Recombinant ADAMTS13 in Congenital Thrombotic Thrombocytopenic Purpura.

BACKGROUND: Congenital thrombotic thrombocytopenic purpura (TTP) results from severe hereditary deficiency of ADAMTS13. The efficacy and safety of recombinant ADAMTS13 and standard therapy (plasma-derived products) administered as routine prophylaxis or on-demand treatment in patients with congenital TTP is not known. METHODS: In this phase 3, open-label, crossover trial, we randomly assigned patients in a 1:1 ratio to two 6-month periods of prophylaxis with recombinant ADAMTS13 (40 IU per kilogram of body weight, administered intravenously) or standard therapy, followed by the alternate treatment; thereafter, all the patients received recombinant ADAMTS13 for an additional 6 months. The trigger for this interim analysis was trial completion by at least 30 patients. The primary outcome was acute TTP events. Manifestations of TTP, safety, and pharmacokinetics were assessed. Patients who had an acute TTP event could receive on-demand treatment. RESULTS: A total of 48 patients underwent randomization; 32 completed the trial. No acute TTP event occurred during prophylaxis with recombinant ADAMTS13, whereas 1 patient had an acute TTP event during prophylaxis with standard therapy (mean annualized event rate, 0.05). Thrombocytopenia was the most frequent TTP manifestation (annualized event rate, 0.74 with recombinant ADAMTS13 and 1.73 with standard therapy). Adverse events occurred in 71% of the patients with recombinant ADAMTS13 and in 84% with standard therapy. Adverse events that were considered by investigators to be related to the trial drug occurred in 9% of the patients with recombinant ADAMTS13 and in 48% with standard therapy. Trial-drug interruption or discontinuation due to adverse events occurred in no patients with recombinant ADAMTS13 and in 8 patients with standard therapy. No neutralizing antibodies developed during recombinant ADAMTS13 treatment. The mean maximum ADAMTS13 activity after recombinant ADAMTS13 treatment was 101%, as compared with 19% after standard therapy. CONCLUSIONS: During prophylaxis with recombinant ADAMTS13 in patients with congenital TTP, ADAMTS13 activity reached approximately 100% of normal levels, adverse events were generally mild or moderate in severity, and TTP events and manifestations were rare. (Funded by Takeda Development Center Americas and Baxalta Innovations; ClinicalTrials.gov number, NCT03393975.).

Interaction of ARRDC4 With GLUT1 Mediates Metabolic Stress in the Ischemic Heart.

BACKGROUND: An ancient family of arrestin-fold proteins, termed alpha-arrestins, may have conserved roles in regulating nutrient transporter trafficking and cellular metabolism as adaptor proteins. One alpha-arrestin, TXNIP (thioredoxin-interacting protein), is known to regulate myocardial glucose uptake. However, the in vivo role of the related alpha-arrestin, ARRDC4 (arrestin domain-containing protein 4), is unknown. METHODS: We first tested whether interaction with GLUTs (glucose transporters) is a conserved function of the mammalian alpha-arrestins. To define the in vivo function of ARRDC4, we generated and characterized a novel Arrdc4 knockout (KO) mouse model. We then analyzed the molecular interaction between arrestin domains and the basal GLUT1. RESULTS: ARRDC4 binds to GLUT1, induces its endocytosis, and blocks cellular glucose uptake in cardiomyocytes. Despite the closely shared protein structure, ARRDC4 and its homologue TXNIP operate by distinct molecular pathways. Unlike TXNIP, ARRDC4 does not increase oxidative stress. Instead, ARRDC4 uniquely mediates cardiomyocyte death through its effects on glucose deprivation and endoplasmic reticulum stress. At baseline, Arrdc4-KO mice have mild fasting hypoglycemia. Arrdc4-KO hearts exhibit a robust increase in myocardial glucose uptake and glycogen storage. Accordingly, deletion of Arrdc4 improves energy homeostasis during ischemia and protects cardiomyocytes against myocardial infarction. Furthermore, structure-function analyses of the interaction of ARRDC4 with GLUT1 using both scanning mutagenesis and deep-learning Artificial Intelligence identify specific residues in the C-terminal arrestin-fold domain as the interaction interface that regulates GLUT1 function, revealing a new molecular target for potential therapeutic intervention against myocardial ischemia. CONCLUSIONS: These results uncover a new mechanism of ischemic injury in which ARRDC4 drives glucose deprivation-induced endoplasmic reticulum stress leading to cardiomyocyte death. Our findings establish ARRDC4 as a new scaffold protein for GLUT1 that regulates cardiac metabolism in response to ischemia and provide insight into the therapeutic strategy for ischemic heart disease.

