Effects of hemodynamic forces on endothelial cell hydraulic conductivity
Abstract (Summary)iii Starling’s hypothesis that fluid movement across the microvascular wall is determined by the transmural differences in hydrostatic and osmotic pressures was tested using an in vitro model comprised of bovine aortic endothelial cells (BAECs) grown on a porous support. In all experiments a 1% bovine serum albumin (BSA) solution was maintained in the abluminal reservoir and the luminal reservoir contained either a 1% or a 5.5% BSA solution. The global osmotic pressure difference across the endothelial layers was thus either 0 or 20.3 cm H2O. When the luminal concentration of BSA was changed from 1% to 5.5% at a hydrostatic pressure differential of 5, 10 or 20 cm H2O, no reverse flow (in the reabsorption direction) was observed even though the hydrostatic pressure differential was far below the global osmotic pressure differential. In another case, the hydrostatic pressure differential was dropped quickly from 20 to 5 cm H2O while a constant osmotic pressure differential was maintained by 5.5% BSA in the luminal reservoir. A strong transient reabsoption flow was observed over a 30 second period which diminished to undetectable levels within 2.5 minutes; then a sustained steady state filtration flow was observed after 20 minutes. These in vitro experiments support other studies in capillaries showing transient reabsorption that decays to steady state filtration at longer times. Human umbilical vein endothelial cells (HUVECs) and BAECs display opposite hydraulic conductivity ( L ) responses to shear stress. In HUVECs, 5, 10, and 20 dyne/cm2 steady shear stress transiently increased P L P of endothelial cells (ECs), and returned to normal after a two hour exposure to shear stress. Pure oscillatory shear stress L P of 0 ± 20 dyne/cm2 had no effect in changing LP . Furthermore, superposition of oscillatory shear stress on steady shear stress suppressed the effect induced by steady shear stress. Shear reversal was not necessary for the oscillatory shear stress to function. This phenomenon is very different from BAEC which showed a significant sustained increase in L P for steady shear stress and only oscillatory shear stress with shear reversal could exert an inhibitory effect on steady shear stress induced increase in LP . Intracellular calcium was first investigated for its role in the signal transduction pathway between shear and L . The addition of BAPTA-AM (10 uM), an intracellular P iv calcium chelator, reduced the baseline LP by 40% over a period of 10 minutes, significantly different from 20% due to the “sealing effect” over the same period, suggesting the involvement of a calcium pathway in the regulation of hydraulic conductivity. The transient increase in L P of HUVEC in response to 1 U/ml thrombin, that is known to be calcium dependent, was delayed by incubation with 10 uM BAPTA- AM for 30 minutes, confirming the requirement of calcium in the thrombin response. However, the transient increase of L P by shear stress does not seem to be calcium dependent, because blocking of intracellular calcium pathway by using BATPA-AM had no effect in blocking the shear response of L . P Shear stress also increased nitric oxide (NO) production in HUVEC. However, the shear response of HUVEC L P did not seem to be NO dependent. Increasing NO concentration with SNAP (500 uM), an exogenous NO donor, had no effect on HUVEC LP , while decreasing of NO production with L-NMMA (100 uM), a nitric oxide synthase (NOS) inhibitor, had no effect on shear response of HUVEC v LP . Other pathways than calcium and NO must be involved in the shear response. Finally the total content and phosphorylation state of the tight junction protein occludin was investigated under experimental conditions. 1 U/ml thrombin and 10 dyne/cm2 shear stress increased occludin phosphorylation significantly at time 30 minutes, which was consistent with the time profile of L P in response to thrombin and shear, respectively. 10 ± 15 dye/cm2 oscillatory shear stress, 500 uM SNAP, and 100 uM L-NMMA did not change occludin phosphorylation while they had no effect on L P as compared with controls, suggesting that occludin phosphorylation is a determining factor for hydraulic conductivity. The total amount of occludin did not change after the application of steady and oscillatory shear stresses, but 1 U/ml thrombin decreased occludin total content at 30 minutes, suggesting that phosphorylation of occludin led to occludin degradation and L P elevation.
School Location:USA - Pennsylvania
Source Type:Master's Thesis
Date of Publication: