Endothelial Surface Glycocalyx Revealed by Stochastic Optical Reconstruction Microscopy (STORM)

Abstract

On the luminal surface of our blood vessels, there is a thin layer called endothelial surface glycocalyx (ESG) which consists of proteoglycans, glycosaminoglycans (GAGs) and glycoproteins. The GAGs in the ESG are heparan sulfate (HS), hyaluronic acid (HA), chondroitin sulfate (CS), and sialic acid (SA). In order to play important roles in vascular functions, such as being a mechanosensor and transducer for the endothelial cells (ECs) to sense the blood flow, a molecular sieve to maintain normal microvessel permeability and a barrier between the circulating cells and endothelial cells forming the vessel wall, the ESG should have an organized structure at the molecular level. Due to the limitations of optical and electrical microscopy, the ultra-structure of ESG has not been revealed until recent development of super high resolution fluorescence optical microscope, STORM. We used newly acquired Nikon-STORM system to observe the ESG on in vitro EC (bEnd3, mouse brain microvascular endothelial cells) monolayers. After confluent, the bEnd3 cells were immunolabeled with anti-HS, followed by an ATTO488 conjugated goat anti-mouse IgG, and with biotinylated HA binding protein, followed by an AF647 conjugated anti-biotin. The ESG was then imaged by the STORM with a 100 x/1.49 oil immersed lens. Multiple Reporters of ATTO488 and AF647 with alternating illumination were used to acquire the 3D images of HS and HA. The field of 256 × 256 (40 × 40 μm2) of HS and HA at the surface of ECs was obtained based on totally 40,000 of EM-CCD captured images for each reporter at a capturing speed of 19 ms/frame. The figure demonstrates the STORM images for the HA (top view, red, top row on the right) and HS (green, top row in the middle) and overlay (top row on the left) of the ESG focused at the bottom surface of the bEnd3 cells. The EC plasma membrane is located at the bottom plane of the 3D images (bottom row). The color bars in the 3D side views are the scale bars in nm. The figure shows that HA is a long molecule weaving into a network, which is horizontal to the EC cell surface. In contrast, HS is a shorter molecule, which is perpendicular to the cell surface. The height of the HS is ~600 nm. HA and HS seem to overlap with each other at the EC surface. The revealed ultra-structure of ESG by STORM suggests that HS plays a major role in mechanosensing and HA plays a major role in forming the molecular sieve. Support or Funding Information NIH 1SC1CA153325-01 and NSF MRI CBET 1337746 STORM images showing distribution of ATTO 488 labeled anti-HS (green, middle) and Alexa Fluor 647 labeled anti-HA (red, right) and overlay (left) in the ESG of bEnd3 cell monolayer, focused on the bottom surface of the cells. The EC plasma membrane is located at the bottom plane of the 3D images (bottom row). The color bars in the 3D side views are the scale bars in nm. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.