Txnip C247S mutation protects the heart against acute myocardial infarction.

RATIONALE: Thioredoxin-interacting protein (Txnip) is a novel molecular target with translational potential in diverse human diseases. Txnip has several established cellular actions including binding to thioredoxin, a scavenger of reactive oxygen species (ROS). It has been long recognized from in vitro evidence that Txnip forms a disulfide bridge through cysteine 247 (C247) with reduced thioredoxin to inhibit the anti-oxidative properties of thioredoxin. However, the physiological significance of the Txnip-thioredoxin interaction remains largely undefined in vivo. OBJECTIVE: A single mutation of Txnip, C247S, abolishes the binding of Txnip with thioredoxin. Using a conditional and inducible approach with a mouse model of a mutant Txnip that does not bind thioredoxin, we tested whether the interaction of thioredoxin with Txnip is required for Txnip's pro-oxidative or cytotoxic effects in the heart. METHODS AND RESULTS: Overexpression of Txnip C247S in cells resulted in a reduction in ROS, due to an inability to inhibit thioredoxin. Hypoxia (1% O2, 24 h)-induced killing effects of Txnip were decreased by lower levels of cellular ROS in Txnip C247S-expressing cells compared with wild-type Txnip-expressing cells. Then, myocardial ischemic injuries were assessed in the animal model. Cardiomyocyte-specific Txnip C247S knock-in mice had better survival with smaller infarct size following myocardial infarction (MI) compared to control animals. The absence of Txnip's inhibition of thioredoxin promoted mitochondrial anti-oxidative capacities in cardiomyocytes, thereby protecting the heart from oxidative damage induced by MI. Furthermore, an unbiased RNA sequencing screen identified that hypoxia-inducible factor 1 signaling pathway was involved in Txnip C247S-mediated cardioprotective mechanisms. CONCLUSION: Txnip is a cysteine-containing redox protein that robustly regulates the thioredoxin system via a disulfide bond-switching mechanism in adult cardiomyocytes. Our results provide the direct in vivo evidence that regulation of redox state by Txnip is a crucial component for myocardial homeostasis under ischemic stress.

Correction: Adipocyte arrestin domain-containing 3 protein (Arrdc3) regulates uncoupling protein 1 (Ucp1) expression in white adipose independently of canonical changes in ß-adrenergic receptor signaling.

[This corrects the article DOI: 10.1371/journal.pone.0173823.].

Adipocyte arrestin domain-containing 3 protein (Arrdc3) regulates uncoupling protein 1 (Ucp1) expression in white adipose independently of canonical changes in ß-adrenergic receptor signaling.

Adaptive thermogenesis and cold-induced activation of uncoupling protein 1 (Ucp1) in brown adipose tissue in rodents is well-described and attributed to sympathetic activation of ß-adrenergic signaling. The arrestin domain containing protein Arrdc3 is a regulator of obesity in mice and also appears linked to obesity in humans. We generated a mouse with conditional deletion of Arrdc3, and here we present evidence that genetic ablation of Arrdc3 specifically in adipocytes results in increased Ucp1 expression in subcutaneous and parametrial adipose tissue. Although this increase in expression did not correspond with significant changes in body weight or energy expenditure, adipocyte-specific Arrdc3-null mice had improved glucose tolerance. It was previously hypothesized that Arrdc3 ablation leads to increased ß-adrenergic receptor sensitivity; however, in vitro experiments show that Arrdc3-null adipocytes responded to ß-adrenergic receptor agonist with decreased Ucp1 levels. Additionally, canonical ß-adrenergic receptor signaling was not different in Arrdc3-null adipocytes. These data reveal a role for Arrdc3 in the regulation of Ucp1 expression in adipocytes. However, this adipocyte effect is insufficient to generate the obesity-resistant phenotype of mice with ubiquitous deletion of Arrdc3, indicating a likely role for Arrdc3 in cells other than adipocytes.

