title: The Krebs cycle and mitochondrial mass are early victims of endothelial dysfunction: proteomic approach.
abstract: 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.
authors.affiliation: Department of Medicine, Renal Research Institute, Division of Nephrology, New York Medical College, Valhalla, NY 10595, USA.
authors.forename: Francesco
authors.initials: F
authors.lastname: Addabbo
authors.forename: Brian
authors.initials: B
authors.lastname: Ratliff
authors.forename: Hyeong-Cheon
authors.initials: HC
authors.lastname: Park
authors.forename: Mei-Chuan
authors.initials: MC
authors.lastname: Kuo
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Ungvari
authors.forename: Anna
authors.initials: A
authors.lastname: Csiszar
authors.forename: Anna
authors.initials: A
authors.lastname: Ciszar
authors.forename: Boris
authors.initials: B
authors.lastname: Krasnikov
authors.forename: Boris
authors.initials: B
authors.lastname: Krasnikof
authors.forename: Komal
authors.initials: K
authors.lastname: Sodhi
authors.forename: Fung
authors.initials: F
authors.lastname: Zhang
authors.forename: Alberto
authors.initials: A
authors.lastname: Nasjletti
authors.forename: Michael S
authors.initials: MS
authors.lastname: Goligorsky
mesh_terms: D000154:Aconitate Hydratase / Q000378:metabolism; D000818:Animals; D002952:Citric Acid Cycle / Q000502:physiology*; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology*; D004746:Enoyl-CoA Hydratase / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000633:toxicity; D006973:Hypertension / Q000139:chemically induced / Q000378:metabolism; D015151:Immunoblotting; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D055806:Microvessels / Q000378:metabolism / Q000473:pathology; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology*; D009569:Nitric Oxide / Q000037:antagonists & inhibitors; D018384:Oxidative Stress / Q000502:physiology; D040901:Proteomics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014652:Vascular Diseases / Q000378:metabolism; D019323:omega-N-Methylarginine / Q000633:toxicity
chemical_list: D004791:Enzyme Inhibitors; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D004746:Enoyl-CoA Hydratase; D000154:Aconitate Hydratase