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A Critical Role for Thioredoxin- Interacting Protein in Diabetes- Related Impairment of Angiogenesis

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A Critical Role for Thioredoxin- Interacting Protein in Diabetes- Related Impairment of Angiogenesis Diabetes Volume 63, February 2014 675 Louise L. Dunn,1,2 Philippa J.L. Simpson,1 Hamish C. Prosser,3 Laura Lecce,1 Gloria S.C. Yuen,1,2 Andrew Buckle,1 Daniel P. Sieveking,1 Laura Z. Vanags,3 Patrick R. Lim,1 Renee W.Y. Chow,1 Yuen Ting Lam,1 Zoe Clayton,1 Shisan Bao,2 Michael J. Davies,2,4 Nadina Stadler,4 David S. Celermajer,2,5,6 Roland Stocker,7 Christina A. Bursill,3 John P. Cooke,8 and Martin K.C. Ng1,2,6 A Critical Role for Thioredoxin- Interacting Protein in Diabetes- Related Impairment of Angiogenesis Impaired angiogenesis in ischemic tissue is production to nondiabetic levels. These data PATHOPHYSIOLOGY a hallmark of diabetes. Thioredoxin-interacting implicate a critical role for TXNIP in diabetes-related protein (TXNIP) is an exquisitely glucose-sensitive impairment of ischemia-mediated angiogenesis and gene that is overexpressed in diabetes. As TXNIP identify TXNIP as a potential therapeutic target for modulates the activity of the key angiogenic the vascular complications of diabetes. cytokine vascular endothelial growth factor (VEGF), Diabetes 2014;63:675–687 | DOI: 10.2337/db13-0417 we hypothesized that hyperglycemia-induced dysregulation of TXNIP may play a role in the pathogenesis of impaired angiogenesis in diabetes. A major part of the morbidity and mortality of diabetes In the current study, we report that high glucose– arises from its vascular complications, of which impaired mediated overexpression of TXNIP induces angiogenesis is an important feature (1). Patients with a widespread impairment in endothelial cell (EC) diabetes have impaired collateral development following function and survival by reducing VEGF production vascular occlusion (2), poor wound healing, and increased and sensitivity to VEGF action, findings that are rates of peripheral limb amputation (3). The impairment rescued by silencing TXNIP with small interfering of ischemia-induced angiogenesis in diabetes is associ- RNA. High glucose–induced EC dysfunction was ated with reduced vascular endothelial growth factor recapitulated in normal glucose conditions by (VEGF) production—a phenotype remedied by VEGF overexpressing either TXNIP or a TXNIP C247S gene therapy (4). Monocytes isolated from diabetic mutant unable to bind thioredoxin, suggesting that individuals exhibit reduced sensitivity to the promi- TXNIP effects are largely independent of thioredoxin gratory effects of VEGF, further highlighting a central activity. In streptozotocin-induced diabetic mice, role for impaired VEGF signaling in the disordered an- TXNIP knockdown to nondiabetic levels rescued giogenesis of diabetes (5). However, to date, the funda- diabetes-related impairment of angiogenesis, mental mechanisms underlying diabetes-related arteriogenesis, blood flow, and functional recovery impairment of endothelial cell (EC) function and in an ischemic hindlimb. These findings were ischemia-mediated angiogenesis, including the role of associated with in vivo restoration of VEGF hyperglycemia, are incompletely understood. 1Translational Research Group, The Heart Research Institute, Sydney, Australia Corresponding author: Martin K.C. Ng, [email protected]. 2Sydney Medical School, University of Sydney, Sydney, Australia 3Immunobiology Group, The Heart Research Institute, Sydney, Australia Received 14 March 2013 and accepted 30 October 2013. 4 Free Radical Group, The Heart Research Institute, Sydney, Australia This article contains Supplementary Data online at http://diabetes 5 Clinical Research Group, The Heart Research Institute, Sydney, Australia .diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0417/-/DC1. 6Department of Cardiology, Royal Prince Alfred Hospital, Sydney, Australia 7Vascular Biology Division, Victor Chang Cardiac Research Institute, Sydney, Australia © 2014 by the American Diabetes Association. See http://creativecommons 8Department of Medicine, Stanford University School of Medicine, Stanford, CA .org/licenses/by-nc-nd/3.0/ for details. 