Deletion of thioredoxin-interacting protein improves cardiac inotropic reserve in the streptozotocin-induced diabetic heart.

Although the precise pathogenesis of diabetic cardiac damage remains unclear, potential mechanisms include increased oxidative stress, autonomic nervous dysfunction, and altered cardiac metabolism. Thioredoxin-interacting protein (Txnip) was initially identified as an inhibitor of the antioxidant thioredoxin but is now recognized as a member of the arrestin superfamily of adaptor proteins that classically regulate G protein-coupled receptor signaling. Here we show that Txnip plays a key role in diabetic cardiomyopathy. High glucose levels induced Txnip expression in rat cardiomyocytes in vitro and in the myocardium of streptozotocin-induced diabetic mice in vivo. While hyperglycemia did not induce cardiac dysfunction at baseline, ß-adrenergic challenge revealed a blunted myocardial inotropic response in diabetic animals (24-wk-old male and female C57BL/6;129Sv mice). Interestingly, diabetic mice with cardiomyocyte-specific deletion of Txnip retained a greater cardiac response to ß-adrenergic stimulation than wild-type mice. This benefit in Txnip-knockout hearts was not related to the level of thioredoxin activity or oxidative stress. Unlike the ß-arrestins, Txnip did not interact with ß-adrenergic receptors to desensitize downstream signaling. However, our proteomic and functional analyses demonstrated that Txnip inhibits glucose transport through direct binding to glucose transporter 1 (GLUT1). An ex vivo analysis of perfused hearts further demonstrated that the enhanced functional reserve afforded by deletion of Txnip was associated with myocardial glucose utilization during ß-adrenergic stimulation. These data provide novel evidence that hyperglycemia-induced Txnip is responsible for impaired cardiac inotropic reserve by direct regulation of insulin-independent glucose uptake through GLUT1 and plays a role in the development of diabetic cardiomyopathy.

FGF21 and the late adaptive response to starvation in humans.

In mice, FGF21 is rapidly induced by fasting, mediates critical aspects of the adaptive starvation response, and displays a number of positive metabolic properties when administered pharmacologically. In humans, however, fasting does not consistently increase FGF21, suggesting a possible evolutionary divergence in FGF21 function. Moreover, many key aspects of FGF21 function in mice have been identified in the context of transgenic overexpression or administration of supraphysiologic doses, rather than in a physiologic setting. Here, we explored the dynamics and function of FGF21 in human volunteers during a 10-day fast. Unlike mice, which show an increase in circulating FGF21 after only 6 hours, human subjects did not have a notable surge in FGF21 until 7 to 10 days of fasting. Moreover, we determined that FGF21 induction was associated with decreased thermogenesis and adiponectin, an observation that directly contrasts with previous reports based on supraphysiologic dosing. Additionally, FGF21 levels increased after ketone induction, demonstrating that endogenous FGF21 does not drive starvation-mediated ketogenesis in humans. Instead, a longitudinal analysis of biologically relevant variables identified serum transaminases--markers of tissue breakdown--as predictors of FGF21. These data establish FGF21 as a fasting-induced hormone in humans and indicate that FGF21 contributes to the late stages of adaptive starvation, when it may regulate the utilization of fuel derived from tissue breakdown.

Delivering heparin-binding insulin-like growth factor 1 with self-assembling peptide hydrogels.

