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Am J Physiol Heart Circ Physiol 283: H750-H759, 2002. First published March 28, 2002; doi:10.1152/ajpheart.00029.2002
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Vol. 283, Issue 2, H750-H759, August 2002

Native LDL and minimally oxidized LDL differentially regulate superoxide anion in vascular endothelium in situ

David W. Stepp1, Jingsong Ou2,4, Allan W. Ackerman2, Scott Welak2,6, David Klick3, and Kirkwood A. Pritchard Jr.2,3,4,5

Departments of 1 Physiology, 2 Surgery (Division of Pediatric Surgery), 3 Pharmacology and Toxicology, 4 Cardiovascular Center, 5 Free Radical Research Center, and 6 Summer Practicum in Undergraduate Research Studies Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Low-density lipoprotein (LDL) and its oxidized derivatives are hypothesized to impair vascular function by increasing superoxide anion (O<UP><SUB>2</SUB><SUP>−</SUP></UP>·). To investigate mechanisms in situ, isolated carotid arteries were incubated with native LDL (nLDL) or minimally oxidized LDL (mmLDL). With the use of en face fluorescent confocal microscopy and hydroethidine, an oxidant-sensitive fluorescent probe, we found that nLDL increased O<UP><SUB>2</SUB><SUP>−</SUP></UP>· in vascular endothelium greater than fourfold by an Nomega -nitro-L-arginine methyl ester (L-NAME)-inhibitable mechanism. In contrast, mmLDL increased O<UP><SUB>2</SUB><SUP>−</SUP></UP>· in vascular endothelium greater than eightfold by mechanisms that were partially inhibited by L-NAME and allopurinol and essentially ablated by diphenyleneiodium. These data indicate that both nLDL and mmLDL uncouple endothelial nitric oxide synthase (eNOS) activity and that mmLDL also activates xanthine oxidase and NADPH oxidoreductase to induce greater increases in O<UP><SUB>2</SUB><SUP>−</SUP></UP>· generation than nLDL. Western analysis revealed that both lipoproteins inhibited A-23187-stimulated association of heat shock protein 90 (HSP90) with eNOS without inhibiting phosphorylation of eNOS at serine-1179 (phospho-eNOS), an immunological index of electron flow through the enzyme. As HSP90 mediates the balance of ·NO and O<UP><SUB>2</SUB><SUP>−</SUP></UP>· generation by eNOS, these data provide new insight into the mechanisms by which oxidative stress, induced by nLDL and mmLDL, uncouple eNOS activity to increase endothelial O<UP><SUB>2</SUB><SUP>−</SUP></UP>· generation.

native low-density lipoprotein; minimally oxidized low-density lipoprotein; smooth muscle cells; superoxide anion; confocal microscopy; hydroethidine


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