PB1282 - Models for identification of endothelial glycocalyx degradation mechanisms during Puumala virus infection

Background: The endothelial glycocalyx (eGLX) cell layer is composed of glycosaminoglycans (e.g., heparan sulfate (HS)) and glycoproteins (e.g., syndecan-1 (SDC1), thrombomodulin). Degradation of vascular eGLX has been implicated in diabetes, coronary atherosclerosis, and kidney failure pathogenesis. Previously, we found an association between plasma levels of SDC-1 and disease severity of hemorrhagic fever with renal syndrome (HFRS) caused by Puumala orthohantavirus.

Aims: Investigate eGLX shedding during orthohantavirus infection; and identify potential mechanisms of eGLX degradation through implementation of in vitro models

Methods: In total 44 HFRS patients with serologically verified HFRS at the Umeå University hospital were included. Using commercial ELISA assays the plasma concentrations of SDC1, MMP9, and HS were quantified. Using Quartz Crystal Microbalance with Dissipation (QCM-D) monitoring (AWSensors), effects of Heparinase (2 units/mL) or MMP-9 (500 ng/mL) were followed on biotinylated HS (b-HS) or SDC1 protein linked to supported lipid bilayers formed on SiO2 sensors. Enzyme immunoassays were performed on confluent GEnC cells on 96-wells. After Heparinases incubation, cells were fixed and stained with anti-HS antibody and HRP-coupled secondary antibody. TBM was added before reading the absorbance at 450nm

Results: In HFRS cohort, HS and SDC1 plasmatic levels were significantly increased during disease compared to follow up (p < 0.01). MMP-9 was also detected as higher concentration in plasma during disease than at follow up (p < 0.001) (Figure.1). QCM-D experiments showed that Heparinase can specifically degrade b-HS (∆f = -28% ±8; ∆D = -41% ±11). MMP-9 sheddase, at plasmatic concentration during acute phase of HFRS, was also effective against SDC1 (∆f = -57% ±12; ∆D = -68% ±18) (Figure.2). In vitro results on GEnC cells showed HS degradation by Heparinase at concentrations ranging from 0.125 units/mL to 2 units/mL

Conclusion(s): We identified two potential candidates that could contribute to cleavage of the eGLX during HFRS and could potentially contribute to disease pathogenesis and severity