Additional file 1 of Heparanase inhibition as a systemic approach to protect the endothelial glycocalyx and prevent microvascular complications in diabetes
Creators: name: Gamez, Monica
name: Elhegni, Hesham E.
name: Fawaz, Sarah
name: Ho, Kwan Ho
name: Campbell, Neill W.
name: Copland, David A.
name: Onions, Karen L.
name: Butler, Matthew J.
name: Wasson, Elizabeth J.
name: Crompton, Michael
name: Ramnath, Raina D.
name: Qiu, Yan
name: Yamaguchi, Yu
name: Arkill, Kenton P.
name: Bates, David O.
name: Turnbull, Jeremy E.
name: Zubkova, Olga V.
name: Welsh, Gavin I.
name: Atan, Denize
name: Satchell, Simon C.
name: Foster, Rebecca R.
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Physiology
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Marine Biology
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Figure S1. Measurements of glomerular filtration parameters and glycocalyx depth. a Area of measurements (dotted lines) for podocyte foot process (PFP) width, slit diagraph (SD) width, basement membrane (BM) thickness, and length of BM. Number of fenestrations were counted as indicated, and divided by the length of BM to obtain fenestrations/µm. b For eGlx measurements, a grid was overlayed over the image. Red arrows point to examples of eGlx staining. Measurements were taken where the intersecting grid lines intersected the lipid bilayer, examples indicated by (x) and measurement example indicated by dotted lines on inset image. Figure S2. Structure of heparanase inhibitor OVZ/HS-1638. OVZ/HS-1638 is a single entity chemical polyvalent dendrimer which allows for multiple target interactions at multiple points on the inhibitor [29]. Figure S3. LEL stains eGlx components in the retina. Mouse retinal vessel on retinal flat mount stained with FITC-LEL (green) and DAPI (blue). Lumen (Lm) of vessel indicated. Arrow points to eGlx staining on surface of endothelial cell. Figure S4. A vascular bolus of heparinase III has no impact on podocyte glycocalyx or other ultrafiltration parameters. Glomerular filtration barrier measurements in mice treated with inactive or active heparinase III. a Podocyte glycocalyx depth. b Basement membrane (BM) thickness. c Fenestration density. d Slit diaphragm width. e Podocyte foot process width. No statistically significant differences found (n = 5 mice, unpaired t- test, not significant). Figure S5. Ext1(ECKO) mice have no gross changes in retinal or glomerular morphology and have reduced eGlx HS. a Representative optical coherence tomography (OCT) images of retinas from LMC and Ext1(ECKO) mice, showing no changes in morphology. Scale bar 50 µm. b Representative images of cortex Haematoxylin and Eosin staining at × 10 and × 40 magnification are shown for LMC and Ext1(ECKO) mice, showing no change in gross morphology. c Representative images of heparan sulphate staining on kidney from LMC and Ext1(ECKO) mice. Inset shows eGlx heparan sulphate staining (green) on luminal side of capillary (arrowhead). Membrane stain R18 (red) used to stain cell membranes and DAPI for nuclear staining (blue). Red blood cells (*) and basement membrane (BM) indicated. Scale bar = 20 µm d Analysis of glomerular capillary eGlx heparan sulphate staining. (n = 6 mice, **p &lt; 0.01, unpaired t-test). e Quantitative PCR performed on FAC sorted glomerular endothelial cells from littermate control (LMC) and Ext1(ECKO) mice. (n = 3 mice, **p &lt; 0.01, unpaired t-test). Figure S6. Ext1(ECKO) mice have no change in podocyte glycocalyx or other ultrafiltration parameters. Glomerular filtration barrier measurements in littermate control (LMC) and Ext1(ECKO) mice. a Podocyte glycocalyx depth. b Basement membrane (BM) thickness. c Fenestration density. d Slit diaphragm width. e Podocyte foot process width (n = 6 mice/group, unpaired t- test, not significant). Figure S7. Treatment with the heparanase inhibitor OVZ/HS-1638 in vitro prevents heparan sulphate shedding by heparanase. a Representative confocal images of control, heparanase (HPSE), or HPSE + OVZ/HS-1638 inhibitor (HPSE + HI) treated GEnCs stained with anti-heparan sulphate in green. b Quantification of total heparan sulphate staining in Control, HPSE, and HPSE + HI treated GEnCs, normalized to cell number (n = 6 technical repeats, P* &lt; 0.05,**P &lt; 0.01, One way ANOVA for normally distributed data, Tukey’s multiple comparison test). Figure S8. Vessel number is not significantly impacted in db/db mice at 11-weeks-of-age Average number of vessels counted per animal in albumin-stained retina per field of view (FOV), minimum of three FOV counted per animal (n = 6 mice/group, One-way ANOVA with Tukey’s multiple comparison test. Not significant). Figure S9. Db/db mice develop early signs of diabetic nephropathy. Glomerular filtration barrier measurements for lean, diabetic (db/db) and diabetic mice treated with OVZ/HS-1638 (db/db + HI). a Podocyte glycocalyx depth. b Basement membrane (BM) thickness. c Fenestration density. d Slit diaphragm width. e Podocyte foot process width (n = 5 mice/group. One way ANOVA for normally distributed data, Tukey’s multiple comparison test. No statistically significant differences found for (a–b). For (d, e), *P &lt; 0.05, **P &lt; 0.01). Figure S10. Treatment with OVZ/HS-1638 prevents glycogen deposition in diabetic nephropathy in db/db mice. a Representative Periodic acid-Schiff (PAS) staining images for lean, diabetic (db/db) and diabetic treated with OVZ/HS-1638 (db/db + HI) b PAS staining analysed to measure fibrosis within glomerulus in lean, diabetic (db/db) and diabetic treated with OVZ/HS-1638 (db/db + HI) mice. (n = 5 mice/group, *P &lt; 0.05 **P &lt; 0.01, One way ANOVA for normally distributed data, Tukey’s multiple comparison test).