Redox Dysregulation of Vascular Smooth Muscle Sirtuin-1 in Thoracic Aortic Aneurysm in Marfan Syndrome.

BACKGROUND: Thoracic aortic aneurysms (TAAs) are abnormal aortic dilatations and a major cardiovascular complication of Marfan syndrome. We previously demonstrated a critical role for vascular smooth muscle (VSM) SirT1 (sirtuin-1), a lysine deacetylase, against maladaptive aortic remodeling associated with chronic oxidative stress and aberrant activation of MMPs (matrix metalloproteinases). METHODS: In this study, we investigated whether redox dysregulation of SirT1 contributed to the pathogenesis of TAA using fibrillin-1 hypomorphic mice (Fbn1mgR/mgR), an established model of Marfan syndrome prone to aortic dissection/rupture. RESULTS: Oxidative stress markers 3-nitrotyrosine and 4-hydroxynonenal were significantly elevated in aortas of patients with Marfan syndrome. Moreover, reversible oxidative post-translational modifications (rOPTM) of protein cysteines, particularly S-glutathionylation, were dramatically increased in aortas of Fbn1mgR/mgR mice, before induction of severe oxidative stress markers. Fbn1mgR/mgR aortas and VSM cells exhibited an increase in rOPTM of SirT1, coinciding with the upregulation of acetylated proteins, an index of decreased SirT1 activity, and increased MMP2/9 activity. Mechanistically, we demonstrated that TGFß (transforming growth factor beta), which was increased in Fbn1mgR/mgR aortas, stimulated rOPTM of SirT1, decreasing its deacetylase activity in VSM cells. VSM cell-specific deletion of SirT1 in Fbn1mgR/mgR mice (SMKO-Fbn1mgR/mgR) caused a dramatic increase in aortic MMP2 expression and worsened TAA progression, leading to aortic rupture in 50% of SMKO-Fbn1mgR/mgR mice, compared with 25% of Fbn1mgR/mgR mice. rOPTM of SirT1, rOPTM-mediated inhibition of SirT1 activity, and increased MMP2/9 activity were all exacerbated by the deletion of Glrx (glutaredoxin-1), a specific deglutathionylation enzyme, while being corrected by overexpression of Glrx or of an oxidation-resistant SirT1 mutant in VSM cells. CONCLUSIONS: Our novel findings strongly suggest a causal role of S-glutathionylation of SirT1 in the pathogenesis of TAA. Prevention or reversal of SirT1 rOPTM may be a novel therapeutic strategy to prevent TAA and TAA dissection/ruptures in individuals with Marfan syndrome, for which, thus far, no targeted therapy has been developed.

Administration of Glutaredoxin-1 Attenuates Liver Fibrosis Caused by Aging and Non-Alcoholic Steatohepatitis.

Liver fibrosis is a sign of non-alcoholic fatty liver disease progression towards steatohepatitis (NASH) and cirrhosis and is accelerated by aging. Glutaredoxin-1 (Glrx) controls redox signaling by reversing protein S-glutathionylation, induced by oxidative stress, and its deletion causes fatty liver in mice. Although Glrx regulates various pathways, including metabolism and apoptosis, the impact of Glrx on liver fibrosis has not been studied. Therefore, we evaluated the role of Glrx in liver fibrosis induced by aging or by a high-fat, high-fructose diet. We found that: (1) upregulation of Glrx expression level inhibits age-induced hepatic apoptosis and liver fibrosis. In vitro studies indicate that Glrx regulates Fas-induced apoptosis in hepatocytes; (2) diet-induced NASH leads to reduced expression of Glrx and higher levels of S-glutathionylated proteins in the liver. In the NASH model, hepatocyte-specific adeno-associated virus-mediated Glrx overexpression (AAV-Hep-Glrx) suppresses fibrosis and apoptosis and improves liver function; (3) AAV-Hep-Glrx significantly inhibits transcription of Zbtb16 and negatively regulates immune pathways in the NASH liver. In conclusion, the upregulation of Glrx is a potential therapeutic for the reversal of NASH progression by attenuating inflammatory and fibrotic processes.