676 Thioredoxin-Interacting Protein and Angiogenesis Diabetes Volume 63, February 2014 Thioredoxin-interacting protein (TXNIP) is a multi- incorporation (Life Technologies). Tubulogenesis on functional protein involved in the regulation of cellular growth factor–reduced Matrigel (BD Biosciences) (16) homeostasis (6). TXNIP is an exquisitely glucose- was assessed by photomicrography and measured by inducible gene as a result of a carbohydrate response tubule area. All results were confirmed by also measuring element in the promoter (7) and it is overexpressed in both tubule length and branch points. Nitric oxide was both diabetic animals and humans (8,9). TXNIP has been assessed by flow cytometry using the probe 4-amino- shown to reversibly bind thioredoxin-1 (TRX1), a ubiq- 5-methylamino-2’,7’-difluorofluorescein diacetate (DAF; uitous oxidoreductase with antioxidant activity, in vitro Life Technologies). (10). However, its ability to antagonize TRX1 in vivo Redox Assays remains unclear (11,12), and it is now appreciated that TXNIP may exert some of its effects via redox- Total TRX activity was determined using the insulin fi independent mechanisms (13,14). More recently, TXNIP disul de reduction assay (17) with removal of the has been shown to bind mitochondrial thioredoxin-2 dithiothreitol and 70°C denaturation steps to enhance ’ (TRX2) to induce apoptosis (15). TXNIP is increasingly the assay s sensitivity to available TRX (18). This assay implicated as a key regulator of angiogenesis (6,10). We does not discriminate between TRX1 and mitochondrial previously identified a central role for TXNIP in VEGF- TRX2, so the results are referred to as total TRX activ- mediated EC migration, which is a key angiogenesis event ity. Glutathione Assay Kit (BioVision) and ThioGlo-5 (10). Indeed, the promigratory effects of angiogenic (Sigma-Aldrich) were used to determine reduced:oxi- growth factors, such as VEGF, are mediated in part by dized glutathione (GSH:GSSG) ratios and total cellular their repression of TXNIP (10). We therefore hypothe- thiols. Iron (III) and total iron were determined by sized that hyperglycemia-mediated overexpression of electron paramagnetic resonance and inductively cou- TXNIP plays a causal role in impaired angiogenesis in pled plasma atomic emission spectroscopy, respectively diabetes. (19,20). Here, we report that elevated glucose concentrations Gene Modulation induce a TXNIP-dependent impairment in EC function Human TXNIP cDNA (Genbank Accession: NM_006472) and VEGF signaling. We also demonstrate that targeted or a TXNIP C247S mutant was overexpressed from the knockdown of TXNIP reverses the high glucose–induced pCI-neo plasmid (Promega). Plasmid containing no insert impairment of EC angiogenic function, VEGF production, was used as a control. Duplex siRNA to human TXNIP and sensitivity to VEGF action. Furthermore, we report and a scrambled (SCR) sequence were used for in vitro that normalization of TXNIP expression to nondiabetic gene silencing experiments (10). Plasmids and siRNA levels by small interfering RNA (siRNA) delivery in vivo were transfected into ECs using Lipofectamine 2000 (Life rescues diabetes-related impairment of ischemia-induced Technologies). For in vivo studies, short-hairpin siRNA to angiogenesis. murine TXNIP was expressed from pRNA-U6.1/neo RESEARCH DESIGN AND METHODS (GenScript) and a SCR hairpin was used as a control. Cell Culture and Angiogenesis Assays Gene Expression Human umbilical vein ECs (HUVECs) and human coro- Quantitative PCR was performed using iQ SYBR-Green nary artery ECs (HCAECs) were cultured in MesoEndo Supermix and iCycler Real-time PCR Detection System Media (Cell Applications) in normal glucose conditions (Bio-Rad) and gene expression calculated using b-actin as (5 mmol/L). For high glucose and mannitol conditions, a reference gene (21). media was supplemented with D-glucose to a final con- Protein Studies centration of 15 or 25 mmol/L, or D-mannitol to a final concentration of 5 mmol/L D-glucose, 20 mmol/L Coimmunoprecipitation and Western blot analyses were D-mannitol. ECs (#passage 4) were treated with glucose performed using commercially available kits (Life Tech- for 24 h, and then plated for migration and proliferation nologies). Membranes were probed for TXNIP (rabbit assays for a further 24 h in their glucose conditions. For anti-VDUP1, 1:500; Life Technologies), TRX1 (rabbit tubulogenesis assays, ECs were pretreated with glucose anti-TRX1, 1:1,000; Abcam), TRX2 (rabbit anti-TRX2, for 42 h, and then assayed for a further 6 h. Therefore, 1:500; Novus Biologicals), VEGF (rabbit anti-VEGF, ECs were exposed to glucose conditions for 48 h unless 1:1,000; Abcam), and endothelial nitric oxide synthase otherwise indicated. (eNOS) (mouse anti-eNOS, 1:1,000; Cell Applications). Cell migration was assessed by a modified Boyden Enhanced chemiluminescence detection was performed chamber assay with cells visualized by DAPI staining using Quantity One (Bio-Rad) and densitometry by a (16). Exogenous human VEGF165 (10 ng/mL, R&D Sys- ImageJ software. VEGF165, tumor necrosis factor- ,in- tems) or VEGF monoclonal antibody (1 mg/mL, Sigma- terleukin (IL)-1b, IL-8, and transforming growth factor-b Aldrich) was added where indicated. Cell proliferation were assessed by ELISA (R&D Systems). Kinase domain was assessed by flow cytometry analysis (FC500; receptor (KDR) (VEGF receptor-2) was assessed by flow Beckman Coulter) of 5-ethynyl-2-deoxyuridine (EdU) cytometry. diabetes.diabetesjournals.org Dunn and Associates 677 Apoptosis Assays analyzed by Mann-Whitney test, three or
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