Endothelial Dysfunction, and A Prothrombotic, Proinflammatory Phenotype Is Caused by Loss of Mitochondrial Thioredoxin Reductase in Endothelium.

OBJECTIVE: Although the investigation on the importance of mitochondria-derived reactive oxygen species (ROS) in endothelial function has been gaining momentum, little is known on the precise role of the individual components involved in the maintenance of a delicate ROS balance. Here we studied the impact of an ongoing dysregulated redox homeostasis by examining the effects of endothelial cell-specific deletion of murine thioredoxin reductase 2 (Txnrd2), a key enzyme of mitochondrial redox control. APPROACH AND RESULTS: We analyzed the impact of an inducible, endothelial cell-specific deletion of Txnrd2 on vascular remodeling in the adult mouse after femoral artery ligation. Laser Doppler analysis and histology revealed impaired angiogenesis and arteriogenesis. In addition, endothelial loss of Txnrd2 resulted in a prothrombotic, proinflammatory vascular phenotype, manifested as intravascular cellular deposits, as well as microthrombi. This phenotype was confirmed by an increased leukocyte response toward interleukin-1 in the mouse cremaster model. In vitro, we could confirm the attenuated angiogenesis measured in vivo, which was accompanied by increased ROS and an impaired mitochondrial membrane potential. Ex vivo analysis of femoral arteries revealed reduced flow-dependent vasodilation in endothelial cell Txnrd2-deficient mice. This endothelial dysfunction could be, at least partly, ascribed to inadequate nitric oxide signaling. CONCLUSIONS: We conclude that the maintenance of mitochondrial ROS via Txnrd2 in endothelial cells is necessary for an intact vascular homeostasis and remodeling and that Txnrd2 plays a vitally important role in balancing mitochondrial ROS production in the endothelium.

TrxR2 deficiencies promote chondrogenic differentiation and induce apoptosis of chondrocytes through mitochondrial reactive oxygen species.

Thioredoxin reductase 2 (TrxR2) is a selenium (Se) containing protein. Se deficiency is associated with an endemic osteoarthropathy characterized by impaired cartilage formation. It is unclear whether TrxR2 have roles in cartilage function. We examined the effects of TrxR2 on chondrogenic ATDC5 cells through shRNA-mediated gene silencing of TrxR2. We demonstrated TrxR2 deficiencies could enhance chondrogenic differentiation and apoptosis of ATDC5 cells. TrxR2 deficiencies increased accumulation of cartilage glycosaminoglycans (GAGs) and mineralization. TrxR2 deficiencies also stimulated expression of extracellular (ECM) gene including Collagen II and Aggrecan. The enhanced chondrogenic properties were further confirmed by activation of Akt signaling which are required for chondrogenesis. In addition, TrxR2 deficiencies promoted chondrocyte proliferation through acceleration of cell cycle progression by increase in both S and G2/M phase cell distribution accompanied with induction of parathyroid hormone-related protein (PTHrP). Moreover, TrxR2 deficiencies induced chondrocyte death via apoptosis and increased cell sensitivity to exogenous oxidative stress. Furthermore, TrxR2 deficiencies induced emission of mitochondrial reactive oxygen species (ROS) without alteration of mitochondrial membrane potential and intracellular ATP content. Finally, treatment of TrxR2 deficiency cells with N-acetylcysteine (NAC) inhibited mitochondrial ROS production and chondrocyte apoptosis. NAC also prevented chondrogenic differentiation of TrxR2 deficiency cells by suppression of ECM gene expression, GAGs accumulation and mineralization, as well as attenuation of Akt signaling. Thus, TrxR2-mediated mitochondrial integrity is indispensable for chondrogenic differentiation of ATDC5 cells. TrxR2 deficiency-induced impaired proliferation and death of chondrocytes may be the pathological mechanism of the osteoarthropathy due to Se deficiency. Notably, this study also uncover the roles of mitochondrial ROS which could stimulate cartilage ECM synthesis that offer novel insights for development of therapeutic agent to prevent cartilage degeneration in human disease.

Heart-Specific Knockout of the Mitochondrial Thioredoxin Reductase (Txnrd2) Induces Metabolic and Contractile Dysfunction in the Aging Myocardium.

BACKGROUND: Ubiquitous deletion of thioredoxin reductase 2 (Txnrd2) in mice is embryonically lethal and associated with abnormal heart development, while constitutive, heart-specific Txnrd2 inactivation leads to dilated cardiomyopathy and perinatal death. The significance of Txnrd2 in aging cardiomyocytes, however, has not yet been examined. METHODS AND RESULTS: The tamoxifen-inducible heart-specific αMHC-MerCreMer transgene was used to inactivate loxP-flanked Txnrd2 alleles in adult mice. Hearts and isolated mitochondria from aged knockout mice were morphologically and functionally analyzed. Echocardiography revealed a significant increase in left ventricular end-systolic diameters in knockouts. Fractional shortening and ejection fraction were decreased compared with controls. Ultrastructural analysis of cardiomyocytes of aged mice showed mitochondrial degeneration and accumulation of autophagic bodies. A dysregulated autophagic activity was supported by higher levels of lysosome-associated membrane protein 1 (LAMP1), microtubule-associated protein 1A/1B-light chain 3-I (LC3-I), and p62 in knockout hearts. Isolated Txnrd2-deficient mitochondria used less oxygen and tended to produce more reactive oxygen species. Chronic hypoxia inducible factor 1, α subunit stabilization and altered transcriptional and metabolic signatures indicated that energy metabolism is deregulated. CONCLUSIONS: These results imply a novel role of Txnrd2 in sustaining heart function during aging and suggest that Txnrd2 may be a modifier of heart failure.

