Interleukin-6 drives endothelial glycocalyx damage in COVID-19 and bacterial sepsis.

Damage of the endothelial glycocalyx (eGC) plays a central role in the development of vascular hyperpermeability and organ damage during systemic inflammation. However, the specific signalling pathways for eGC damage remain poorly defined. Aim of this study was to combine sublingual video-microscopy, plasma proteomics and live cell imaging to uncover further pathways of eGC damage in patients with coronavirus disease 2019 (COVID-19) or bacterial sepsis. This secondary analysis of the prospective multicenter MICROCODE study included 22 patients with COVID-19 and 43 patients with bacterial sepsis admitted to intermediate or intensive care units and 10 healthy controls. Interleukin-6 (IL-6) was strongly associated with damaged eGC and correlated both with eGC dimensions (rs=0.36, p = 0.0015) and circulating eGC biomarkers. In vitro, IL-6 reduced eGC height and coverage, which was inhibited by blocking IL-6 signalling with the anti-IL-6 receptor antibody tocilizumab or the Janus kinase inhibitor tofacitinib. Exposure of endothelial cells to 5% serum from COVID-19 or sepsis patients resulted in a significant decrease in eGC height, which was attenuated by co-incubation with tocilizumab. In an external COVID-19 cohort of 219 patients from Massachusetts General Hospital, a previously identified proteomic eGC signature correlated with IL-6 (rs=-0.58, p < 0.0001) and predicted the combined endpoint of 28-day mortality and/or intubation (ROC-AUC: 0.86 [95% CI: 0.81-0.91], p < 0.001). The data suggest that IL-6 may significantly drive eGC damage in COVID-19 and bacterial sepsis. Our findings provide valuable insights into pathomechanisms of vascular dysfunction during systemic inflammation and highlight the need for further in vivo studies.

Analysis of Urinary Glycosaminoglycans to Predict Outcome in COVID-19 and Community-Acquired Pneumonia-A Proof-of-Concept Study.

Although coronavirus disease 2019 (COVID-19) is considered a systemic disease associated with vascular inflammation and eventual destruction of the protective endothelial glycocalyx (eGC), biomarkers of eGC damage are not yet available in the clinic. The most prominent components of eGC are sulphated glycosaminoglycans (sGAGs) attached to core proteoglycans. We hypothesised that the amount of sGAG fragments shed in urine (as a surrogate for systemic eGC damage) would correlate with disease severity and outcome. Total urinary sGAG concentration was measured using an in-house optimised 1,9-dimethylmethylene blue (DMMB) assay, which is highly accurate and insensitive to interferences. The median urinary sGAG concentration was significantly higher in 67 hospitalised patients with COVID-19 compared to 72 hospitalised patients with community-acquired pneumonia (CAP). In both groups, urinary sGAG concentrations predicted a combined endpoint (including intubation and death) with an area under the receiver operator characteristic curve of 0.72 (95% CI 0.55-0.88, p = 0.01) and 0.70 (95% CI 0.57-0.83, p = 0.007), respectively. In conclusion, the inexpensive and easy-to-perform DMMB assay provides a surrogate parameter for eGC damage that may be useful for risk stratification of patients with COVID-19 and CAP.

A Dietary Supplement Containing Fucoidan Preserves Endothelial Glycocalyx through ERK/MAPK Signaling and Protects against Damage Induced by CKD Serum.

(1) Damage to the endothelial glycocalyx (eGC), a protective layer lining the endothelial luminal surface, is associated with chronic kidney disease (CKD), which leads to a worsening of cardiovascular outcomes in these patients. Currently, there are no targeted therapeutic approaches. Whether the dietary supplement EndocalyxTM (ECX) protects against endothelial damage caused by uremic toxins is unknown. (2) We addressed this question by performing atomic force microscopy measurements on living endothelial cells. We examined the effect of ECX on eGC thickness at baseline and with pooled serum from hemodialysis patients. ECX was also successfully administered in vivo in mice, in which eGC was assessed using perfused boundary region measurements by intravital microscopy of cremasteric vessels. (3) Both ECX and fucoidan significantly improved baseline eGC thickness. Our data indicate that these effects are dependent on ERK/MAPK and PI3K signaling. After incubation with eGC damaging serum from dialysis patients, ECX increased eGC height. Intravital microscopy in mice revealed a relevant increase in baseline eGC dimensions after feeding with ECX. (4) We identified a dietary supplement containing glycocalyx substrates and fucoidan as potential mediators of eGC preservation in vitro and in vivo. Our findings suggest that fucoidan may be an essential component responsible for protecting the eGC in acute settings. Moreover, ECX might contribute to both protection and rebuilding of the eGC in the context of CKD.

The Soluble Fms-like Tyrosine Kinase-1 Contributes to Structural and Functional Changes in Endothelial Cells in Chronic Kidney Disease.

Endothelial cells are a critical target of the soluble Fms-like tyrosine kinase-1 (sFlt-1), a soluble factor increased in different diseases with varying degrees of renal impairment and endothelial dysfunction, including chronic kidney disease (CKD). Although the mechanisms underlying endothelial dysfunction are multifactorial and complex, herein, we investigated the damaging effects of sFlt-1 on structural and functional changes in endothelial cells. Our results evidenced that sera from patients with CKD stiffen the endothelial cell cortex in vitro, an effect correlated with sFlt-1 levels and prevented by sFlt-1 neutralization. Besides, we could show that recombinant sFlt-1 leads to endothelial stiffening in vitro and in vivo. This was accompanied by cytoskeleton reorganization and changes in the endothelial barrier function, as observed by increased actin polymerization and endothelial cell permeability, respectively. These results depended on the activation of the p38 MAPK and were blocked by the specific inhibitor SB203580. However, sFlt-1 only minimally affected the expression of stiffness-sensitive genes. These findings bring new insight into the mechanism of action of sFlt-1 and its biological effects that cannot be exclusively ascribed to the regulation of angiogenesis.

