Fig 2: HPSE1 aggravates endothelial cell response to LPS. (a) HMEC-1 cells were treated with active HPSE1 for 1 h prior to LPS stimulation. Then, cells were treated with 100 ng/ml LPS for 3 hrs. Cells were fixed and stained with 10E4 antibody for heparan sulfate (Alexa 594). Nuclei were stained with DAPI. Scale Bar 50 µm. (b) Quantification of heparan sulfate stainings. Percentage of stained areas was calculated using ImageJ. (c) Primary endothelial cells treated as in a were stained with Alexa 488-Phalloidin (green) and DAPI. White arrows show loss of cellular contacts. Scale Bar 50 µm. (d) Quantification of phalloidin images. Percentage of cell-coated areas was calculated using ImageJ. (e) HMEC-1 cells were treated with catalytically active HPSE1 and then stimulated with LPS. IL-6 expression was assessed by RT-PCR. (f) Cell were treated with conditioned medium from control (LV-Ctrl) and HPSE1 overexpression (LV-HPSE1) lentivirus-transduced HMEC-1 cells for 1 h prior to LPS stimulation. IL-6 expression was assessed by RT-PCR. (g) Expression of 50 kD active HPSE1 after LPS stimulation of endothelial cells assessed in cell lysates by western blotting. (h) Expression of 65 kD isoform of HPSE1 was assessed in conditioned medium of LPS-stimulated endothelial cells. (i) Quantification of three independent western blotting experiments as shown in (g) (Cell lysate) and (h) (Cond. Medium). Data were quantified using QuantityOne software (BioRad).

Fig 3: HPSE2 prevents HPSE1 activity. (a,b) Endothelial cells were pre-treated with inhibitor of HPSE1 catalytic activity, OGT 2115, and then stimulated with 100 ng/ml LPS for 3 hrs (a) and overnight (b). IL-6 expression was analyzed by RT-PCR (a) and ELISA (b). (c) Endothelial cells were pre-incubated with mouse IgG or anti- HPSE2 antibody 1c7 for 30 min prior to LPS stimulation and then stimulated with 100 ng/ml LPS for 3 hrs. IL-6 expression was assessed by TaqMan RT-PCR.

Fig 4: HPSE2 overexpression protects endothelial heparan sulfate glycocalyx in a microfluidic chip model. (a) 4-channels PDMS-microfluidic chip. (b) Development of 3D heparan sulfate glycocalyx layer after endothelial cells cultivation under flow conditions (3 days, 0.1 dyn/cm2) was assessed by 3D confocal microscopy using 10E4 antibody to heparan sulfate followed by image reconstruction using ImageJ software. DraQ5 was used as a nuclear stain. Yellow lines show position of orthogonal slices. Bottom and right panels show corresponding orthogonal slices of the sum images. (c) Endothelial cells transduced with Control and HPSE2 overexpression-lentivirus were cultivated in the microfluidic chip for 3 days, and then perfused with recombinant HPSE1 for 4 hrs. Cells were fixed by perfusion with PFA and stained with 10E4 antibody as described in the methods section. Heparan sulfate content was quantified using ImageJ software after 3D confocal microscopy. Experiment was independently repeated in 4 chips. (d) 3D reconstruction of the heparan sulfate glycocalyx layer images of lentivirus-transduced endothelial cells after incubation in the microfluidic chip and recombinant HPSE1 treatment. The 3D reconstruction was performed using ImageJ software. Scale bar 20 µm. (e) Endothelial cells incubated in microfluidic chips under static and flow conditions were stimulated with 100 ng/ml LPS for 3 hrs. IL-6 expression was assessed by TaqMan RT-PCR. (f) 50 kD HPSE1 expression in lysates of LPS-stimulated endothelial cells.