A bispecific protein capable of engaging CTLA-4 and MHCII protects non-obese diabetic mice from autoimmune diabetes.

Crosslinking ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) with a bispecific fusion protein (BsB) comprised of a mutant mouse CD80 and lymphocyte activation antigen-3 (LAG-3) has been shown to attenuate TCR signaling and to direct T-cell differentiation toward Foxp3(+) regulatory T cells (Tregs) in an allogenic mixed lymphocyte reaction (MLR). Here, we show that antigen-specific Tregs can also be induced in an antigen-specific setting in vitro. Treatment of non-obese diabetic (NOD) female mice between 9-12 weeks of age with a short course of BsB elicited a transient increase of Tregs in the blood and moderately delayed the onset of autoimmune type 1 diabetes (T1D).

Ligation of cytotoxic T lymphocyte antigen-4 to T cell receptor inhibits T cell activation and directs differentiation into Foxp3+ regulatory T cells.

Cross-linking of ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) during the early phase of T cell activation attenuates TCR signaling, leading to T cell inhibition. To promote this event, a bispecific fusion protein comprising a mutant mouse CD80 (CD80w88a) and lymphocyte activation antigen-3 was engineered to concurrently engage CTLA-4 and cross-link it to the TCR. Cross-linking is expected to be attained via ligation of CTLA-4 first to MHCII and then indirectly to the TCR, generating a CTLA-4-MHCII-TCR trimolecular complex that forms between T cells and antigen-presenting cells during T cell activation.

Adeno-associated virus-mediated expression of acid sphingomyelinase decreases atherosclerotic lesion formation in apolipoprotein E(-/-) mice.

Background: The secretory form of acid sphingomyelinase (ASM) is postulated to play a key role in the retention and aggregation of lipoproteins in the subendothelial space of the arterial wall by converting sphingomyelin in lipoproteins into ceramide. The present study aimed to determine whether the level of circulating ASM activity affects lesion development in mouse model of atherosclerosis.

Methods: Apolipoprotein E deficient (ApoE(-/-) ) mice were injected intravenously with a recombinant adeno-associated virus (AAV8-ASM) that constitutively expressed high levels of human ASM in liver and plasma.

Lowering glycosphingolipid levels in CD4+ T cells attenuates T cell receptor signaling, cytokine production, and differentiation to the Th17 lineage.

Lipid rafts reportedly have a role in coalescing key signaling molecules into the immunological synapse during T cell activation, thereby modulating T cell receptor (TCR) signaling activity. Recent findings suggest that a correlation may exist between increased levels of glycosphingolipids (GSLs) in the lipid rafts of T cells and a heightened response of those T cells toward activation. Here, we show that lowering the levels of GSLs in CD4(+) T cells using a potent inhibitor of glucosylceramide synthase (Genz-122346) led to a moderation of the T cell response toward activation.

Increased hepatic insulin action in diet-induced obese mice following inhibition of glucosylceramide synthase.

Background: Obesity is characterized by the accumulation of fat in the liver and other tissues, leading to insulin resistance. We have previously shown that a specific inhibitor of glucosylceramide synthase, which inhibits the initial step in the synthesis of glycosphingolipids (GSLs), improved glucose metabolism and decreased hepatic steatosis in both ob/ob and diet-induced obese (DIO) mice. Here we have determined in the DIO mouse model the efficacy of a related small molecule compound, Genz-112638, which is currently being evaluated clinically for the treatment of Gaucher disease, a lysosomal storage disorder.

Reducing glycosphingolipid biosynthesis in airway cells partially ameliorates disease manifestations in a mouse model of asthma.

Lipid rafts reportedly play an important role in modulating the activation of mast cells and granulocytes, the primary effector cells of airway hyperresponsiveness and asthma. Activation is mediated through resident signaling molecules whose activity, in part, may be modulated by the composition of glycosphingolipids (GSLs) in membrane rafts. In this study, we evaluated the impact of inhibiting GSL biosynthesis in mast cells and in the ovalbumin (OVA)-induced mouse model of asthma using either a small molecule inhibitor or anti-sense oligonucleotides (ASOs) directed against specific enzymes in the GSL pathway.

Inhibition of osteoclastogenesis by prolyl hydroxylase inhibitor dimethyloxallyl glycine.

Studies examining the effects of hypoxia upon osteoclast biology have consistently revealed a stimulatory effect; both osteoclast differentiation and resorption activity have been shown to be enhanced in the presence of hypoxia. In the present study we examined the effects of the hypoxia mimetics dimethyloxallyl glycine (DMOG) and desferrioxamine (DFO) upon osteoclastogenesis. In contrast to hypoxia, our studies revealed a dose-dependent inhibition of osteoclast formation from macrophages treated with DMOG and DFO.

Inhibiting glycosphingolipid synthesis ameliorates hepatic steatosis in obese mice.

Unlabelled: Steatosis in the liver is a common feature of obesity and type 2 diabetes and the precursor to the development of nonalcoholic steatohepatitis (NASH), cirrhosis, and liver failure. It has been shown previously that inhibiting glycosphingolipid (GSL) synthesis increases insulin sensitivity and lowers glucose levels in diabetic rodent models. Here we demonstrate that inhibiting GSL synthesis in ob/ob mice not only improved glucose homeostasis but also markedly reduced the development of hepatic steatosis.

A hypoxic inducible factor-1 alpha hybrid enhances collateral development and reduces vascular leakage in diabetic rats.

Background: Diabetes mellitus is a common comorbidity of atherosclerosis. Hypoxia-inducible factor-1 (HIF-1) is the master regulator of the angiogenic response to hypoxia.

Methods: We studied the effects of adenoviral vectors expressing a constitutively active HIF-1 alpha hybrid (Ad2/HIF-1 alpha/VP16) or vascular endothelial growth factor (Ad2/VEGF) on collateral development and vascular leakiness in a diabetic rat model of hindlimb ischemia.

Glycoengineered acid alpha-glucosidase with improved efficacy at correcting the metabolic aberrations and motor function deficits in a mouse model of Pompe disease.

Improving the delivery of therapeutics to disease-affected tissues can increase their efficacy and safety. Here, we show that chemical conjugation of a synthetic oligosaccharide harboring mannose 6-phosphate (M6P) residues onto recombinant human acid alpha-glucosidase (rhGAA) via oxime chemistry significantly improved its affinity for the cation-independent mannose 6-phosphate receptor (CI-MPR) and subsequent uptake by muscle cells. Administration of the carbohydrate-remodeled enzyme (oxime-neo-rhGAA) into Pompe mice resulted in an approximately fivefold higher clearance of lysosomal glycogen in muscles when compared to the unmodified counterpart.

Hypoxia-inducible factor 1 mediates hypoxia-induced cardiomyocyte lipid accumulation by reducing the DNA binding activity of peroxisome proliferator-activated receptor alpha/retinoid X receptor.

In response to cellular hypoxia, cardiomyocytes adapt to consume less oxygen by shifting ATP production from mitochondrial fatty acid beta-oxidation to glycolysis. The transcriptional activation of glucose transporters and glycolytic enzymes by hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1). In this study, we examined whether HIF-1 was involved in the suppression of mitochondrial fatty acid beta-oxidation in hypoxic cardiomyocytes.

A constitutively active hypoxia-inducible factor-1alpha/VP16 hybrid factor activates expression of the human B-type natriuretic peptide gene.

Hypoxia-inducible factor-1 (HIF-1) is a primary regulator of the physiological response to hypoxia. A recombinant adenovirus expressing a constitutively active hybrid form of the HIF-1alpha subunit (Ad2/HIF-1alpha/VP16) is being evaluated as a gene therapy for the treatment of peripheral vascular disease. Ad2/HIF-1alpha/VP16 up-regulates known HIF-1-responsive genes, including those involved in angiogenesis.

Carbohydrate-remodelled acid alpha-glucosidase with higher affinity for the cation-independent mannose 6-phosphate receptor demonstrates improved delivery to muscles of Pompe mice.

