Cysteine/cystine redox signaling in cardiovascular disease

Abstract

Extracellular thiol/disulfide redox environments are highly regulated in healthy individuals. The major thiol/disulfide redox couple in human plasma is cysteine (Cys) and its disulfide form, cystine (CySS). Oxidation of this redox couple, measured as a more positive steady-state redox potential (E(h)), is associated with risk factors for cardiovascular disease (CVD), including aging, smoking, obesity, and alcohol abuse. Rodent and vascular cell studies show that the extracellular redox state of Cys/CySS (E(h)CySS) can play a vital role in controlling CVD through proinflammatory signaling. This inflammatory signaling is regulated by cell-surface protein redox state and involves mitochondrial oxidation, nuclear factor-κB activation, and elevated expression of genes for monocyte recruitment to endothelial cells. Gene array and proteomics studies reveal the global nature of redox effects, and different cell types, e.g., endothelial cells, monocytes, fibroblasts, and epithelial cells, show cell-specific redox responses with different phenotypic traits, e.g., proliferation and apoptosis, which can contribute to CVD. The critical nature of the proinflammatory redox signaling and cell biology associated with E(h)CySS supports the use of plasma levels of Cys, CySS, and E(h)CySS as key indicators of vascular health. Plasma redox-state-based pharmacologic interventions to control or improve E(h)CySS may be effective in preventing CVD onset or progression.

Figure 1

Proposed scheme for inflammatory signaling in response to oxidized extracellular E_hCySS. Extracellular E_hCySS-induced oxidation of plasma membrane and cytoskeleton proteins stimulates ROS generation in mitochondria that is blocked by Trx2. This signaling mechanism was also blocked by inhibiting plasma membrane thiol/disulfide regulation by AMS and qBBr. H₂O₂ from the mitochondria triggers inflammatory signaling including NF-κB activation and increased expression of cell adhesion molecules. H₂O₂ can affect cell structure by affecting actin dynamics. Monocytes stimulate proinflammatory signaling by generating inflammatory cytokine, IL-1β and H₂O₂ in respond to extracellular E_hCySS. Changes in the endothelial cell structure and increases in cell adhesion molecules and cytokines result in an increase in monocyte recruitment as an early event of atherosclerosis [24-26, 47]. GPx, glutathione peroxidases; ICAM, intercellular cell adhesion molecule; IL-1β, interleukin 1β; PECAM; platelet endothelial cell adhesion molecule; Prx, peroxiredoxins

Figure 2

Steady-state redox potentials of the major redox couples in subcellular compartments. Results from multiple cell types provide evidence for stably non-equilibrium relationships between redox couples in different compartments. Representative values for NADP⁺/NADPH, GSH/GSSG, Trx(SH)2/Trx(SS), and Cys/CySS in cytoplasm, mitochondria, nucleus, cytoplasm, endoplasmic reticulum, and extracellular space are based upon data reviewed in detail [92]. The presence of these couples in each distinct compartment allows for the independent, specific control of redox processes that occur in these regions.

Figure 3

Proteins oxidized by extracellular E_hCySS control cell signaling and morphology. Redox ICAT-based mass spectrometry identified endothelial proteins that alter redox state in response to extracelluar E_hCySS (supplementary data from the previous study [26]). Ingenuity Pathway Analysis shows that proteins oxidized by more positive extracellular E_hCySS (red) are in networks for cell signaling and cell morphology in association with angiotensin- and retinoic acid-dependent pathways. Protein names of symbols are as follows: AGT, angiotensinogen; ATP1B1, ATPase, Na⁺/K⁺ transporting, beta-1 polypeptide; ATP5C1; ATP synthase, H⁺ transporting, mitochondrial F₁ complex, gamma polypeptide 1; ATP5H, ATP synthase, H⁺ transporting, mitochondrial F₀ complex, subunit d; CKAP4, cytoskeleton-associated protein 4; DYNC1H1, dynein, cytoplasmic 1, heavy chain 1; EEF1A1, eukaryotic translation elongation factor 1 alpha 1; EEF1G, eukaryotic translation elongation factor 1 gamma; HNRPA3, heterogeneous nuclear ribonucleoprotein A3; MAP2, microtubule-associated protein 2; MYH9, myosin, heavy chain 9, non-muscle; MYO1C, myosin IC; NDUFS1, NADH dehydrogenase (ubiquinone) Fe-S protein 1; POSTN, periostin, osteoblast specific factor; RPL4, ribosomal protein L4; RPS16, ribosomal protein S16; SDHA, succinate dehydrogenase complex, subunit A; SLC5A3, solute carrier family 5, member 3; VCP, valosin-containing protein.

Figure 4

Cys/CySS redox interactions with sulfur amino acid metabolism. Metabolic regulation of plasma Cys, CySS, and E_hCySS is controlled by diet, body protein turnover, sulfur amino acid metabolism in peripheral tissues and cells, and Cys/CySS shuttle mechanism. 3), 4), 5), and 11) indicate a key process in controlling systemic E_hCySS in plasma. See text for additional details.

Figure 5

Interrelationships between Cys and GSH systems. Cysteine (Cys) and glutathione (GSH) are interconnected by both carbon skeleton changes and oxidation-reduction of thiols. The carbon skeleton changes involve extracellular hydrolysis of GSH to CysGly and then to Cys (large and hatch style arrows, left) and intracellular synthesis of GSH in 2 steps from glutamate, cysteine and glycine (large and hatch style arrow, right). The oxidation-reduction reactions in the plasma involve GSH and CysGly reactions with CySS. The products are hydrolyzed by the same enzymes that hydrolyze GSH and CysGly. The cellular reactions are less clear. No enzyme is known that reduces CySS in mammalian tissues. Reaction with GSH can occur, but the non-catalyzed rate appears to be too slow to account for the rate of CySS reduction [88]. The product of this reaction is CySSG, which is a relatively poor substrate for GSSG reductase (GR). CySSG can also transfer the GSH moiety to glutaredoxin (Grx) with release of Cys. Reaction of the GSH-Grx with GSH results in release of GSSG, which is a substrate for GSSG reductase. Thioredoxin (Trx) also has a relatively low capacity to reduce CySS. The x_C⁻, Asc, and MRP1 transporter systems for CySS, Cys, and GSH respectively are shown, and there are multiple transporters for each.

Figure 6

Diverse signaling responses by extracellular E_hCySS. Reduced E_hCySS stimulates growth and proliferation mechanisms in Caco2 epithelial cells [27, 42, 98] and vascular cells, monocytes [24]. Oxidized E_hCySS stimulates detoxification and proliferation mechanisms in NIH3T3 fibroblasts [118, 135]. Oxidized E_hCySS stimulates tBH-induced cell death mechanism in human retina pigment epithelial cells [28] and stimulate inflammatory mechanisms in vascular cells including human monocytes and endothelial cells [24-26, 47].

Figure 7

Integration of extracellular E_hCySS with other oxidative stimuli in cellular responses of CVD. Responses of endothelial cells and fibroblasts to oxidative stimuli of hypoxia and angiotensin II have been well described [128, 129, 131]. Accumulating evidence suggests that oxidized E_hCySS can contribute to the cellular responses of CVD. Possible effects in endothelial cells (left) include activation of proinflammatory signaling via NF-κB and result in increased VCAM-1, P-selectin and proinflmmatory cytokines. Additional cell stress responses are mediated through mitochondria and possibly p53, which can result in apoptosis. Effects on fibroblasts (right) include proliferative signaling and activation of profibrotic signaling by TGF-β1. This increased activity can contribute to wound healing and also to fibrous cap formation.