Matrix metalloproteinase-9 mediates endothelial glycocalyx degradation and correlates with severity of hemorrhagic fever with renal syndrome

Abstract

Hemorrhagic fever with renal syndrome (HFRS) caused by Puumala virus (PUUV) leads to vascular dysfunction contributing to acute kidney injury (AKI) and pulmonary complications. The endothelial glycocalyx (eGLX) is crucial for vascular integrity, and its degradation may exacerbate disease severity. In this study, we examined the association between eGLX degradation and renal and pulmonary dysfunction in 44 patients with laboratory-confirmed PUUV infection. We measured plasma levels of eGLX degradation markers-syndecan-1, heparan sulfate, soluble thrombomodulin, and albumin-and found that these correlated with severe AKI and the need for oxygen therapy. In vitro experiments showed that matrix metalloproteinase-9 (MMP-9) and heparanase can degrade eGLX components, but albumin at physiological concentrations can mitigate this degradation and protect endothelial barrier function. These findings indicate that eGLX degradation contributes to HFRS pathogenesis and suggest that targeting the eGLX could be a therapeutic strategy to improve patient outcomes.

Keywords: biochemistry; cell biology; microbiology.

Graphical abstract

Figure 1

Endothelial glycocalyx degradation in HFRS patients in plasma and association with acute severe injury or need of oxygen HFRS patients were stratified into two groups of severe AKI (stage 2/3) or not (AKI stage 0/1) according to KDIGO criteria and in two groups need of oxygen or not. The estimated mean and standard error for each group were calculated using generalized estimating equation (GEE) and adjusted for sex and age. Receiver operating characteristics curve (ROC) analysis were performed using maximum values of eGLX markers and minimal values of albumin. The timeline kinetics and ROC curves are shown for syndecan-1 (A), heparan sulfate (B), soluble thrombomodulin (C), albumin (D) MMP-9 (E), and NGAL (F). Significant differences within the same time point between marker levels are indicated by asterisks (∗∗∗p < 0.001; ∗∗p < 0.01; and ∗p < 0.05). AKI, acute kidney injury; HFRS, hemorrhagic fever with renal syndrome; HS, heparan sulfate; KDIGO, Kidney Disease Improval Global Outcomes; SDC-1, syndecan-1; P, plasma; sTM, soluble thrombomodulin; MMP9, matrix metalloproteinase 9; NGAL, neutrophil gelatinase-associated lipocalin.

Figure 2

In vitro glycocalyx degradation mediated by heparanase and MMP-9 followed by QCM-D (A) b-HS and (B) rhSDC-1 protein were absorbed on synthetic membrane composed by POPC-DOPE (95:5) lipids and streptavidin, plus Tris-NTA linker for rhSDC-1. After incubation with albumin at 40, 20, or 10 mg/mL, real-time degradation of b-HS by HPSE at 250 ng/mL (C) and rhSDC1 by MMP-9 at 500 ng/mL (D). Arrow indicates when the enzyme is injected. Shift in frequency (Δf) and dissipation (ΔD) due to enzyme activity was measured after enzyme activity in enzyme buffer. Percentage of Δf and ΔD signal reduction for b-HS (E) and rhSDC1 (F) after enzymatic treatment (n = 4) were calculated from the b-HS or rhSDC-1 signal in enzyme buffer (corresponding to 100%). Data are represented as mean ± SD. Significant differences are indicated by asterisks (∗∗∗p < 0.001; ∗∗p < 0.01; and ∗p < 0.05). QCM-D, quartz crystal microbalance with dissipation; b-HS, biotinylated heparan sulfate; rhSDC-1, recombinant human syndecan-1; MMP-9, matrix metalloproteinase 9; HPSE, human heparanase.

Figure 3

In vitro endothelial glycocalyx degradation and endothelial barrier disruption followed by impedance measurement (A) SDC-1 staining on ciGEnC cells after incubation with MMP-9 enzyme and increased concentration of albumin (scale bars 20 μm). (B) SDC-1 fluorescence quantification after treatment compared to control (no treatment) (averaged values of 3 sections per condition, n = 3 assays). (C) HS staining on ciGEnC cells after incubation with HSPE enzyme and increased concentration of albumin (scale bars 20 μm). (D) HS fluorescence quantification after treatment compared to control (no treatment) (averaged values of 3 sections per condition, n = 3 assays). (E) Endothelial barrier resistance followed by impedance measurement (ECIS, APBiophysic) on ciGEnC after treatment with MMP-9 and albumin (averaged of 2 values per conditions, n = 3 assays). (F) Endothelial barrier resistance followed by impedance measurement (ECIS, APBiophysic) on ciGEnC after treatment with HSPE and albumin (averaged of 2 values per conditions, n = 3 assays). Data are represented as mean ± SD. Significant differences are indicated by asterisks (∗∗∗p < 0.001; ∗∗p < 0.01; and ∗p < 0.05). HS, heparan sulfate; SDC-1, syndecan-1; MMP-9, matrix metalloproteinase 9; HPSE, human heparanase.

Figure 4

Immune cells levels in plasma for HFRS patients and association with acute severe injury or need of oxygen HFRS patients were stratified into two groups of severe AKI (stage 2/3) or not (AKI stage 0/1) according to KDIGO criteria or in two groups need of oxygen or no oxygen. The estimated mean and standard error for each group were calculated using generalized estimating equation (GEE) and adjusted for sex and age. Receiver operating characteristics curve (ROC) analysis were performed using maximum values of eGLX markers and minimal values of albumin. The timeline kinetics and ROC curves are shown for leukocytes (A), lymphocytes (B), monocytes (C), neutrophils (D), basophils (E), and eosinophils (F). Significant differences within the same time point between marker levels are indicated by asterisks (∗∗∗p < 0.001; ∗∗p < 0.01; and ∗p < 0.05). AKI, acute kidney injury.