Restricted diffusion of macromolecules by endothelial monolayers and small-pore filters

We studied the size-selective permeability and restricted diffusion (Rd) properties of macromolecules across bovine pulmonary artery endothelial cell (BPAEC) or epithelial (LLC-PK1) monolayers grown on polycarbonate (PC) filter supports using fluorescein isothiocyanate-hydroxyethyl starch (FITC-HES, 16 A less than ae less than 180 A). Most BPAEC seeded at subconfluent density and grown for 3-6 days produced barriers with marked Rd. This characteristic was similar to that measured across PC filters with pore radii (rp) of 150 and 250 A without cells. Rd of LLC-PK1 monolayers was consistent with a transport pathway rp of much less than 75A. BPAEC monolayers prepared by supraconfluent seeding showed convective solute transport due to a significant number of incompletely formed intracellular junctions. Most monolayers grown to confluence, or a thin layer of collagen type I prevented this effect, Rd was enhanced when BPAEC monolalyers were grown on this collagen network. These data suggest that the subendothelial layers, which includes basal lamina, pericyte, and interstitial collagen, may act as series resistors with the endothelium to provide the Rd observed in the microvascular wall in vivo. This may explain why BPAEC monolayers grown to confluence without subendothelial layers in vitro showed Rd consistent with large (150-250 A) rp that was significantly greater than those modeled as the small (approximately 50 A) "pore" or 6-A fiber matrix of the in vivo capillary wall.