To enhance the delivery of rhGAA (recombinant GAA, where GAA stands for acid alpha-glucosidase) to the affected muscles in Pompe disease, the carbohydrate moieties on the enzyme were remodelled to exhibit a high affinity ligand for the CI-MPR (cation-independent M6P receptor, where M6P stands for mannose 6-phosphate). This was achieved by chemically conjugating on to rhGAA, a synthetic oligosaccharide ligand bearing M6P residues in the optimal configuration for binding the receptor. The carbonyl chemistry used resulted in the conjugation of approx.

Adenovirus-mediated expression of beta-adrenergic receptor kinase C-terminus reduces intimal hyperplasia and luminal stenosis of arteriovenous polytetrafluoroethylene grafts in pigs.

Background: Hemodialysis vascular access dysfunction is the single most important cause of morbidity in kidney hemodialysis patients. Failure of an arteriovenous polytetrafluoroethylene (PTFE) graft, the most common form of hemodialysis access, is primarily due to intimal hyperplasia and thrombosis at the venous anastomosis.

Methods And Results: This study was aimed at evaluating the efficacy and safety of an adenoviral vector (Ad2/betaARKct) encoding the carboxyl terminus of beta-adrenergic receptor kinase (betaARKct) in a pig model of arteriovenous PTFE graft failure.

Expression of constitutively stable hybrid hypoxia-inducible factor-1alpha protects cultured rat cardiomyocytes against simulated ischemia-reperfusion injury.

Preconditioning in cultured cardiomyocytes elevates the expression of several protective genes including Glut-4 and heat shock protein (HSP)70. Hypoxia-inducible factor-1 (HIF-1) is known to mediate the transcriptional activation of hypoxia-responsive genes. In this study, we examined the effect of adenovirus-mediated expression of constitutively stable hybrid forms of HIF-1alpha on cardiomyocyte viability and gene expression.

Catheter-mediated delivery of adenoviral vectors expressing beta-adrenergic receptor kinase C-terminus inhibits intimal hyperplasia and luminal stenosis in rabbit iliac arteries.

Background: Previous studies have shown that incubation of balloon-injured rat carotid arteries with adenoviral vectors encoding the carboxyl terminus of the beta-adrenergic receptor kinase (Ad2/betaARKct) for 30 min reduces neointima formation. However, it is unclear whether this beneficial effect of betaARKct could be achieved using a catheter-based vector delivery system and whether the observed inhibition of neointima formation translated into a reduction of vessel stenosis.

Methods: In this study, Ad2/betaARKct was infused into the balloon-injured site of rabbit iliac arteries using a porous infusion catheter over 2 min.

Expression of angiopoietins in renal epithelial and clear cell carcinoma cells: regulation by hypoxia and participation in angiogenesis.

The hereditary von Hippel-Lindau (VHL) syndrome predisposes sufferers to highly vascularized tumors such as renal clear cell carcinoma (RCC) and central nervous system hemangioblastoma. In RCC4 and RCC786-0 VHL- cells with VHL mutations, the protein of hypoxia-inducible factor-1alpha (HIF-1alpha) is constitutively stabilized and the mRNA levels of HIF target genes, including vascular endothelial growth factor (VEGF), are elevated. However, the expression of angiopoietins in these cells and their involvement in angiogenesis are not well known.

Fas ligand gene therapy for vascular intimal hyperplasia.

Fas, a member of the tumor necrosis factor receptor super-family, is expressed in all cell types examined, while physiologic expression of Fas ligand (FasL) is found predominantly in activated T-lymphocytes, vascular endothelial cells, and "immune-privileged" tissues. Activation of Fas following FasL binding activates caspases, which results in apoptosis. In the vasculature, there may be a delicate balance between cell proliferation and apoptosis in vascular smooth muscle cells.

Hypoxia-inducible factor-1 mediates activation of cultured vascular endothelial cells by inducing multiple angiogenic factors.

Hypoxia-inducible factor-1 (HIF-1) mediates transcriptional activation of vascular endothelial growth factor (VEGF) and other hypoxia-responsive genes. Transgenic expression of a constitutively stable HIF-1alpha mutant increases the number of vascular vessels without vascular leakage, tissue edema, or inflammation. This study aimed to investigate the molecular basis by which HIF-1 mediates the angiogenic response to hypoxia.