Heparin-binding insulin-like growth factor 1 (HB-IGF-1) is a fusion protein of IGF-1 with the HB domain of heparin-binding epidermal growth factor-like growth factor. A single dose of HB-IGF-1 has been shown to bind specifically to cartilage and to promote sustained upregulation of proteoglycan synthesis in cartilage explants. Achieving strong integration between native cartilage and tissue-engineered cartilage remains challenging. We hypothesize that if a growth factor delivered by the tissue engineering scaffold could stimulate enhanced matrix synthesis by both the cells within the scaffold and the adjacent native cartilage, integration could be enhanced. In this work, we investigated methods for adsorbing HB-IGF-1 to self-assembling peptide hydrogels to deliver the growth factor to encapsulated chondrocytes and cartilage explants cultured with growth factor-loaded hydrogels. We tested multiple methods for adsorbing HB-IGF-1 in self-assembling peptide hydrogels, including adsorption prior to peptide assembly, following peptide assembly, and with/without heparan sulfate (HS, a potential linker between peptide molecules and HB-IGF-1). We found that HB-IGF-1 and HS were retained in the peptide for all tested conditions. A subset of these conditions was then studied for their ability to stimulate increased matrix production by gel-encapsulated chondrocytes and by chondrocytes within adjacent native cartilage. Adsorbing HB-IGF-1 or IGF-1 prior to peptide assembly was found to stimulate increased sulfated glycosaminoglycan per DNA and hydroxyproline content of chondrocyte-seeded hydrogels compared with basal controls at day 10. Cartilage explants cultured adjacent to functionalized hydrogels had increased proteoglycan synthesis at day 10 when HB-IGF-1 was adsorbed, but not IGF-1. We conclude that delivery of HB-IGF-1 to focal defects in cartilage using self-assembling peptide hydrogels is a promising technique that could aid cartilage repair via enhanced matrix production and integration with native tissue.

Loss of white adipose hyperplastic potential is associated with enhanced susceptibility to insulin resistance.

Fat mass expansion occurs by adipocyte hypertrophy or recruitment of differentiating adipocyte progenitors, the relative balance of which may impact systemic metabolism. We measured adipogenesis in murine subcutaneous (sWAT) and visceral white adipose tissue (vWAT) using stable isotope methodology and then modeled adipocyte turnover. Birth and death rates were similar within depots; however, turnover was higher in vWAT relative to sWAT. In juvenile mice, obesity increased adipogenesis, but in adults, this was only seen in vWAT after prolonged high-fat feeding. Statistical modeling suggests differentiation of adipocyte progenitors without an accompanying self-renewing division step may partially explain the age-dependent decline in hyperplastic potential. Additional metabolic interrogation of obese mice demonstrated an association between adipocyte turnover and insulin sensitivity. These data therefore identify adipocyte hypertrophy as the dominant mechanism of adult fat mass expansion and support the paradoxical concept that metabolic disease ensues due to a failure of adipose tissue plasticity.

Thioredoxin-interacting protein regulates protein disulfide isomerases and endoplasmic reticulum stress.

The endoplasmic reticulum (ER) is responsible for protein folding, modification, and trafficking. Accumulation of unfolded or misfolded proteins represents the condition of ER stress and triggers the unfolded protein response (UPR), a key mechanism linking supply of excess nutrients to insulin resistance and type 2 diabetes in obesity. The ER harbors proteins that participate in protein folding including protein disulfide isomerases (PDIs). Changes in PDI activity are associated with protein misfolding and ER stress. Here, we show that thioredoxin-interacting protein (Txnip), a member of the arrestin protein superfamily and one of the most strongly induced proteins in diabetic patients, regulates PDI activity and UPR signaling. We found that Txnip binds to PDIs and increases their enzymatic activity. Genetic deletion of Txnip in cells and mice led to increased protein ubiquitination and splicing of the UPR regulated transcription factor X-box-binding protein 1 (Xbp1s) at baseline as well as under ER stress. Our results reveal Txnip as a novel direct regulator of PDI activity and a feedback mechanism of UPR signaling to decrease ER stress.

Targeted delivery to cartilage is critical for in vivo efficacy of insulin-like growth factor 1 in a rat model of osteoarthritis.

OBJECTIVE: Acute articular injuries lead to an increased risk of progressive joint damage and osteoarthritis (OA), and no therapies are currently available to repair or protect the injured joint tissue. Intraarticular delivery of therapeutic proteins has been limited by their rapid clearance from the joint space and lack of retention within cartilage. The aim of this study was to test whether targeted delivery to cartilage by fusion with a heparin-binding domain would be sufficient to prolong the in vivo function of the insulin-like growth factor 1 (IGF-1). METHODS: We produced a humanized and optimized recombinant HB-IGF-1 fusion protein. By injecting HB-IGF-1, IGF-1, or saline alone into the knee joints of adult Lewis rats, we tested whether fusion with a heparin-binding domain 1) altered the kinetics of retention in joint tissues, 2) prolonged functional stimulation as measured by radiolabel incorporation, and 3) enhanced efficacy in a rat model of surgically induced OA, using weekly injections. RESULTS: Fusion of heparin-binding domain with IGF-1 prolonged retention in articular and meniscal cartilage from <1 day to 8 days after injection. Unmodified IGF-1 had no functional effect 2 days after injection, whereas HB-IGF-1 stimulated meniscal cartilage at least 4 days after injection. HB-IGF-1, but not IGF-1, significantly slowed cartilage damage in a rat model of OA. CONCLUSION: Heparin-binding domain fusions can transform rapidly cleared proteins into potential intraarticular therapies by targeting them to cartilage.

