The endothelial cell layer which lines all blood vessels provides the principal barrier to the transport of water and solutes between the blood and underlying tissue. This barrier is extremely important in maintaining normal tissue homeostasis and its breakdown becomes critical in various disease states such as atherosclerosis (leakage of lipid-bound cholesterol), diabetic retinopathy (leakage of albumin), and in the inflammatory response (leakage of water leading to tissue edema).
To study the endothelial transport barrier we have developed cell culture models using bovine aortic and microvascular endothelial cells, human umbilical vein endothelial cells, and human coronary artery endothelial cells. The endothelial cells are plated on a porous, polycarbonate substrate which is strong enough to support the cells mechanically but which offers negligible resistance to transport of water and solutes. By measuring the water flux across the cell-filter system under a known pressure gradient we are able to determine the hydraulic conductivity of the endothelial monolayer, and by measuring the solute flux across the monolayer under a known concentration gradient we are able to measure the solute permeability coefficient of the monolayer.
A novel apparatus was developed in our laboratory to simultaneously measure water and solute flux across BAEC monolayers after exposure to a transmural pressure gradient. Using this apparatus we have shown that transmural pressure induces mechanical and biological adaptive responses (known as the sealing effect) in endothelial cells characterized by a reduction in hydraulic conductivity and permeability. Our results suggest that BAECs adapt to elevated transmural pressure by mobilizing ZO-1 to intercellular junctions. A mechanical component of the sealing effect appears to reduce the size of a small pore system that allows the transport of water but not dextran or albumin.
Using the same apparatus, we have investigated the transport of LDL across BAEC monolayers under pressurized conditions. The results show that leaky junctions associated with dying or dividing cells are the dominant pathway for LDL transport, while receptor-mediated transcellular transport makes a minor contribution to overall transport. Additional studies focused on the leaky junction pathway of LDL transport, revealed that, while both apoptosis and mitosis contributed to the leaky junction pathway, the rate of endothelial cell apoptosis had a much larger effect on the BAEC monolayer permeability. Current in vitro and animal studies in our lab focus on reducing the permeability of the leaky junction pathway pharmacologically using apoptosis inhibitors as well as drugs that enhance the glycocalyx.
A unique apparatus developed in our laboratory allows us to impose fluid shear stresses on the endothelial monolayer surface while we simultaneously measure the transport properties. Using this apparatus we have been able to show that step changes in fluid shear stress on the order of 10 dynes/cm2 can induce a 10-fold increase in albumin permeability and a 5-fold increase in hydraulic conductivity within 3 hours. The hydraulic conductivity response can be blocked with nitric oxide synthase inhibitors whereas the albumin permeability response cannot be blocked in this manner indicating that there are different transport pathways across the endothelium for water and solutes which are controlled by different signal transduction pathways. We are actively investigating the chemical signaling pathways which control these transport property responses to shear stress as they are of fundamental importance in normal function and pathophysiology. A recent study focused on the effect of shear stress on LDL permeability and found that a short term-shear application (3hr) enhanced LDL permeability of BAEC monloayers while a longer time shear exposure (12hr) reduced LDL permeability to baseline levels. These results were positively correlated to the apoptosis rates of the monolayers after shear exposure.