Myocardial expression of baculoviral p35 alleviates doxorubicin-induced cardiomyopathy in rats.

The clinical use of doxorubicin, one of the most effective antitumor drugs, is limited by its cardiotoxicity, which results in irreversible cardiomyopathy and congestive heart failure. This study aimed to evaluate a gene therapy approach using adenovirus-mediated expression of p35, a baculoviral antiapoptotic gene, for alleviating doxorubicin-induced cardiomyopathy. In cultured neonatal rat cardiomyocytes, transduction with a recombinant adenoviral vector expressing p35 (Ad2/CMVp35) but not a control adenoviral vector expressing no transgene (Ad2/CMVEV) significantly inhibited doxorubicin-induced increase in cellular reactive oxygen species (ROS), the activity of caspases 8 and 3, cytochrome c release, and apoptosis.

Differential effects of membrane and soluble Fas ligand on cardiomyocytes: role in ischemia/reperfusion injury.

Cardiomyocyte apoptosis by Fas ligand (FasL)/Fas signaling is associated with various pathophysiological conditions, such as ischemia/reperfusion injury and congestive heart failure. In this study, we tested the hypothesis that shedding of membrane FasL is a mechanism for downregulating FasL/Fas signaling and both membrane and soluble FasL are involved in cardiomyocyte hypoxia/reoxygenation (H/R) injury. We also examined the relative importance of mitochondrial damage and direct cleavage of the executioner caspases by activated initiator caspase 8 in the propagation of FasL/Fas signaling activated by either recombinant membrane FasL or H/R.

Intracellular accumulation of mercury enhances P450 CYP1A1 expression and Cl- currents in cultured shark rectal gland cells.

The effects of acute and subchronic exposure to mercury on the Cl- current (ICl) were investigated in cultured shark rectal gland (SRG) cells. The effects of intracellular accumulation of mercury on cytochrome P450 (P450) were also assessed. Bath perfusion of a cocktail solution containing forskolin, 1-isobutyl-3-methylxanthine, and 8-bromoadenosine monophosphate enhanced ICl.

Adenovirus-mediated expression of p35 prevents hypoxia/reoxygenation injury by reducing reactive oxygen species and caspase activity.

Objective: This study aimed to examine the effects of adenovirus-mediated expression of p35, a baculovirus gene, on apoptosis induced by hypoxia/reoxygenation (H/R) in cardiomyocytes.

Methods: Neonatal rat cardiomyocytes were infected with recombinant adenoviral vectors expressing p35 (Ad2/CMVp35) or no transgene (Ad2/CMVEV) and were then subjected to H/R. Separate groups of non-infected cardiomyocytes were treated with pharmacological caspase inhibitors or antioxidants.

Hypoxia up-regulates expression of peroxisome proliferator-activated receptor gamma angiopoietin-related gene (PGAR) in cardiomyocytes: role of hypoxia inducible factor 1alpha.

Peroxisome proliferator-activated receptors (PPAR), especially the PPARalpha and PPARgamma, are associated with an extraordinary diverse spectrum of cardiovascular diseases including hypertension, angiogenesis, cardiac hypertrophy, and atherosclerosis. PGAR (for PPAR gamma angiopoietin-related gene) is a recently identified PPAR target gene which is associated with adipose differentiation, systemic lipid metabolism, energy homeostasis, and possibly angiogenesis. We report here that WY-14643, a selective PPARalpha ligand up-regulated PGAR expression in neonatal rat cardiomyocytes.

Stabilization of vascular endothelial growth factor mRNA by hypoxia-inducible factor 1.

Hypoxia regulates expression of vascular endothelial growth factor (VEGF) by increasing its transcription and by stabilizing its mRNA. Despite the pivotal role of hypoxia-inducible factor 1 (HIF-1) in transcriptional activation of hypoxia-responsive genes, it is not known whether HIF-1 mediates hypoxia-induced stabilization of VEGF mRNA. We constructed adenoviral vectors expressing either the wild-type HIF-1 alpha (Ad2/HIF-1 alpha/FL), a constitutively stable hybrid form of HIF-1 alpha (Ad2/HIF-1 alpha/VP16), or no transgene (Ad2/CMVEV).