ABSTRACT
Mechanical forces and the activation of the renin-angiotensin system (RAS) may alter the NO/O2(*-) balance, imparing endothelial nitric oxide (NO) availability. This study investigates the link between RAS and NO/O2(*-) balance in human aortic endothelial cells (HAEC) exposed to pulsatile stretch with and without ACE inhibitor quinaprilat or angiotensin II type 1 (AT(1)) receptor antagonist losartan. Pulsatile stretch increased Ang II levels and O2(*-) production, reducing NO release. RAS blockade with quinaprilat or losartan restored the balance between NO and O2(*-). These results provide a molecular basis for understanding the vascular protective effects of ACE inhibition and AT(1) receptor antagonism.
Subject(s)
Angiotensin II Type 1 Receptor Blockers/pharmacology , Angiotensin II/metabolism , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Endothelial Cells/drug effects , Endothelial Cells/physiology , Oxidative Stress , Biomechanical Phenomena , Cells, Cultured , Endothelial Cells/metabolism , Hemorheology , Humans , Losartan/pharmacology , Models, Biological , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/metabolism , Pulsatile Flow , Renin-Angiotensin System/drug effects , Renin-Angiotensin System/physiology , Stress, Mechanical , Superoxides/metabolism , Tetrahydroisoquinolines/pharmacologyABSTRACT
BACKGROUND: Enhanced production of reactive oxygen species (ROS) has been recognized as the major determinant of age-related endothelial dysfunction. The p66shc protein controls cellular responses to oxidative stress. Mice lacking p66shc (p66shc-/-) have increased resistance to ROS and a 30% prolonged life span. The present study investigates age-dependent changes of endothelial function in this model. METHODS AND RESULTS: Aortic rings from young and old p66shc-/- or wild-type (WT) mice were suspended for isometric tension recording. Nitric oxide (NO) release was measured by a porphyrinic microsensor. Expression of endothelial NO synthase (eNOS), inducible NOS (iNOS), superoxide dismutase, and nitrotyrosine-containing proteins was assessed by Western blotting. Nitrotyrosine residues were also identified by immunohistochemistry. Superoxide (O2-) production was determined by coelenterazine-enhanced chemiluminescence. Endothelium-dependent relaxation in response to acetylcholine was age-dependently impaired in WT mice but not in p66shc-/- mice. Accordingly, an age-related decline of NO release was found in WT but not in p66shc-/- mice. The expression of eNOS and manganese superoxide dismutase was not affected by aging either in WT or in p66shc-/- mice, whereas iNOS was upregulated only in old WT mice. It is interesting that old WT mice displayed a significant increase of O2- production as well as of nitrotyrosine expression compared with young animals. Such age-dependent changes were not found in p66shc-/- mice. CONCLUSIONS: We report that inactivation of the p66shc gene protects against age-dependent, ROS-mediated endothelial dysfunction. These findings suggest that the p66shc is part of a signal transduction pathway also relevant to endothelial integrity and may represent a novel target to prevent vascular aging.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Aging/physiology , Endothelium, Vascular/physiopathology , Adaptor Proteins, Signal Transducing/deficiency , Adaptor Proteins, Signal Transducing/genetics , Animals , Aorta , Endothelium, Vascular/metabolism , Isometric Contraction , Longevity/genetics , Male , Mice , Mice, Knockout , Nitric Oxide/metabolism , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Nitric Oxide Synthase Type III , Oxidative Stress , Proteins/chemistry , Reactive Oxygen Species , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 1 , Superoxide Dismutase/metabolism , Tyrosine/analogs & derivatives , Tyrosine/analysis , VasoconstrictionABSTRACT
BACKGROUND: Prostaglandins generated by cyclooxygenase (COX) have been implicated in hyperglycemia-induced endothelial dysfunction. However, the role of individual COX isoenzymes as well as the molecular mechanisms linking oxidative stress and endothelial dysfunction in diabetes remains to be clarified. METHODS AND RESULTS: Human aortic endothelial cells were exposed to normal (5.5 mmol/L) and high (22.2 mmol/L) glucose. Glucose selectively increased mRNA and protein expression of COX-2. Its upregulation was associated with an increase of thromboxane A2 and a reduction of prostacyclin (PGI2) release. Glucose-induced activation of PKC resulted in the formation of peroxynitrite and tyrosine nitration of PGI2 synthase. NO release was reduced despite 2-fold increase of endothelial NO synthase expression. Phorbol ester caused an increase of COX-2 and endothelial NO synthase expression similar to that elicited by glucose. These effects were prevented by the PKC inhibitor calphostin C. N-acetylcysteine, vitamin C, and calphostin C prevented ROS formation, restored NO release, and reduced colocalization of nitrotyrosine and PGI2 synthase. Expression of p22(phox), a subunit of NAD(P)H oxidase, was increased, and diphenyleneiodonium inhibited ROS formation. By contrast, indomethacin did not affect glucose-induced ROS generation. CONCLUSIONS: Thus, high glucose, via PKC signaling, induces oxidative stress and upregulation of COX-2, resulting in reduced NO availability and altered prostanoid profile.
