluorescent staining with anti-syndecan-1 Ab and Alexa Flour 488-conjugated secondary Ab (D, E). Cell nuclei were stained with Hoche 33258. Bars represent 200 nm in (C) and 25 µm in (D). HA (F) and syndecan-1 (G) levels in the culture supernatants collected from the different groups were measured at the indicated time points after HUVECs were incubated with H₂O₂. Data are mean ± SD from six separate experiments per time point. **p<0.01 compared with the control group, ##p<0.01 compared with the HS group or H₂O₂ group. NAC, N-acetylcysteine; EGCX, endothelial glycocalyx; HUVECs, human umbilical vein endothelial cells; H₂O₂, hydrogen peroxide; ROS, reactive oxygen species; HA, hyaluronic acid.