Published online before print December 18, 2008

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(American Journal of Pathology. 2009;174:34-43.)
© 2009 American Society for Investigative Pathology
DOI: 10.2353/ajpath.2009.080650

The Krebs Cycle and Mitochondrial Mass Are Early Victims of Endothelial Dysfunction

Proteomic Approach

Francesco Addabbo^*, Brian Ratliff^*, Hyeong-Cheon Park^*, Mei-Chuan Kuo^*, Zoltan Ungvari, Anna Ciszar, Boris Krasnikof, Komal Sodhi^*, Fung Zhang^*, Alberto Nasjletti^* and Michael S. Goligorsky^*

From the Department of Medicine and Pharmacology,^* the Renal Research Institute and Division of Nephrology, the Department of Physiology, and the Department of Biochemistry, New York Medical College, Valhalla, New York

Endothelial cell dysfunction is associated with bioavailable nitric oxide deficiency and an excessive generation of reactive oxygen species. We modeled this condition by chronically inhibiting nitric oxide generation with subpressor doses of N^G-monomethyl-L-arginine (L-NMMA) in C57B6 and Tie-2/green fluorescent protein mouse strains. L-NMMA-treated mice exhibited a slight reduction in vasorelaxation ability, as well as detectable abnormalities in soluble adhesion molecules (soluble intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1, and matrix metalloproteinase 9), which represent surrogate indicators of endothelial dysfunction. Proteomic analysis of the isolated microvasculature using 2-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy revealed abnormal expression of a cluster of mitochondrial enzymes, which was confirmed using immunodetection. Aconitase-2 and enoyl-CoA-hydratase-1 expression levels were decreased in L-NMMA-treated animals; this phenotype was absent in nitric oxide synthase-1 and -3 knockout mice. Depletion of aconitase-2 and enoyl-CoA-hydratase-1 resulted in the inhibition of the Krebs cycle and enhanced pyruvate shunting toward the glycolytic pathway. To assess mitochondrial mass in vivo, co-localization of green fluorescent protein and MitoTracker fluorescence was detected by intravital microscopy. Quantitative analysis of fluorescence intensity showed that L-NMMA-treated animals exhibited lower fluorescence of MitoTracker in microvascular endothelia as a result of reduced mitochondrial mass. These findings provide conclusive and unbiased evidence that mitochondriopathy represents an early manifestation of endothelial dysfunction, shifting cell metabolism toward "metabolic hypoxia" through the selective depletion of both aconitase-2 and enoyl-CoA-hydratase-1. These findings may contribute to an early preclinical diagnosis of endothelial dysfunction.

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