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. 2010 Jan 15;48(2):275-83.
doi: 10.1016/j.freeradbiomed.2009.10.050. Epub 2009 Oct 30.

A key role for mitochondria in endothelial signaling by plasma cysteine/cystine redox potential (V体育平台登录)

Young-Mi Go  1 , Heonyong Park, Michael Koval, Michael Orr, Matthew Reed, Yongliang Liang, Debra Smith, Jan Pohl, Dean P Jones
Affiliations

A key role for mitochondria in endothelial signaling by plasma cysteine/cystine redox potential

Young-Mi Go et al. Free Radic Biol Med. .

Abstract

The redox potential of the plasma cysteine/cystine couple (E(h)CySS) is oxidized in association with risk factors for cardiovascular disease (CVD), including age, smoking, type 2 diabetes, obesity, and alcohol abuse. Previous in vitro findings support a cause-effect relationship for extracellular E(h)CySS in cell signaling pathways associated with CVD, including those controlling monocyte adhesion to endothelial cells. In this study, we provide evidence that mitochondria are a major source of reactive oxygen species (ROS) in the signaling response to a more oxidized extracellular E(h)CySS. This increase in ROS was blocked by overexpression of mitochondrial thioredoxin-2 (Trx2) in endothelial cells from Trx2-transgenic mice, suggesting that mitochondrial thiol antioxidant status plays a key role in this redox signaling mechanism. Mass spectrometry-based redox proteomics showed that several classes of plasma membrane and cytoskeletal proteins involved in inflammation responded to this redox switch, including vascular cell adhesion molecule, integrins, actin, and several Ras family GTPases VSports手机版. Together, the data show that the proinflammatory effects of oxidized plasma E(h)CySS are due to a mitochondrial signaling pathway that is mediated through redox control of downstream effector proteins. .

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Figures

Fig. 1
Fig. 1
Oxidized extracellular EhCySS (0 mV) stimulates mitochondrial Trx2 oxidation in MAEC. (A) MAEC from Trx2 transgenic and littermate control mice were verified by AcLDL labeling. (B) Trx2 overexpression in Tg MAEC was verified by PCR (top left) and Western blotting (bottom left) with no effect on endogenous mouse Trx2 (mTrx2) or Trx1 (mTrx1) expression. Western blots were probed with antibodies specific to V5 epitope, mTrx2, and mTrx1. Mitochondria-localized expression of V5-hTrx2 in Tg is shown (right; red, V5-hTrx2; blue, Hoechst). (C) Trx2 (top) and Trx1 (bottom) redox states at their respective extracellular Eh values. Cytoplasmic (Trx1) and nuclear (NLS-Trx1) Trx1 redox states were examined in cells transfected with or without NLS-Trx1. DTT- and H2O2-treated samples were used for fully reduced and oxidized controls, respectively. (D) Cellular GSH (bottom), GSSG (bottom), and GSH/GSSG redox states (top) at various Eh. Data presented are means±SEM of triplicates from two experiments.
Fig. 2
Fig. 2
Extracellular EhCySS changed with time. Confluent WT MAEC were incubated with various initial EhCySS (intended redox values −150, −80, 0 mV) and assayed for Cys and CySS in culture medium as a function of time. EhCySS calculated from the Nernst equation are shown as means±SEM; n=3.
Fig. 3
Fig. 3
Proinflammatory response of MAEC to oxidized extracellular Eh is signaled through a pathway dependent upon membranal thiols and mitochondrial ROS. WT and Tg MAEC exposed to EhCySS (−150, −80, 0 mV) for 3 h were examined for cellular and mitochondrial ROS by quantifying fluorescence of (A) DCF and (B) MitoSOX, respectively. Cell images (B, right) to visualize mitochondrial ROS increase are representative of WT MAEC treated with −150 mV (1) and 0 mV (2) and Tg MAEC treated with 0 mV (3) from three experiments. (C) WT MAEC pretreatment with the membrane-nonpermeative thiol-alkylating reagents qBBr and AMS (500 μM each) prevented Eh-dependent mitochondrial ROS increase measured by MitoSOX fluorescence (left) and visualized by fluorescence microscopy (right, compare 5 and 6 to 4). Data are means±SEM (n=8). *P<0.05 vs group of WT treated with 0 mV. (D) Increase in mitochondrial ROS by 0 mV was verified by increase in MitoTracker fluorescence (red, 0 mV), whereas AMS pretreatment (red, AMS+0 mV) and Trx2 overexpression (red, Tg+0 mV) inhibited ROS increase. No changes in actin filaments (F-actin) or nuclei were observed with Alexa Fluor 488 phalloidin and Hoechst 33342 staining, respectively.
Fig. 4
Fig. 4
Increase in ROS level by oxidized EhCySS is regulated by mitochondrial electron transport complex I. WT MAEC were pretreated with rotenone (2 μM) or antimycin A (5 μM) for 2 h before oxidized EhCySS treatment. Cells were then examined for ROS levels by measuring DCF fluorescence. (A) ROS levels induced by various EhCySS (−150, −80, 0 mV) were quantified without (None) or with rotenone or antimycin A. (B) Rotenone (left) and antimycin A (right)-stimulated levels of ROS at various EhCySS were quantified.
Fig. 5
Fig. 5
Extracellular Eh-dependent redox changes in plasma-membrane- and actin-associated proteins. The membrane-enriched fraction isolated from MAEC (WT) treated with −150 or 0 mV was analyzed by redox ICAT-based mass spectrometry. (A) Distribution of the oxidized states of the peptides (1756 peptides at −150 mV; 1888 peptides at 0 mV) relevant to 144 proteins (−150 and 0 mV) from the membrane-enriched fraction. The mean values of oxidation were 33 (SD of 17%) and 42% (SD of 20%) for the −150 and 0 mV treatments, respectively. (B) Redox changes in 34 actin and actin-related proteins (416 peptides at −150 mV; 453 peptides at 0 mV) were examined. Mean values of the percentage of oxidized state for −150 and 0 mV were 30 (SD of 14%) and 42% (SD of 15%), respectively.
Fig. 6
Fig. 6
Proposed scheme for mitochondrial redox signaling in response to oxidized extracellular Eh. Eh-induced oxidation of plasma membrane (PM) and cytoskeleton proteins stimulates ROS generation in mitochondria that is blocked by Trx2. H2O2 from the mitochondria triggers inflammatory signaling including NF-κB activation and subsequent gene expression (cell adhesion molecules, integrins, cytoskeletal proteins). H2O2 can affect cell structure by affecting actin dynamics. Changes in the endothelial cell structure and increases in cell adhesion molecules result in an increase in monocyte recruitment as an early event of atherosclerosis.
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