An expanded family of arrestins regulate metabolism.

The classical visual and ß-arrestins belong to a larger family of proteins that likely share structural similarity. Humans have an additional six related proteins sometimes termed the α-arrestins, whose functions are now emerging. Surprisingly, several α-arrestins play prominent roles in the regulation of metabolism and obesity. One α-arrestin, thioredoxin-interacting protein (Txnip), has crucial functions in regulating glucose uptake and glycolytic flux through the mitochondria. Another α-arrestin, Arrdc3, is linked to obesity in men and was recently identified in mice as a regulator of body mass, adiposity, and energy expenditure. Here we discuss recent evidence suggesting potential common themes for all arrestins, including physiological roles for classical arrestins in metabolism and the functions of α-arrestins in receptor signaling and endocytosis.

The arrestin domain-containing 3 protein regulates body mass and energy expenditure.

A human genome-wide linkage scan for obesity identified a linkage peak on chromosome 5q13-15. Positional cloning revealed an association of a rare haplotype to high body-mass index (BMI) in males but not females. The risk locus contains a single gene, "arrestin domain-containing 3" (ARRDC3), an uncharacterized α-arrestin. Inactivating Arrdc3 in mice led to a striking resistance to obesity, with greater impact on male mice. Mice with decreased ARRDC3 levels were protected from obesity due to increased energy expenditure through increased activity levels and increased thermogenesis of both brown and white adipose tissues. ARRDC3 interacted directly with ß-adrenergic receptors, and loss of ARRDC3 increased the response to ß-adrenergic stimulation in isolated adipose tissue. These results demonstrate that ARRDC3 is a gender-sensitive regulator of obesity and energy expenditure and reveal a surprising diversity for arrestin family protein functions.

Intraarticular injection of heparin-binding insulin-like growth factor 1 sustains delivery of insulin-like growth factor 1 to cartilage through binding to chondroitin sulfate.

OBJECTIVE: Insulin-like growth factor 1 (IGF-1) stimulates cartilage repair but is not a practical therapy due to its short half-life. We have previously modified IGF-1 by adding a heparin-binding domain and have shown that this fusion protein (HB-IGF-1) stimulates sustained proteoglycan synthesis in cartilage. This study was undertaken to examine the mechanism by which HB-IGF-1 is retained in cartilage and to test whether HB-IGF-1 provides sustained growth factor delivery to cartilage in vivo and to human cartilage explants. METHODS: Retention of HB-IGF-1 and IGF-1 was analyzed by Western blotting. The necessity of heparan sulfate (HS) or chondroitin sulfate (CS) glycosaminoglycans (GAGs) for binding was tested using enzymatic removal and cells with genetic deficiency of HS. Binding affinities of HB-IGF-1 and IGF-1 proteins for isolated GAGs were examined by surface plasmon resonance and enzyme-linked immunosorbent assay. RESULTS: In cartilage explants, chondroitinase treatment decreased binding of HB-IGF-1, whereas heparitinase had no effect. Furthermore, HS was not necessary for HB-IGF-1 retention on cell monolayers. Binding assays showed that HB-IGF-1 bound both CS and HS, whereas IGF-1 did not bind either. After intraarticular injection in rat knees, HB-IGF-1 was retained in articular and meniscal cartilage, but not in tendon, consistent with enhanced delivery to CS-rich cartilage. Finally, HB-IGF-1 was retained in human cartilage explants but IGF-1 was not. CONCLUSION: Our findings indicate that after intraarticular injection in rats, HB-IGF-1 is specifically retained in cartilage through its high abundance of CS. Modification of growth factors with heparin-binding domains may be a new strategy for sustained and specific local delivery to cartilage.