Oxidized GAPDH transfers S-glutathionylation to a nuclear protein Sirtuin-1 leading to apoptosis.

AIMS: S-glutathionylation is a reversible oxidative modification of protein cysteines that plays a critical role in redox signaling. Glutaredoxin-1 (Glrx), a glutathione-specific thioltransferase, removes protein S-glutathionylation. Glrx, though a cytosolic protein, can activate a nuclear protein Sirtuin-1 (SirT1) by removing its S-glutathionylation. Glrx ablation causes metabolic abnormalities and promotes controlled cell death and fibrosis in mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a key enzyme of glycolysis, is sensitive to oxidative modifications and involved in apoptotic signaling via the SirT1/p53 pathway in the nucleus. We aimed to elucidate the extent to which S-glutathionylation of GAPDH and glutaredoxin-1 contribute to GAPDH/SirT1/p53 apoptosis pathway. RESULTS: Exposure of HEK 293T cells to hydrogen peroxide (H2O2) caused rapid S-glutathionylation and nuclear translocation of GAPDH. Nuclear GAPDH peaked 10-15 min after the addition of H2O2. Overexpression of Glrx or redox dead mutant GAPDH inhibited S-glutathionylation and nuclear translocation. Nuclear GAPDH formed a protein complex with SirT1 and exchanged S-glutathionylation to SirT1 and inhibited its deacetylase activity. Inactivated SirT1 remained stably bound to acetylated-p53 and initiated apoptotic signaling resulting in cleavage of caspase-3. We observed similar effects in human primary aortic endothelial cells suggesting the GAPDH/SirT1/p53 pathway as a common apoptotic mechanism. CONCLUSIONS: Abundant GAPDH with its highly reactive-cysteine thiolate may function as a cytoplasmic rheostat to sense oxidative stress. S-glutathionylation of GAPDH may relay the signal to the nucleus where GAPDH trans-glutathionylates nuclear proteins such as SirT1 to initiate apoptosis. Glrx reverses GAPDH S-glutathionylation and prevents its nuclear translocation and cytoplasmic-nuclear redox signaling leading to apoptosis. Our data suggest that trans-glutathionylation is a critical step in apoptotic signaling and a potential mechanism that cytosolic Glrx controls nuclear transcription factors.

Negative-pressure pulmonary Hemorrhaging Due to Severe Obstructive Sleep Apnea.

A 24-year-old man with a history of bloody sputum for 6 months was referred to our hospital with suspected alveolar hemorrhaging due to vasculitis. Chest computed tomography showed ground-glass opacities in both lungs, and an examination of his bronchoalveolar lavage fluid showed alveolar hemorrhaging. However, no evidence of vasculitis was found, and subsequent polysomnographic testing confirmed that he had severe obstructive sleep apnea (OSA). Since the alveolar hemorrhaging improved after the initiation of continuous positive airway pressure treatment, the diagnosis was negative-pressure alveolar hemorrhaging due to severe OSA.

Role of Glutaredoxin-1 and Glutathionylation in Cardiovascular Diseases.

Cardiovascular diseases are the leading cause of death worldwide, and as rates continue to increase, discovering mechanisms and therapeutic targets become increasingly important. An underlying cause of most cardiovascular diseases is believed to be excess reactive oxygen or nitrogen species. Glutathione, the most abundant cellular antioxidant, plays an important role in the body's reaction to oxidative stress by forming reversible disulfide bridges with a variety of proteins, termed glutathionylation (GSylation). GSylation can alter the activity, function, and structure of proteins, making it a major regulator of cellular processes. Glutathione-protein mixed disulfide bonds are regulated by glutaredoxins (Glrxs), thioltransferase members of the thioredoxin family. Glrxs reduce GSylated proteins and make them available for another redox signaling cycle. Glrxs and GSylation play an important role in cardiovascular diseases, such as myocardial ischemia and reperfusion, cardiac hypertrophy, peripheral arterial disease, and atherosclerosis. This review primarily concerns the role of GSylation and Glrxs, particularly glutaredoxin-1 (Glrx), in cardiovascular diseases and the potential of Glrx as therapeutic agents.

