Energy depletion causes endothelial hyperpermeability in hyperglycemia

Diabetes is strongly correlated to an increased risk of cardiovascular disease (CVD). Reduced endothelial barrier function has been identified as a result of unregulated hyperglycemia in diabetes, and as risk factor in the development of atherosclerosis. Endothelial barrier dysfunction is measured by hyperpermeability to water and solutes, and by reduced dynamic response to a change in pressure. To investigate the relationship between endothelial dysfunction and high blood glucose we cultured bovine aortic endothelial cells (BAEC) in confluent monolayers on polycarbonate filters in a series of increasing glucose concentrations from 5mM (normal) to 30mM (hyperglycemic) for use in a pressure-flow apparatus, with which we measured the hydraulic conductivity of water, and the convective and diffusive transport of low-density lipoprotein (LDL) through the monolayers in response to a 10-cmH2O transmural pressure gradient. We developed a mathematical model of intracellular protein diffusion to extract parameters from this data to quantify the initial water volume flow rate (flux), the rate of attenuation of flow by the endothelial sealing effect, and the steady-state flux. Analysis of these extracted parameters revealed that endothelial monolayers grown in high glucose media have a statistically higher permeability to water and solute compared to cells grown in normal glucose media. Steady state hydraulic conductivity increased by 57.9 +/- 10.0% at 30 mM glucose, and by 30.1 +/- 12.3% at the physiologically relevant level of 10 mM glucose, compared to normal. A similar trend was observed for the initial water flux for the same conditions. Permeability to LDL increased by 46.8 +/- 14.1% at 30 mM glucose, and 42.1 +/- 8.1% at 10 mM glucose. Further analysis revealed that the dynamic response of cells grown in high glucose was significantly slower at physiological levels of hyperglycemia. We conducted parallel investigations into the effect of the depletion of adenosine tri-phosphate (ATP), caused either by (1) high glucose mediated activation of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), or (2) antimycin-A induced chemical impairment of the mitochondria. Our results support the hypothesis that increased steady-state hydraulic conductivity and LDL permeability are strongly linked to ATP depletion independent of mechanism.