Chengjin Gao1, Jiajun Tang, Rongrong Li, Jingning Huan. 1. Department of Burn and Plastic Surgery, Ninth People's Hospital, Rui Jin Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, China.
Abstract
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.
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.
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