Endothelial cell-specific redox gene modulation inhibits angiogenesis but promotes B16F0 tumor growth in mice.

Glutaredoxin-1 (Glrx) is a small cytosolic enzyme that removes S-glutathionylation, glutathione adducts of protein cysteine residues, thus modulating redox signaling and gene transcription. Although Glrx up-regulation prevented endothelial cell (EC) migration and global Glrx transgenic mice had impaired ischemic vascularization, the effects of cell-specific Glrx overexpression remained unknown. Here, we examined the role of EC-specific Glrx up-regulation in distinct models of angiogenesis; namely, hind limb ischemia and tumor angiogenesis. EC-specific Glrx transgenic (EC-Glrx TG) overexpression in mice significantly impaired EC migration in Matrigel implants and hind limb revascularization after femoral artery ligation. Additionally, ECs migrated less into subcutaneously implanted B16F0 melanoma tumors as assessed by decreased staining of EC markers. Despite reduced angiogenesis, EC-Glrx TG mice unexpectedly developed larger tumors compared with control mice. EC-Glrx TG mice showed higher levels of VEGF-A in the tumors, indicating hypoxia, which may stimulate tumor cells to form vascular channels without EC, referred to as vasculogenic mimicry. These data suggest that impaired ischemic vascularization does not necessarily associate with suppression of tumor growth, and that antiangiogenic therapies may be ineffective for melanoma tumors because of their ability to implement vasculogenic mimicry during hypoxia.-Yura, Y., Chong, B. S. H., Johnson, R. D., Watanabe, Y., Tsukahara, Y., Ferran, B., Murdoch, C. E., Behring, J. B., McComb, M. E., Costello, C. E., Janssen-Heininger, Y. M. W., Cohen, R. A., Bachschmid, M. M., Matsui, R. Endothelial cell-specific redox gene modulation inhibits angiogenesis but promotes B16F0 tumor growth in mice.

Production of adeno-associated virus vectors for in vitro and in vivo applications.

Delivering and expressing a gene of interest in cells or living animals has become a pivotal technique in biomedical research and gene therapy. Among viral delivery systems, adeno-associated viruses (AAVs) are relatively safe and demonstrate high gene transfer efficiency, low immunogenicity, stable long-term expression, and selective tissue tropism. Combined with modern gene technologies, such as cell-specific promoters, the Cre/lox system, and genome editing, AAVs represent a practical, rapid, and economical alternative to conditional knockout and transgenic mouse models. However, major obstacles remain for widespread AAV utilization, such as impractical purification strategies and low viral quantities. Here, we report an improved protocol to produce serotype-independent purified AAVs economically. Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation with subsequent aqueous two-phase partitioning. Furthermore, we then implemented an iodixanol gradient purification, which resulted in preparations with purities adequate for in vivo use. Of note, we achieved titers of 1010-1011 viral genome copies per µl with a typical production volume of up to 1 ml while requiring five times less than the usual number of HEK293T cells used in standard protocols. For proof of concept, we verified in vivo transduction via Western blot, qPCR, luminescence, and immunohistochemistry. AAVs coding for glutaredoxin-1 (Glrx) shRNA successfully inhibited Glrx expression by ~66% in the liver and skeletal muscle. Our study provides an improved protocol for a more economical and efficient purified AAV preparation.

IL-33 induction and signaling are controlled by glutaredoxin-1 in mouse macrophages.