Heparanase Is a Putative Mediator of Endothelial Glycocalyx Damage in COVID-19 - A Proof-of-Concept Study.

Coronavirus disease 2019 (COVID-19) is a systemic disease associated with injury (thinning) of the endothelial glycocalyx (eGC), a protective layer on the vascular endothelium. The aim of this translational study was to investigate the role of the eGC-degrading enzyme heparanase (HPSE), which is known to play a central role in the destruction of the eGC in bacterial sepsis. Excess activity of HPSE in plasma from COVID-19 patients correlated with several markers of eGC damage and perfused boundary region (PBR, an inverse estimate of glycocalyx dimensions of vessels with a diameter 4-25 µm). In a series of translational experiments, we demonstrate that the changes in eGC thickness of cultured cells exposed to COVID-19 serum correlated closely with HPSE activity in concordant plasma samples (R = 0.82, P = 0.003). Inhibition of HPSE by a nonanticoagulant heparin fragment prevented eGC injury in response to COVID-19 serum, as shown by atomic force microscopy and immunofluorescence imaging. Our results suggest that the protective effect of heparin in COVID-19 may be due to an eGC-protective off-target effect.

Microvascular and proteomic signatures overlap in COVID-19 and bacterial sepsis: the MICROCODE study.

AIMS: Although coronavirus disease 2019 (COVID-19) and bacterial sepsis are distinct conditions, both are known to trigger endothelial dysfunction with corresponding microcirculatory impairment. The purpose of this study was to compare microvascular injury patterns and proteomic signatures in COVID-19 and bacterial sepsis patients. METHODS AND RESULTS: This multi-center, observational study included 22 hospitalized adult COVID-19 patients, 43 hospitalized bacterial sepsis patients, and 10 healthy controls from 4 hospitals. Microcirculation and glycocalyx dimensions were quantified via intravital sublingual microscopy. Plasma proteins were measured using targeted proteomics (Olink). Coregulation and cluster analysis of plasma proteins was performed using a training-set and confirmed in a test-set. An independent external cohort of 219 COVID-19 patients was used for validation and outcome analysis. Microcirculation and plasma proteome analysis found substantial overlap between COVID-19 and bacterial sepsis. Severity, but not disease entity explained most data variation. Unsupervised correlation analysis identified two main coregulated plasma protein signatures in both diseases that strictly counteract each other. They were associated with microvascular dysfunction and several established markers of clinical severity. The signatures were used to derive new composite biomarkers of microvascular injury that allow to predict 28-day mortality or/and intubation (area under the curve 0.90, p < 0.0001) in COVID-19. CONCLUSION: Our data imply a common biological host response of microvascular injury in both bacterial sepsis and COVID-19. A distinct plasma signature correlates with endothelial health and improved outcomes, while a counteracting response is associated with glycocalyx breakdown and high mortality. Microvascular health biomarkers are powerful predictors of clinical outcomes.

Protection and rebuilding of the endothelial glycocalyx in sepsis - Science or fiction?

The endothelial glycocalyx (eGC), a delicate carbohydrate-rich structure lining the luminal surface of the vascular endothelium, is vital for maintenance of microvascular homeostasis. In sepsis, damage of the eGC triggers the development of vascular hyperpermeability with consecutive edema formation and organ failure. While there is evidence that protection or rebuilding of the eGC might counteract sepsis-induced vascular leakage and improve outcome, approved therapeutics are not yet available. This narrative review aims to outline possible therapeutic strategies to ameliorate organ dysfunction caused by eGC impairment.

Tie2 Activation Promotes Protection and Reconstitution of the Endothelial Glycocalyx in Human Sepsis.

The endothelial glycocalyx (eGC), a carbohydrate-rich layer lining the luminal surface of the endothelium, provides a first vasoprotective barrier against vascular leakage in sepsis. We hypothesized that angiopoietin-2 (Angpt-2), antagonist of the endothelium-stabilizing receptor Tie2, induces a rapid loss of the eGC in human sepsis. Using intravital microscopy, we measured the perfused boundary region (PBR), an inverse parameter of eGC dimensions in sublingual microvessels, in patients with sepsis and age-matched nonseptic subjects. Median PBR values were significantly higher in patients compared with controls and correlated with serum Angpt-2 levels. To transfer and further explore these findings in a cell culture system, we exposed endothelial cells (ECs) to serum (5%) from a subgroup of septic patients and nonseptic controls. Confocal and atomic force microscopy revealed that sepsis serum, but not control serum, induced thinning of the eGC on human ECs in vitro, which correlated with paired PBR values obtained in vivo (r = 0.96, p < 0.01). Inhibition of Angpt-2 or Tie2 activation completely abolished eGC damage. Mechanistically, sepsis-induced eGC breakdown required the loss of its main constituent heparan sulfate; a result of heparan sulfate-specific enzyme heparanase, which was suppressed by Tie2 activation. Finally, Tie2 activation, but not Angpt-2 inhibition, initiated after septic or enzymatic damage provoked rapid refurbishment of the eGC. Our data indicate that eGC breakdown in human sepsis is mediated via Tie2 deactivation by Angpt-2. Activation of Tie2 seems to accelerate recovery of the eGC and might hold promise as a therapeutic target in human sepsis.