Heparan sulfate mimetic fucoidan restores the endothelial glycocalyx and protects against dysfunction induced by serum of COVID-19 patients in the intensive care unit

Loss of glycocalyx in primary human pulmonary microvascular endothelial cells in the presence of serum of coronavirus disease 2019 (COVID-19) patients in the intensive care unit (ICU). a) Representative fluorescence (FL) confocal images of Lycopersicon esculentum (LEA)-fluorescein isothiocyanate (FITC) or anti-thrombomodulin (TM) staining on the surface of primary human pulmonary microvascular endothelial cells (HPMECs) in the presence of 10% serum of pooled healthy controls (n=12), COVID-19 non-ICU (n=8) and COVID-19 ICU (n=26) samples for 24 h. HPMEC surface expression quantification of b) LEA-FITC and c) TM in the presence of 10% pooled healthy control, COVID-19 non-ICU and COVID-19 ICU serum for 24 h. All values are given as mean±sd of four independent experiments. One-way ANOVA followed by Tukey's multiple comparisons test was performed. ***: p<0.001.

Serum mediators of coronavirus disease 2019 (COVID-19) patients in the intensive care unit (ICU) induce microvascular barrier disruption. Barrier integrity parameter, resistance of a and b) primary human pulmonary microvascular endothelial cells (HPMECs) and c and d) primary human glomerular microvascular endothelial cells (GEnCs) assessed by electric cell-substrate impedance sensing system (ECIS Zθ) in response to stimulation with 10% serum (at t=0 h) of healthy controls (n=12), COVID-19 non-ICU (n=8) and COVID-19 ICU (n=26). Cell–cell contact parameter, Rb of e and f) HPMECs and g and h) GEnCs assessed by ECIS Zθ in response to stimulation with 10% serum (at t=0 h) of healthy controls (n=12), COVID-19 non-ICU (n=8) and COVID-19 ICU (n=26). In some cases, multiple samples (in ICU and out of ICU) from the same patient at different time points were obtained. Data of raw resistance and Rb values were shown and presented as mean±sem. Quantification of barrier integrity was based on the measurements of area under the curve (AUC) over the final 15 h of b) HPMECs and d) GEnCs. Quantification of cell–cell contact was based on the measurements of AUC over the final 15 h of f) HPMECs and h) GEnCs. i) Representative confocal images of VE-cadherin (green) and F-actin (red) staining on HPMECs in the presence of 10% pooled healthy control (n=12), COVID-19 non-ICU (n=8) and COVID-19 ICU (n=26) serum for 24 h (scale bar=20 µm). j) Quantification of adhesion junction percentage of HPMECs in the presence of 10% pooled healthy control, COVID-19 non-ICU and COVID-19 ICU serum for 24 h of four independent experiments. Graphs represent the mean±sd. k) Pearson's correlation heatmap between endothelial dysfunction and glycocalyx shedding-related markers (angiopoietin 2 (ANG2), soluble thrombomodulin (sTM) and soluble syndecan-1 (sSDC1)) and barrier function-related parameters (last 15 h AUC of R and Rb in HPMECs and GEnCs). Colours represent correlation, blue means negative correlation and red means positive correlation. Blank means no significance. One-way ANOVA followed by Tukey's multiple comparisons test and Kruskal–Wallis test followed by Dunn's multiple comparisons test were performed. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

Fucoidan restores glycocalyx thickness on human pulmonary microvascular endothelial cells (HPMECs) and reduces endothelial activation. a) Representative confocal images of Lycopersicon esculentum (LEA)-fluorescein isothiocyanate (FITC) or anti-thrombomodulin (TM) staining on the surface of primary HPMECs in the presence of 10% pooled coronavirus disease 2019 (COVID-19) intensive care unit (ICU) (n=26) serum with and without fucoidan (10 µg mL⁻¹) for 24 h. Quantification of HPMECs surface b) LEA and c) TM expression in the presence of 10% pooled COVID-19 ICU serum with and without fucoidan (10 µg mL⁻¹) for 24 h of four independent experiments. d) Western blot images of intercellular adhesion molecule (ICAM)1, p-P65, t-P65 protein expression. Quantification of e) ICAM1/GAPDH ratio and f) p-P65/t-P65 ratio in HPMECs in response to 10% pooled healthy control (n=12), COVID-19 non-ICU (n=8) and COVID-19 ICU (n=26) serum with and without fucoidan (10 µg mL⁻¹) for 24 h of four independent experiments, presented as fold change expression normalised to healthy control. Secreted g) von Willebrand factor (VWF), h) angiopoietin 2, i) interleukin-6 (IL-6) and (j) soluble thrombomodulin (sTM) of HPMECs stimulated with 10% individual COVID-19 ICU sera (n=26) with and without fucoidan (10 µg·mL⁻¹) for 24 h. FL: fluorescence. One-way ANOVA followed by Tukey's multiple comparisons test, Kruskal–Wallis test followed by Dunn's multiple comparisons test and paired two-tailed t-test were performed. **: p<0.01; ***: p<0.001; ****: p<0.0001.

Fucoidan could ameliorate endothelial cell barrier function in presence of serum of coronavirus disease 2019 (COVID-19) intensive care unit (ICU) patients. a) Barrier integrity parameter, resistance of primary human pulmonary microvascular endothelial cells (HPMECs) assessed by electric cell-substrate impedance sensing system (ECIS) in response to stimulation with 10% serum (at t=0 h) of healthy controls (n=12, grey line), COVID-19 ICU (n=26, red line) and COVID-19 ICU with fucoidan (n=26, blue line). Cell–cell contact parameter, Rb of c) HPMECs assessed by ECIS in response to stimulation with 10% serum (at t=0 h) of healthy controls (n=12, grey line), COVID-19 ICU (n=26, red line) and COVID-19 ICU with fucoidan (n=26, blue line). Data were normalised to the baseline resistance or Rb to calculate the relative resistance or Rb to baseline (%) and presented as mean±sem. b) Quantification of barrier integrity was based on the measurements of area under the curve (AUC) of final 15 h. d) Quantification of cell–cell contact was based on the measurements of AUC of final 15 h. e) Representative confocal images of VE-cadherin (green) and F-actin (red) staining on HPMECs in the presence of 10% pooled COVID-19 ICU serum with and without fucoidan (10 µg mL⁻¹) for 24 h (scale bar=20 µm). f) Quantification of adhesion junction percentage of HPMECs in the presence of 10% pooled COVID-19 ICU serum with and without fucoidan (10 µg mL⁻¹) for 24 h of four independent experiments. Graphs represent the mean±sd. Nonpaired and paired two-tailed t-test were performed. ****: p<0.0001.