Methods for measurement of permeability coefficient of pulmonary microvascular endothelial cells in rat

Abstract

To explore an optimal method for the measurement of pulmonary microvascular endothelial cell (PMVEC) permeability coefficient. A monolayer of rat PMVEC model was constructed by culturing a cell suspension on transwell filter or polycarbonate filter membrane. After the state of confluence of cells was affirmed with epithelial volt-ohm meter or inverted microscope, the permeability coefficient was determined by means of transendothelial electrical resistance (TER), fluoresceinisothiocyanate-dextran (Pd), and permeation of Hanks solution (Lp) across monolayers. Meanwhile,changes in PMVEC permeability expressed by the ratio of the observed value and the original value were observed after lipopolysaccharide (LPS) challenge for 0. 5 hour or 2 hours. The cells reached the state of confluence as observed under inverted microscope on the third day post-seeding, and the TER and Pdat this time-point were C(39. 45 ± 3. 96) ( 2 cm2] and [(8. 52 + 0. 50) X 10-6cm/s], respectively. After PMVEC were seeded on transwell filters, the TER increased steadily in a time-dependent manner after seeding of PMVEC, reaching the summit at the fourth day post-seeding C(49. 84 ± 3. 93)f " cm2].Under the natural state, the TER, Pd and Lp of confluent PMVEC monolayers were (49.84 ±3.93) ·.* cm2, (6.15±0.63) X 106 cm/s and (6.80 + 0.62) X10< cm * s-' * cm HZO-', respectively.After PMVEC monolayers were challenged with 10 mg/L LPS for both 0. 5 hour and 2 hours, there was significant decrease in the permeability coefficient as measured by TER (0. 87+ 0. 03, 0.45 0. 04 vs. 1.00+0.08, respectively, both P< 0.05), and an increase in the permeability coefficient measured by Pd (1.33±0. 11, 2.43±0. 14 vs. 1.00+0.10, respectively, both P<0. 05) and the permeability coefficient measured by Lp (1.30± 0.07, 2.38 0.15 vs. 1.00 + 0.11, respectively, both P< 0.05) when compared with the normal group. Three methods, namely TER, Pd and Lp are available to use to assess PMVEC permeability coefficient. The combination of an inverted microscope, TER and Pd enhances the accuracy in determining PMVEC permeability coefficient, and it provides an experimental technique for studying the pathogenesis of acute lung injury in vitro.