P176Distribution of endotheltial surface layer anchoring proteins analyzed with super resolution microscopy

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Abstract

Introduction: The endothelial surface layer (ESL, glycocalyx) is known to be anchored to membrane bound proteins (syndecan-1,-2,-4 and glypican-1), glycoproteins (e.g. CD44) and enzymes (e.g. hyaluronan synthase-1,-2,-3). Little is known about the concentration and distribution of these anchoring elements on the endothelial surface.

methods: Human umbilical vein endothelial cells (HUVEC) cultured under static conditions or in the presence of shear stress (24h, non-pulsatile 8dyn/cm2) were fixed and immunostained against syndecan-1,-4 and CD44. To analyze the frequency and location of anchoring points on the endothelial cells, the samples were imaged with confocal microscopy and with gated stimulated emission depletion (STED) microscopy. Gated STED was used to resolve individual syndecan-1, and -4 anchoring sites and assess possible clustering of these individual anchoring molecules.

Results: Syndecan-1 was found to be distributed on the endothelial cell surface with density of 0.13 foci/μm2 and with 2.6±0.9 μm average distances between two foci. Shear stress treatment had only slight effect on these values (after shear stress 0.07 foci/μm2 and 2.7±1 μm). Syndecan-4 was detected on the endothelial cell surface with density of 0.6 foci/μm2 and with distance between two foci of 1.3±0.4 μm. These values were unaffected by shear stress treatment (after shear stress 0.6 foci/μm2 and 1.2±0.3 μm). In some cases confocal scanning showed clusters of syndecan-1 and -4 but gated STED could resolve these sites and could show that these cluster-like structures are composed of individual foci situated close to each other. Based on that the observed foci may corresponding to non-clustered syndecan-1 or -4 molecules. CD44 was present on the endothelial cell surface with 1.3 foci/μm2 density and with 0.8±0.3 μm distance between the foci which values weren't altered by shear stress (after shear stress 1.5 foci/μm2 and 0.8±0.2 μm).

Conclusion: The amount and distribution of ESL anchoring proteins on the surface of static or shear stress treated HUVECs was determined with confocal and gated STED microscopy. This information helps to better understand the spatial organization of the ESL.

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