Visualization of three pathways for macromolecule transport across cultured endothelium and their modification by flow

Abstract

Transport of macromolecules across vascular endothelium and its modification by fluid mechanical forces are important for normal tissue function and in the development of atherosclerosis. However, the routes by which macromolecules cross endothelium, the hemodynamic stresses that maintain endothelial physiology or trigger disease, and the dependence of transendothelial transport on hemodynamic stresses are controversial. We visualized pathways for macromolecule transport and determined the effect on these pathways of different types of flow. Endothelial monolayers were cultured under static conditions or on an orbital shaker producing different flow profiles in different parts of the wells. Fluorescent tracers that bound to the substrate after crossing the endothelium were used to identify transport pathways. Maps of tracer distribution were compared with numerical simulations of flow to determine effects of different shear stress metrics on permeability. Albumin-sized tracers dominantly crossed the cultured endothelium via junctions between neighboring cells, high-density lipoprotein-sized tracers crossed at tricellular junctions, and low-density lipoprotein-sized tracers crossed through cells. Cells aligned close to the angle that minimized shear stresses across their long axis. The rate of paracellular transport under flow correlated with the magnitude of these minimized transverse stresses, whereas transport across cells was uniformly reduced by all types of flow. These results contradict the long-standing two-pore theory of solute transport across microvessel walls and the consensus view that endothelial cells align with the mean shear vector. They suggest that endothelial cells minimize transverse shear, supporting its postulated proatherogenic role. Preliminary data show that similar tracer techniques are practicable in vivo. Solutes of increasing size crossed cultured endothelium through intercellular junctions, through tricellular junctions, or transcellularly. Cells aligned to minimize the shear stress acting across their long axis. Paracellular transport correlated with the level of this minimized shear, but transcellular transport was reduced uniformly by flow regardless of the shear profile.

Understanding mechanobiology in cultured endothelium: A review of the orbital shaker method

“…Hence there are low magnitude, multidirectional shear stresses at the centre and high magnitude, uniaxial shear stresses at the edge. This behaviour has been captured by polar plots of instantaneous WSS vectors [ 37 , 47 , 48 ], by a combination of the OSI and transWSS metrics, or by a metric termed the Directional OSI [DOSI] [ 37 , 49 ].…”

“…That can be achieved by comparing their radial profiles with the radial profile of the cellular property of interest, and/or by changing the physical variables that influence flow patterns. For example, in 12-well plates having a static medium depth of 2 mm, rotated at 150 rpm with an orbital radius of 5 mm, TAWSS decreases from the centre to a radial distance of 7.8 mm before rapidly increasing as it approaches the edge, whereas in the larger 6-well plates with the same medium depth and orbital parameters, TAWSS is uniform until a radial distance of 10 mm before the rapid increase [ 47 , 48 ]. This type of diversity can be used to separate the effects of different metrics.…”

“…Transendothelial transport of macromolecules has also been investigated in the swirling well [ 47 ]. Transport was measured using a modification of the method of Dubrovskyi et al [ 64 ], who grew endothelial cells on biotinylated gelatin and used FITC-labelled avidin (69 kDa) as a tracer; the FITC-avidin is added to the medium and binds to the biotin immediately on crossing the endothelium, thus remaining under – and hence identifying – the structure through which it travelled.…”

“…The dominant transport pathways for the tracers of increasing size were, respectively, junctions between neighbouring pairs of cells, tricellular junctions, and a transcellular route. Paracellular transport was elevated in the centre of the well and reduced at the edge, but there was no difference in transcellular transport [ 47 ].…”

Rho Flares Repair Local Tight Junction Leaks

“…Recently developed ''sandwich assays'' employ avidin-and biotin-labeled molecules applied to either side of the tissue to capture the macromolecules as they cross the tight junction barrier. These studies have revealed that barrier function to macromolecules is not uniform across the cell and tissue level and that the leak pathway opens and closes over time (Dubrovskyi et al, 2013;Ghim et al, 2017;Richter et al, 2016).…”