Substrate stiffness modulates migration and local intercellular membrane motion in pulmonary endothelial cell monolayers

Abstract

The pulmonary artery endothelium forms a semipermeable barrier that limits macromolecular flux through intercellular junctions. This barrier is maintained by an intrinsic forward protrusion of the interacting membranes between adjacent cells. However, dynamic interactions of these membranes have been incompletely quantified. Here, we present a novel technique to quantify motion of the peripheral membrane of cells, called paracellular morphological fluctuations (PMFs), and to assess the impact of substrate stiffness on PMFs. Substrate stiffness impacted large length scale morphological changes such as cell size and motion. Cell size was larger on stiffer substrates, whereas the speed of cell movement was decreased on hydrogels with stiffness either larger or smaller than 1.25 kPa, consistent with cells approaching a jammed state. Pulmonary artery endothelial cells moved fastest on 1.25 kPa hydrogel, stiffness consistent with a healthy pulmonary artery. Unlike these large length scale morphological changes, the baseline of PMFs was largely insensitive to substrate stiffness on which cells were cultured. Activation of store-operated calcium channels using thapsigargin treatment triggered a transient increase in PMFs beyond control treatment. However, in hypocalcemic conditions, such an increase in PMFs was absent on 1.25 kPa hydrogel but was present on 30 kPa hydrogel - a stiffness consistent with that of a hypertensive pulmonary artery. These findings indicate: (i) PMFs occur in cultured endothelial cell clusters, irrespective of the substrate stiffness; (ii) PMFs increase in response to calcium influx through store-operated calcium entry channels; and (iii) stiffer substrate promotes PMFs through a mechanism that does not require calcium influx.

Selective Blood Cell Hitchhiking in Whole Blood with Ionic Liquid-Coated PLGA Nanoparticles to Redirect Biodistribution After Intravenous Injection

“…This suggests not direct protein-avoidance but stronger RBC membrane hitchhiking to avoid opsonization. This is because the rst site of accumulation is not a gradual distribution of shear within chambers of the rst pulmonary capillary bed (CA2HA 1:2), but likely a sudden NP "drop-off" of oxygenated RBCs from the lung circulation into the hepatic artery at 1 hour after IL-NP:RBC weakening from enduring multiple levels of matrix stiffness, shear stress, and arterial pressure [105][106][107][108][109] . Additionally, the location of liver accumulation is likely in the parenchyma and portal triad ducts, as some enterohepatic recycling from the liver to the bloodstream and intestines was observed (2.4 ± 1.1%), without further CAHPA 1:1 PLGA NP loss from circulation 6-24 hours 13 .…”