Jie Fan1, Yi Sun2, Yifan Xia1, John M Tarbell1, Bingmei M Fu1. 1. Department of Biomedical Engineering, The City College of the City University of New York, \unskip\break New York, NY, USA. 2. Department of Electrical Engineering, The City College of the City University of New York, New York, NY, USA.
Abstract
BACKGROUND: In order to play different roles in vascular functions as a mechanosensor to blood flows and as a barrier to transvascular exchange, the endothelial surface glycocalyx (ESG) should have an organized structure. Due to the limitations of optical and electron microscopy, the ultra-structure of ESG has not been revealed until the recent development of super-resolution optical microscopy, STORM. OBJECTIVES: To investigate the ESG components and their organization on bEnd3 (mouse brain microvascular endothelial cells) monolayer. METHODS: ESG was immunolabeled with anti-heparan sulfate (HS), followed by an ATTO488 conjugated goat anti-mouse IgG, and with biotinylated hyaluronic acid (HA) binding protein, followed by an AF647 conjugated anti-biotin. The ESG was then imaged by the STORM. RESULTS: HA is a long molecule weaving into a network which covers the endothelial luminal surface. In contrast, HS is a shorter molecule, perpendicular to the cell surface. HA and HS are partially overlapped with each other at the endothelial luminal surface. We also quantified the length, diameter, orientation, and density of HS at the top, middle and bottom regions of the endothelial surface. CONCLUSIONS: Our results suggest that HS plays a major role in mechanosensing and HA plays a major role in the molecular sieve.
BACKGROUND: In order to play different roles in vascular functions as a mechanosensor to blood flows and as a barrier to transvascular exchange, the endothelial surface glycocalyx (ESG) should have an organized structure. Due to the limitations of optical and electron microscopy, the ultra-structure of ESG has not been revealed until the recent development of super-resolution optical microscopy, STORM. OBJECTIVES: To investigate the ESG components and their organization on bEnd3 (mouse brain microvascular endothelial cells) monolayer. METHODS: ESG was immunolabeled with anti-heparan sulfate (HS), followed by an ATTO488 conjugated goat anti-mouse IgG, and with biotinylated hyaluronic acid (HA) binding protein, followed by an AF647 conjugated anti-biotin. The ESG was then imaged by the STORM. RESULTS:HA is a long molecule weaving into a network which covers the endothelial luminal surface. In contrast, HS is a shorter molecule, perpendicular to the cell surface. HA and HS are partially overlapped with each other at the endothelial luminal surface. We also quantified the length, diameter, orientation, and density of HS at the top, middle and bottom regions of the endothelial surface. CONCLUSIONS: Our results suggest that HS plays a major role in mechanosensing and HA plays a major role in the molecular sieve.