Extensive blood transcriptome analysis reveals cellular signaling networks activated by circulating glycocalyx components reflecting vascular injury in COVID-19

Abstract

Background: Degradation of the endothelial protective glycocalyx layer during COVID-19 infection leads to shedding of major glycocalyx components. These circulating proteins and their degradation products may feedback on immune and endothelial cells and activate molecular signaling cascades in COVID-19 associated microvascular injury. To test this hypothesis, we measured plasma glycocalyx components in patients with SARS-CoV-2 infection of variable disease severity and identified molecular signaling networks activated by glycocalyx components in immune and endothelial cells.

Methods: We studied patients with RT-PCR confirmed COVID-19 pneumonia, patients with COVID-19 Acute Respiratory Distress Syndrome (ARDS) and healthy controls (wildtype, n=20 in each group) and measured syndecan-1, heparan sulfate and hyaluronic acid. The in-silico construction of signaling networks was based on RNA sequencing (RNAseq) of mRNA transcripts derived from blood cells and of miRNAs isolated from extracellular vesicles from the identical cohort. Differentially regulated RNAs between groups were identified by gene expression analysis. Both RNAseq data sets were used for network construction of circulating glycosaminoglycans focusing on immune and endothelial cells.

Results: Plasma concentrations of glycocalyx components were highest in COVID-19 ARDS. Hyaluronic acid plasma levels in patients admitted with COVID-19 pneumonia who later developed ARDS during hospital treatment (n=8) were significantly higher at hospital admission than in patients with an early recovery. RNAseq identified hyaluronic acid as an upregulator of TLR4 in pneumonia and ARDS. In COVID-19 ARDS, syndecan-1 increased IL-6, which was significantly higher than in pneumonia. In ARDS, hyaluronic acid activated NRP1, a co-receptor of activated VEGFA, which is associated with pulmonary vascular hyperpermeability and interacted with VCAN (upregulated), a proteoglycan important for chemokine communication.

Conclusions: Circulating glycocalyx components in COVID-19 have distinct biologic feedback effects on immune and endothelial cells and result in upregulation of key regulatory transcripts leading to further immune activation and more severe systemic inflammation. These consequences are most pronounced during the early hospital phase of COVID-19 before pulmonary failure develops. Elevated levels of circulating glycocalyx components may early identify patients at risk for microvascular injury and ARDS. The timely inhibition of glycocalyx degradation could provide a novel therapeutic approach to prevent the development of ARDS in COVID-19.

Keywords: COVID-19; acute respiratory distress syndrome; cell-free microRNAs; endothelial dysfunction; extracellular vesicles; glycocalyx; small RNA.

Figure 1

Step by step illustration of the study procedure. After recruitment of the study participants and blood sampling, plasma concentrations of glycocalyx components were measured by ELISA, EVs were extracted from serum and RNA extracted from EVs and whole blood cells followed by high-throughput RNA sequencing. The last step consisted of bioinformatic analysis resulting in the construction of glycocalyx component signaling networks in COVID-19. ADAMTS13 = von Willebrand factor-cleaving protease, DGE, differential gene expression analysis.

Figure 2

Plasma concentrations of syndecan-1, ADAMTS13, hyaluronic acid and heparan sulfate in healthy controls, patients with COVID-19 pneumonia and those who progressed to ARDS presented as boxplots. Horizontal lines across the boxes show significant differences with p-values between groups. Boxes represent Q1 and Q3 with median in between. Whiskers are Q1-1.5*IQR and Q3+1.5*IQR. Outliers are shown as dots.

Figure 3

Network illustrating hyaluronic acid and syndecan-1 (SDC1) signaling in immune and endothelial cells for patients with COVID-19 pneumonia with healthy controls serving as baseline. RNAseq data resulted from miRNAs (extracellular vesicles) and mRNA transcripts (blood cells). The significantly downregulated miR-32-5p is shown in green and the activated molecules SDC1 and hyaluronic acid are shown in red. The network was filtered for experimentally observed findings only, solid lines indicate direct and dashed lines indirect relationships.

Figure 4

Network for immune and endothelial cells comparing COVID-19 ARDS to the healthy state based on experimentally observed findings. Upregulated molecules are shown in red and downregulated ones in green. Significantly increased plasma concentrations of hyaluronic acid, heparan sulfate, syndecan-1 (SDC1) and downregulated signaling of ADAMTS13 are colored in darker red/green. Solid lines indicate direct and dashed lines indirect relationships.

Figure 5

Immune and endothelial cell signaling in COVID-19 pneumonia compared to patients progressing to COVID-ARDS. Upregulated molecules are shown in red and downregulated in green. Network is based on experimentally observed findings and highly predicted interactions.