Deletion of the alpha-arrestin protein Txnip in mice promotes adiposity and adipogenesis while preserving insulin sensitivity.

OBJECTIVE: Thioredoxin interacting protein (Txnip), a regulator of cellular oxidative stress, is induced by hyperglycemia and inhibits glucose uptake into fat and muscle, suggesting a role for Txnip in type 2 diabetes pathogenesis. Here, we tested the hypothesis that Txnip-null (knockout) mice are protected from insulin resistance induced by a high-fat diet. RESEARCH DESIGN AND METHODS: Txnip gene-deleted (knockout) mice and age-matched wild-type littermate control mice were maintained on a standard chow diet or subjected to 4 weeks of high-fat feeding. Mice were assessed for body composition, fat development, energy balance, and insulin responsiveness. Adipogenesis was measured from ex vivo fat preparations, and in mouse embryonic fibroblasts (MEFs) and 3T3-L1 preadipocytes after forced manipulation of Txnip expression. RESULTS: Txnip knockout mice gained significantly more adipose mass than controls due to a primary increase in both calorie consumption and adipogenesis. Despite increased fat mass, Txnip knockout mice were markedly more insulin sensitive than controls, and augmented glucose transport was identified in both adipose and skeletal muscle. RNA interference gene-silenced preadipocytes and Txnip(-/-) MEFs were markedly adipogenic, whereas Txnip overexpression impaired adipocyte differentiation. As increased adipogenesis and insulin sensitivity suggested aspects of augmented peroxisome proliferator-activated receptor-gamma (PPARgamma) response, we investigated Txnip's regulation of PPARgamma function; manipulation of Txnip expression directly regulated PPARgamma expression and activity. CONCLUSIONS: Txnip deletion promotes adiposity in the face of high-fat caloric excess; however, loss of this alpha-arrestin protein simultaneously enhances insulin responsiveness in fat and skeletal muscle, revealing Txnip as a novel mediator of insulin resistance and a regulator of adipogenesis.

Thioredoxin-independent regulation of metabolism by the alpha-arrestin proteins.

Thioredoxin-interacting protein (Txnip), originally characterized as an inhibitor of thioredoxin, is now known to be a critical regulator of glucose metabolism in vivo. Txnip is a member of the alpha-arrestin protein family; the alpha-arrestins are related to the classical beta-arrestins and visual arrestins. Txnip is the only alpha-arrestin known to bind thioredoxin, and it is not known whether the metabolic effects of Txnip are related to its ability to bind thioredoxin or related to conserved alpha-arrestin function. Here we show that wild type Txnip and Txnip C247S, a Txnip mutant that does not bind thioredoxin in vitro, both inhibit glucose uptake in mature adipocytes and in primary skin fibroblasts. Furthermore, we show that Txnip C247S does not bind thioredoxin in cells, using thiol alkylation to trap the Txnip-thioredoxin complex. Because Txnip function was independent of thioredoxin binding, we tested whether inhibition of glucose uptake was conserved in the related alpha-arrestins Arrdc4 and Arrdc3. Both Txnip and Arrdc4 inhibited glucose uptake and lactate output, while Arrdc3 had no effect. Structure-function analysis indicated that Txnip and Arrdc4 inhibit glucose uptake independent of the C-terminal WW-domain binding motifs, recently identified as important in yeast alpha-arrestins. Instead, regulation of glucose uptake was intrinsic to the arrestin domains themselves. These data demonstrate that Txnip regulates cellular metabolism independent of its binding to thioredoxin and reveal the arrestin domains as crucial structural elements in metabolic functions of alpha-arrestin proteins.

Mechanical control of tissue morphogenesis.

Mechanical forces participate in morphogenesis from the level of individual cells to whole organism patterning. This article reviews recent research that has identified specific roles for mechanical forces in important developmental events. One well defined example is that dynein-driven cilia create fluid flow that determines left-right patterning in the early mammalian embryo. Fluid flow is also important for vasculogenesis, and evidence suggests that fluid shear stress rather than fluid transport is primarily required for remodeling the early vasculature. Contraction of the actin cytoskeleton, driven by nonmuscle myosins and regulated by the Rho family GTPases, is a recurring mechanism for controlling morphogenesis throughout development, from gastrulation to cardiogenesis. Finally, novel experimental approaches suggest critical roles for the actin cytoskeleton and the mechanical environment in determining differentiation of mesenchymal stem cells. Insights into the mechanisms linking mechanical forces to cell and tissue differentiation pathways are important for understanding many congenital diseases and for developing regenerative medicine strategies.

