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

Abstract

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.

Transendothelial insulin transport is not saturable in vivo. No evidence for a receptor-mediated process.

“…Most notably, endothelial cells grown in vitro do not actually form capillary-like structures but rather are confluent monolayers. The permeability characteristics of such monolayers depend on many factors (28); how the layer was originally seeded (monolayers seeded at supraconfluent densities do not form as tight cell-cell junctions as those seeded at lower densities and allowed to grow to confluence), the type of culture media (the presence of albumin or histamine may alter permeability), and how long the cell culture is incubated. It is possible that, in vitro, cells form such tight junctions as to eliminate the diffusion pathway, leaving all transport to be via receptors; whereas, in vivo, insulin transport may be dominated by diffusion with only a small component transported by the receptormediated pathway.…”

Effect of advanced glycation end products on oxidative stress in endothelial cells in culture: a warning on the use of cells studied in serum-free media

“…BPAECs at passage 6 were obtained from R. C. Schaeffer, Jr. at the Sidney Kimmel Cancer Center, San Diego, Calif., USA [15]. They were used from passages 7±13.…”

“…They were used from passages 7±13. The cells were grown to confluence in T-75 flasks, detached by exposure to 0.1 % trypsin (Worthington, Worthington Biochemical, Lakewood, NJ, USA)-EDTA solution and then seeded on 100 mg/ml gelatin-and 25 mg/ml fibronectin-coated CorningCostar transwell (TW) filters (200 nm radius pores, 0.33 cm 2 surface area) (Corning, Corning, NY, USA) at a subconfluent cell density of 75 000 cells per well [15]. Monolayers were grown in a medium consisting of DMEM (Cellgro, Mediatech, Herndon, VA, USA) with 10 % fetal bovine serum (FBS, At-lanta Biological, Atlanta, Ga., USA), 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mg/ml fungizone.…”

“…At 1.5±3 h, the transport was stopped by rapidly transferring the top wells back to the original plate. The volumes used avoided any convective transport [15] due to hydrostatic pressure gradients between the two wells. Aliquots (30 ml) of the solution in each top well were pipetted into another 24-well plate and 570 ml of the HEPES-EMEM added to each of the 12 wells to total 600 ml.…”

Studies on Breast Cancer Cell Interactions with Aged Endothelial Cells in Culture and Rat Models

“…These were: (1) bovine pulmonary artery endothelial cells (BPAECs) from a personal stock, which had been previously characterized [10], (2) MCF-7 breast cancer cells (American Type Culture Collection, Manassas, VA), and (3) 184A1 cells (American Type Culture Collection, Manassas, VA), a non-malignant human mammary duct epithelial cell line. Cells were grown in 75 cm 2 culture flasks at 37 8C in 7.5% CO 2 .…”