Oxidative stress and redox-signal pathways are known to be involved in endothelial apoptosis induced by serum depletion. However, the associated mechanism is not well understood and thus, was investigated in the prese...Oxidative stress and redox-signal pathways are known to be involved in endothelial apoptosis induced by serum depletion. However, the associated mechanism is not well understood and thus, was investigated in the present study focusing on NADPH oxidases (NOX). Serum removal from the culture medium led to an increase in reactive oxygen species (ROS) production and apoptotic death of human umbilical vein endothelial cells. Serum depletion also increased the gene expression of the NOX2 and NOX4 subunits. The selective suppression of NOX4 expression by small interfering RNA (siRNA) attenuated ROS production and cell death due to serum-depletion whereas siRNA for NOX2 increased cell death. Expression of exogenous NOX2 or NOX4 subunit alone had no significant effects on ROS production or cell death. Coexpression of the subunits of the NOX4 complex (NOX4 and p22phox) or the NOX2 complex (NOX2, p22phox, p47phox and p67phox) increased ROS production and cell death under serum-depleted conditions. This study suggests that endothelial cell survival and death are differentially regulated by expression levels of the subunits of NOX2 and NOX4 complexes.展开更多
Laminar shear stress (LSS) due to pulsatile blood flow enhances endothelial function by multiple mechanisms including NO production. Red wine and its constituent, resveratrol, have also been postulated to provide vasc...Laminar shear stress (LSS) due to pulsatile blood flow enhances endothelial function by multiple mechanisms including NO production. Red wine and its constituent, resveratrol, have also been postulated to provide vascular protective effects. The aim of the present study was to compare the effects of mechanical LSS and pharmacological resveratrol treatments on the endothelial citrulline-NO cycle. Human umbilical vein endothelial cells (HUVECs) were treated with LSS (12 dyn·cm-2) or resveratrol (25 - 100 μM). The expressions of argininosuccinate synthetase 1 (ASS1), argininosuccinate lyase (ASL), nitric oxide synthase 3 (NOS3) and cationic amino acid transporter 1 (CAT1), and the production of NO were determined. The expressions of Kruppel-like factor (KLF) 2 and KLF4 as upstream regulators of ASS1 and NOS3 were also analyzed. LSS strongly increased the mRNA levels of ASS1 (8.3 fold) and NOS3 (5.4 fold) without significant effects on ASL and CAT1 mRNAs. Resveratrol increased the ASS1 mRNA level in a dose-dependent manner up to 3.8 fold at 100 μM. The effects of resveratrol on the expressions of KLF2 and KLF4 mRNAs were smaller than those of LSS. Protein levels of ASS1 and NOS3, and NO production were markedly increased by LSS but resveratrol (50 μM) increased only ASS1 protein level. The results of the current study showed that LSS had greater effects on the citrulline-NO cycle activity leading to NO production, compared to resveratrol. Because resveratrol was not so effective at stimulating the endothelial citrulline-NO cycle, further studies are needed to find more potent drugs that increase the expression of ASS1 and NOS3 genes.展开更多
文摘Oxidative stress and redox-signal pathways are known to be involved in endothelial apoptosis induced by serum depletion. However, the associated mechanism is not well understood and thus, was investigated in the present study focusing on NADPH oxidases (NOX). Serum removal from the culture medium led to an increase in reactive oxygen species (ROS) production and apoptotic death of human umbilical vein endothelial cells. Serum depletion also increased the gene expression of the NOX2 and NOX4 subunits. The selective suppression of NOX4 expression by small interfering RNA (siRNA) attenuated ROS production and cell death due to serum-depletion whereas siRNA for NOX2 increased cell death. Expression of exogenous NOX2 or NOX4 subunit alone had no significant effects on ROS production or cell death. Coexpression of the subunits of the NOX4 complex (NOX4 and p22phox) or the NOX2 complex (NOX2, p22phox, p47phox and p67phox) increased ROS production and cell death under serum-depleted conditions. This study suggests that endothelial cell survival and death are differentially regulated by expression levels of the subunits of NOX2 and NOX4 complexes.
文摘Laminar shear stress (LSS) due to pulsatile blood flow enhances endothelial function by multiple mechanisms including NO production. Red wine and its constituent, resveratrol, have also been postulated to provide vascular protective effects. The aim of the present study was to compare the effects of mechanical LSS and pharmacological resveratrol treatments on the endothelial citrulline-NO cycle. Human umbilical vein endothelial cells (HUVECs) were treated with LSS (12 dyn·cm-2) or resveratrol (25 - 100 μM). The expressions of argininosuccinate synthetase 1 (ASS1), argininosuccinate lyase (ASL), nitric oxide synthase 3 (NOS3) and cationic amino acid transporter 1 (CAT1), and the production of NO were determined. The expressions of Kruppel-like factor (KLF) 2 and KLF4 as upstream regulators of ASS1 and NOS3 were also analyzed. LSS strongly increased the mRNA levels of ASS1 (8.3 fold) and NOS3 (5.4 fold) without significant effects on ASL and CAT1 mRNAs. Resveratrol increased the ASS1 mRNA level in a dose-dependent manner up to 3.8 fold at 100 μM. The effects of resveratrol on the expressions of KLF2 and KLF4 mRNAs were smaller than those of LSS. Protein levels of ASS1 and NOS3, and NO production were markedly increased by LSS but resveratrol (50 μM) increased only ASS1 protein level. The results of the current study showed that LSS had greater effects on the citrulline-NO cycle activity leading to NO production, compared to resveratrol. Because resveratrol was not so effective at stimulating the endothelial citrulline-NO cycle, further studies are needed to find more potent drugs that increase the expression of ASS1 and NOS3 genes.