Interleukin (IL)-33 is an interleukin-1 like cytokine that enhances Th2 responses and mediates mucosal immunity and allergic inflammation but the mechanism regulating endogenous IL-33 production are still under investigation. In macrophages, lipopolysaccharide (LPS) administration resulted in marked induction of IL-33 mRNA that was blunted in macrophages from glutaredoxin-1 (Glrx) knockout mice and in RAW264.7 macrophages with Glrx knockdown by siRNA. Glutaredoxin-1 is a small cytosolic thioltransferase that controls a reversible protein thiol modification, S-glutationylation (protein-GSH adducts), thereby regulating redox signaling. In this study, we examined the mechanism of Glrx regulation of endogenous IL-33 induction in macrophages. Glrx knockdown resulted in impaired de-glutathionylation of TRAF6, which is required for TRAF6 activation, and inhibited downstream IKKß and NF-κB activation. Inhibitors of NF-κB suppressed IL-33 induction and chromatin IP sequencing data analysis confirmed that IL-33 is an NF-κB-responsive gene. Since TRAF6-NF-κB activation is also essential for IL-33 signaling through its receptor, ST2L, we next tested the involvement of Glrx in exogenous IL-33 responses in RAW264.7 cells. Recombinant IL-33 (rIL-33) administration induced IL-33 mRNA expression in RAW264.7 macrophages, and this was inhibited by Glrx knockdown. Interestingly, rIL-33-induced IL-33 protein was identified as the 20 kDa cleaved form whereas LPS-induced IL-33 protein was identified as full-length IL-33, which may be less active than the cleaved form. In a clinically-relevant mouse model of asthma, intra-tracheal cockroach antigen treatment induced Glrx protein in wild type mouse lungs but Glrx induction was attenuated in IL-33 knockout mouse lungs, suggesting that IL-33 may regulate Glrx induction in vivo in response to allergen challenge. In summary, our data reveal a novel mechanism by which Glrx controls both LPS- and IL-33-mediated NF-κB activation leading to IL-33 production, and paracrine IL-33 can induce Glrx to further regulate inflammatory reactions.

Sall1 transiently marks undifferentiated heart precursors and regulates their fate.

Cardiac progenitor cells (CPCs) are a crucial source of cells in cardiac development and regeneration. However, reported CPCs are heterogeneous, and no gene has been identified to transiently mark undifferentiated CPCs throughout heart development. Here we show that Spalt-like gene 1 (Sall1), a zing-finger transcription factor, is expressed in undifferentiated CPCs giving rise to both left and right ventricles. Sall1 was transiently expressed in precardiac mesoderm contributing to the first heart field (left ventricle precursors) but not in the field itself. Similarly, Sall1 expression was maintained in the second heart field (outflow tract/right ventricle precursors) but not in cardiac cells. In vitro, high levels of Sall1 at mesodermal stages enhanced cardiomyogenesis, whereas its continued expression suppressed cardiac differentiation. Our study demonstrates that Sall1 marks CPCs in an undifferentiated state and regulates cardiac differentiation. These findings provide fundamental insights into CPC maintenance, which can be instrumental for CPC-based regenerative medicine.

TROP2 expressed in the trunk of the ureteric duct regulates branching morphogenesis during kidney development.

TROP2, a cell surface protein structurally related to EpCAM, is expressed in various carcinomas, though its function remains largely unknown. We examined the expression of TROP2 and EpCAM in fetal mouse tissues, and found distinct patterns in the ureteric bud of the fetal kidney, which forms a tree-like structure. The tip cells in the ureteric bud proliferate to form branches, whereas the trunk cells differentiate to form a polarized ductal structure. EpCAM was expressed throughout the ureteric bud, whereas TROP2 expression was strongest at the trunk but diminished towards the tips, indicating the distinct cell populations in the ureteric bud. The cells highly expressing TROP2 (TROP2(high)) were negative for Ki67, a proliferating cell marker, and TROP2 and collagen-I were co-localized to the basal membrane of the trunk cells. TROP2(high) cells isolated from the fetal kidney failed to attach and spread on collagen-coated plates. Using MDCK cells, a well-established model for studying the branching morphogenesis of the ureteric bud, TROP2 was shown to inhibit cell spreading and motility on collagen-coated plates, and also branching in collagen-gel cultures, which mimic the ureteric bud's microenvironment. These results together suggest that TROP2 modulates the interaction between the cells and matrix and regulates the formation of the ureteric duct by suppressing branching from the trunk during kidney development.