Engineering insulin-like growth factor-1 for local delivery.

Insulin-like growth factor-1 (IGF-1) is a small protein that promotes cell survival and growth, often acting over long distances. Although for decades IGF-1 has been considered to have therapeutic potential, systemic side effects of IGF-1 are significant, and local delivery of IGF-1 for tissue repair has been a long-standing challenge. In this study, we designed and purified a novel protein, heparin-binding IGF-1 (Xp-HB-IGF-1), which is a fusion protein of native IGF-1 with the heparin-binding domain of heparin-binding epidermal growth factor-like growth factor. Xp-HB-IGF-1 bound selectively to heparin as well as the cell surfaces of 3T3 fibroblasts, neonatal cardiac myocytes and differentiating ES cells. Xp-HB-IGF-1 activated the IGF-1 receptor and Akt with identical kinetics and dose response, indicating no compromise of biological activity due to the heparin-binding domain. Because cartilage is a proteoglycan-rich environment and IGF-1 is a known stimulus for chondrocyte biosynthesis, we then studied the effectiveness of Xp-HB-IGF-1 in cartilage. Xp-HB-IGF-1 was selectively retained by cartilage explants and led to sustained chondrocyte proteoglycan biosynthesis compared to IGF-1. These data show that the strategy of engineering a "long-distance" growth factor like IGF-1 for local delivery may be useful for tissue repair and minimizing systemic effects.

Thioredoxin-interacting protein (Txnip) is a critical regulator of hepatic glucose production.

Thioredoxin-interacting protein (Txnip) has been recently described as a possible link between cellular redox state and metabolism; Txnip binds thioredoxin and inhibits its disulfide reductase activity in vitro, while a naturally occurring strain of Txnip-deficient mice has hyperlipidemia, hypoglycemia, and ketosis exacerbated by fasting. We generated Txnip-null mice to investigate the role of Txnip in glucose homeostasis. Txnip-null mice were hypoglycemic, hypoinsulinemic, and had blunted glucose production following a glucagon challenge, consistent with a central liver glucose-handling defect. Glucose release from isolated Txnip-null hepatocytes was 2-fold lower than wild-type hepatocytes, whereas beta-hydroxybutyrate release was increased 2-fold, supporting an intrinsic defect in hepatocyte glucose metabolism. While hepatocyte-specific gene deletion of Txnip did not alter glucose clearance compared with littermate controls, Txnip expression in the liver was required for maintaining normal fasting glycemia and glucose production. In addition, hepatic overexpression of a Txnip transgene in wild-type mice resulted in elevated serum glucose levels and decreased ketone levels. Liver homogenates from Txnip-null mice had no significant differences in the glutathione oxidation state or in the amount of available thioredoxin. However, overexpression of wild-type Txnip in Txnip-null hepatocytes rescued cellular glucose production, whereas overexpression of a C247S mutant Txnip, which does not bind thioredoxin, had no effect. These data demonstrate that Txnip is required for normal glucose homeostasis in the liver. While available thioredoxin is not changed in Txnip-null mice, the effects of Txnip on glucose homeostasis are abolished by a single cysteine mutation that inhibits binding to thioredoxin.

Thioredoxins, mitochondria, and hypertension.

Endothelial dysfunction, often demonstrated by the loss of the endothelial cell's ability to cause vasodilation in response to appropriate stimuli, is one of the earliest events in the development of atherosclerosis. This has led to intense investigation of the factors affecting both the production and the degradation of NO, the endothelium-derived relaxing factor and a primary mediator of endothelial function. Reactive oxygen species (ROS), particularly superoxide anion, are well known to inhibit NO, and therefore the mechanisms by which endothelium regulates production of ROS are also of high interest. In this issue of The American Journal of Pathology, Zhang et al( 1) demonstrate regulation of such events by a mitochondria-specific thioredoxin, which reduces oxidative stress and increases NO bioavailability, thus preserving vascular endothelial cell function and preventing atherosclerosis development.