BACKGROUND: Aquaporin (AQP)-1 is expressed in most microvasculature endothelial cells forming water channels that play major roles in a variety of physiologic processes. Our aim was to investigate the regulatory functions of AQP1 on trancellular and paracellular permeability.
METHODS:
We designed, synthesized, and used small interfering RNAs (siRNAs) selective for AQP1 and investigated their effectiveness in altering AQP1-mediated permeability in human pulmonary microvascular endothelial cells.
RESULTS:
Twenty-four hours after transfection of ECs with siRNAs targeting two different regions of the AQP1 transcript, AQP1 protein was inhibited by 47.8% to 74.6%. siRNAs containing the same percent of base pairs as... More
BACKGROUND: Aquaporin (AQP)-1 is expressed in most microvasculature endothelial cells forming water channels that play major roles in a variety of physiologic processes. Our aim was to investigate the regulatory functions of AQP1 on trancellular and paracellular permeability.
METHODS:
We designed, synthesized, and used small interfering RNAs (siRNAs) selective for AQP1 and investigated their effectiveness in altering AQP1-mediated permeability in human pulmonary microvascular endothelial cells.
RESULTS:
Twenty-four hours after transfection of ECs with siRNAs targeting two different regions of the AQP1 transcript, AQP1 protein was inhibited by 47.8% to 74.6%. siRNAs containing the same percent of base pairs as the AQP1-siRNAs but in random sequence (i.e., scrambled siRNAs) had no effect. Suppression of AQP1 expression in ECs resulted in decreases in epithelial Na+ channel (ENaC) and Na-K ATPase of ECs, and the suppression ENaC α, β, γ, and Na-K ATPase were 43.1% to 48.2%,70.0% to 76.0%, 52.6% to 55.0%, and 72.7% to 79.3%, respectively. The reduced AQP1expression also resulted in decreased cell-cell junction protein level of VE-cadherin, which was suppressed by 36.5% to 59.5% but had no effect on occludin protein. Tube formation assay and tranwell assay showed AQP1 siRNAs induced high permeability of human pulmonary microvascular endothelial cells. Rho-kinase (ROCK) I and ROCK II were increased by 46.0% to 50.0% and 59% to 81%, respectively, AQP1 siRNA treatment accelerated the formation of F-actin bundles, demonstrating the activation of Rho/ROCK signaling pathway, and decreased mitochondrial membrane potential after AQP1 siRNA treatment, showing an important event of apoptosis process.
CONCLUSIONS:
The data demonstrate that AQP1 is a critical participate in regulating endothelial permeability and barrier function and provide direct evidence of the contribution of AQP1 to blood vessel formation.