Deletion of thioredoxin reductase and effects of selenite and selenate toxicity in Caenorhabditis elegans.

Thioredoxin reductase-1 (TRXR-1) is the sole selenoprotein in C. elegans, and selenite is a substrate for thioredoxin reductase, so TRXR-1 may play a role in metabolism of selenium (Se) to toxic forms. To study the role of TRXR in Se toxicity, we cultured C. elegans with deletions of trxr-1, trxr-2, and both in axenic media with increasing concentrations of inorganic Se. Wild-type C. elegans cultured for 12 days in Se-deficient axenic media grow and reproduce equivalent to Se-supplemented media. Supplementation with 0-2 mM Se as selenite results in inverse, sigmoidal response curves with an LC50 of 0.20 mM Se, due to impaired growth rather than reproduction. Deletion of trxr-1, trxr-2 or both does not modulate growth or Se toxicity in C. elegans grown axenically, and (75)Se labeling showed that TRXR-1 arises from the trxr-1 gene and not from bacterial genes. Se response curves for selenide (LC50 0.23 mM Se) were identical to selenite, but selenate was 1/4(th) as toxic (LC50 0.95 mM Se) as selenite and not modulated by TRXR deletion. These nutritional and genetic studies in axenic media show that Se and TRXR are not essential for C. elegans, and that TRXR alone is not essential for metabolism of inorganic Se to toxic species.

Thioredoxin reductase deficiency potentiates oxidative stress, mitochondrial dysfunction and cell death in dopaminergic cells.

Mitochondria are considered major generators of cellular reactive oxygen species (ROS) which are implicated in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD). We have recently shown that isolated mitochondria consume hydrogen peroxide (H2O2) in a substrate- and respiration-dependent manner predominantly via the thioredoxin/peroxiredoxin (Trx/Prx) system. The goal of this study was to determine the role of Trx/Prx system in dopaminergic cell death. We asked if pharmacological and lentiviral inhibition of the Trx/Prx system sensitized dopaminergic cells to mitochondrial dysfunction, increased steady-state H2O2 levels and death in response to toxicants implicated in PD. Incubation of N27 dopaminergic cells or primary rat mesencephalic cultures with the Trx reductase (TrxR) inhibitor auranofin in the presence of sub-toxic concentrations of parkinsonian toxicants paraquat; PQ or 6-hydroxydopamine; 6OHDA (for N27 cells) resulted in a synergistic increase in H2O2 levels and subsequent cell death. shRNA targeting the mitochondrial thioredoxin reductase (TrxR2) in N27 cells confirmed the effects of pharmacological inhibition. A synergistic decrease in maximal and reserve respiratory capacity was observed in auranofin treated cells and TrxR2 deficient cells following incubation with PQ or 6OHDA. Additionally, TrxR2 deficient cells showed decreased basal mitochondrial oxygen consumption rates. These data demonstrate that inhibition of the mitochondrial Trx/Prx system sensitizes dopaminergic cells to mitochondrial dysfunction, increased steady-state H2O2, and cell death. Therefore, in addition to their role in the production of cellular H2O2 the mitochondrial Trx/Prx system serve as a major sink for cellular H2O2 and its disruption may contribute to dopaminergic pathology associated with PD.

System x(c)- and thioredoxin reductase 1 cooperatively rescue glutathione deficiency.

GSH is the major antioxidant and detoxifier of xenobiotics in mammalian cells. A strong decrease of intracellular GSH has been frequently linked to pathological conditions like ischemia/reperfusion injury and degenerative diseases including diabetes, atherosclerosis, and neurodegeneration. Although GSH is essential for survival, the deleterious effects of GSH deficiency can often be compensated by thiol-containing antioxidants. Using three genetically defined cellular systems, we show here that forced expression of xCT, the substrate-specific subunit of the cystine/glutamate antiporter, in gamma-glutamylcysteine synthetase knock-out cells rescues GSH deficiency by increasing cellular cystine uptake, leading to augmented intracellular and surprisingly high extracellular cysteine levels. Moreover, we provide evidence that under GSH deprivation, the cytosolic thioredoxin/thioredoxin reductase system plays an essential role for the cells to deal with the excess amount of intracellular cystine. Our studies provide first evidence that GSH deficiency can be rescued by an intrinsic genetic mechanism to be considered when designing therapeutic rationales targeting specific redox enzymes to combat diseases linked to GSH deprivation.