Knockdown of paraoxonase 1 expression influences the aging of human dermal microvascular endothelial cells

Abstract

Vascular endothelial cells participate in the regulation of both blood homeostasis and the interaction of blood with surrounding tissues. Skin is one of the most commonly studied tissues for microcirculation research due to its close correlation of cutaneous vascular function, aging and age-related cardiovascular events. To elucidate proteins that determine this correlation between endothelial cell function and aging in the vascular environment of the skin, two-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry were carried out on the plasma samples from six donors in their 20s (young) and six donors in their 60s (old). Two-dimensional gel spots from old donors were compared with two-dimensional gel spots from young donors, and 496 paired spots were observed. Among the 496 paired spots analyzed, 45 proteins, including paraoxonase 1 (PON1) and transthyretin, were downregulated by more than two-fold, and 34 proteins, including Zinc finger protein 774, were upregulated by more than two-fold in the plasma of old donors compared to their amounts in the plasma of young donors. PON1 was selected in this study for further characterization due to its primary location of synthesis in the liver and its association with high-density lipoprotein (HDL) metabolism after secretion into plasma. HDL-associated PON1 has been reported to reduce oxidative stress in lipoproteins and atherosclerotic lesions. To elucidate the role of PON1 on skin aging and determine how it controls cellular senescence, the characteristics of PON1 in human dermal microvascular endothelial cells (HDMECs) were determined. When the expression of endogenous PON1 was knocked-down by small interfering RNA targeting PON1, HDMECs showed characteristic features of cellular senescence such as increases in senescence-associated β-galactosidase stained cells and enlarged and flattened cell morphology. These features were found in aged HDMECs at passage 20 as well. At 48 h post-transfection, the protein expression of p16 in PON1 siRNA-treated HDMECs was higher than that in scrambled siRNA-treated HDMECs. In addition, the expressions of moesin and rho GTP dissociation inhibitor, additional age-related candidate biomarkers, were decreased by PON1 knock-down in HDMECs. In conclusion, these results suggest that PON1 functions as an aging-related protein and plays an important role in the cellular senescence of HDMECs.