Mouse hepatoblasts at distinct developmental stages are characterized by expression of EpCAM and DLK1: drastic change of EpCAM expression during liver development.

Hepatoblasts are hepatic progenitor cells that expand and give rise to either hepatocyte or cholangiocytes during liver development. We previously reported that delta-like 1 homolog (DLK1) is expressed in the mouse liver primordium at embryonic day (E) 10.5 and that DLK1(+) cells in E14.5 liver contain high proliferative and bipotential hepatoblasts. While the expression of epithelial cell adhesion molecule (EpCAM) in hepatic stem/progenitor cells has been reported, its expression profile at an early stage of liver development remains unknown. In this study, we show that EpCAM is expressed in mouse liver bud at E9.5 and that EpCAM(+)DLK1(+) hepatoblasts form hepatic cords at the early stage of hepatogenesis. DLK1(+) cells of E11.5 liver were fractionated into EpCAM(+) and EpCAM(-) cells; one forth of EpCAM(+)DLK1(+) cells formed a colony in vitro whereas EpCAM(-)DLK1(+) cells rarely did it. Moreover, EpCAM(+)DLK1(+) cells contained cells capable of forming a large colony, indicating that EpCAM(+)DLK1(+) cells in E11.5 liver contain early hepatoblasts with high proliferation potential. Interestingly, EpCAM expression in hepatoblasts was dramatically reduced along with liver development and the colony-forming capacities of both EpCAM(+)DLK1(+) and EpCAM(-)DLK1(+) cells were comparable in E14.5 liver. It strongly suggested that most of mouse hepatoblasts are losing EpCAM expression at this stage. Moreover, we provide evidence that EpCAM(+)DLK1(+) cells in E11.5 liver contain extrahepatic bile duct cells as well as hepatoblasts, while EpCAM(-)DLK1(+) cells contain mesothelial cell precursors. Thus, the expression of EpCAM and DLK1 suggests the developmental pathways of mouse liver progenitors.

Potential hepatic stem cells reside in EpCAM+ cells of normal and injured mouse liver.

Hepatic oval cells are considered to be facultative hepatic stem cells (HSCs) that differentiate into hepatocytes and cholangiocytes in severely injured liver. Hepatic oval cells have also been implicated in tumorigenesis. However, their nature and origin remain elusive. To isolate and characterize mouse oval cells, we searched for cell surface molecules expressed on oval cells and analyzed their nature at the single-cell level by flow cytometric analysis and in the in vitro colony formation assay. We demonstrate that epithelial cell adhesion molecule (EpCAM) is expressed in both mouse normal cholangiocytes and oval cells, whereas its related protein, TROP2, is expressed exclusively in oval cells, establishing TROP2 as a novel marker to distinguish oval cells from normal cholangiocytes. EpCAM(+) cells isolated from injured liver proliferate to form colonies in vitro, and the clonally expanded cells differentiate into hepatocytes and cholangiocytes, suggesting that the oval cell fraction contains potential HSCs. Interestingly, such cells with HSC characteristics exist among EpCAM(+) cells of normal liver. Intriguingly, comparison of the colony formation of EpCAM(+) cells in normal and injured liver reveals little difference in the number of potential HSCs, strongly suggesting that most proliferating mouse oval cells represent transit-amplifying cells rather than HSCs.