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). However, a longer course of treatment (10 weeks) of 4-13 weeks-old female NOD animals with BsB significantly delayed the onset of disease or protected animals from developing diabetes, with only 13% of treated animals developing diabetes by 35 weeks of age compared to 80% of the animals in the control group. Histopathological analysis of the pancreata of the BsB-treated mice that remained non-diabetic revealed the preservation of insulin-producing ß-cells despite the presence of different degrees of insulitis. Thus, a bifunctional protein capable of engaging CTLA-4 and MHCII and indirectly co-ligating CTLA-4 to the TCR protected NOD mice from developing 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. Treating T cells with this bispecific fusion protein inhibited T cell activation. In addition, it induced the production of IL-10 and TGF-ß and attenuated AKT and mTOR signaling. Intriguingly, treatment with the bispecific fusion protein also directed early T cell differentiation into Foxp3-positive regulatory T cells (Tregs). This process was dependent on the endogenous production of TGF-ß. Thus, bispecific fusion proteins that engage CTLA-4 and co-ligate it to the TCR during the early phase of T cell activation can negatively regulate the T cell response. Bispecific biologics with such dual functions may therefore represent a novel class of therapeutics for immune modulation. These findings presented here also reveal a potential new role for CTLA-4 in Treg differentiation.

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. RESULTS: Plasma sphingomyelin levels were reduced at early but not later time points after the administration of AAV8-ASM despite persistently elevated circulating ASM. No change in serum lipoprotein levels was observed. Thirteen or 17 weeks after the administration of AAV8-ASM, the amount of plaque formation in the aortic sinus was comparable to that of mice treated with a control AAV. CONCLUSIONS: Unexpectedly, the lesion area of the entire aorta was reduced significantly in the AAV8-ASM virus-treated group. Hepatic expression and secretion of ASM into the circulation did not accelerate or exacerbate, but rather decreased, lesion formation in ApoE(-/-) mice. Thus, plasma ASM activity does not appear to be rate limiting for plaque formation during atherogenesis.

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. TCR proximal signaling events, such as phosphorylation of Lck, Zap70 and LAT, as well as early Ca(2+) mobilization, were attenuated by treatment with Genz-122346. Concomitant with these events were significant reductions in IL-2 production and T cell proliferation. Similar findings were obtained with CD4(+) T cells isolated from transgenic mice genetically deficient in GM3 synthase activity. Interestingly, lowering the GSL levels in CD4(+) T cells by either pharmacological inhibition or disruption of the gene for GM3 synthase also specifically inhibited the differentiation of T cells to the Th(17) lineage but not to other Th subsets in vitro. Taken together with the recently reported effects of Raftlin deficiency on Th(17) differentiation, these results strongly suggest that altering the GSL composition of lipid rafts modulates TCR signaling activity and affects Th(17) differentiation.

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. METHODOLOGY/PRINCIPAL FINDINGS: DIO mice were treated with the Genz-112638 for 12 to 16 weeks by daily oral gavage. Genz-112638 lowered HbA1c levels and increased glucose tolerance. Whole body adiposity was not affected in normal mice, but decreased in drug-treated obese mice. Drug treatment also significantly lowered liver triglyceride levels and reduced the development of hepatic steatosis. We performed hyperinsulinemic-euglycemic clamps on the DIO mice treated with Genz-112638 and showed that insulin-mediated suppression of hepatic glucose production increased significantly compared to the placebo treated mice, indicating a marked improvement in hepatic insulin sensitivity. CONCLUSIONS/SIGNIFICANCE: These results indicate that GSL inhibition in obese mice primarily results in an increase in insulin action in the liver, and suggests that GSLs may have an important role in hepatic insulin resistance in conditions of obesity.

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. Lowering GSL levels in mast cells through inhibition of glucosylceramide synthase (GCS) reduced phosphorylation of Syk tyrosine kinase and phospholipase C gamma 2 (PLC-gamma2) as well as cytoplasmic Ca(2+) levels. Modulating these intracellular signaling events also resulted in a significant decrease in mast cell degranulation. Primary mast cells isolated from a GM3 synthase (GM3S) knockout mouse exhibited suppressed activation-induced degranulation activity further supporting a role of GSLs in this process. In previously OVA-sensitized mice, intra-nasal administration of ASOs to GCS, GM3S or lactosylceramide synthase (LCS) significantly suppressed metacholine-induced airway hyperresponsiveness and pulmonary inflammation to a subsequent local challenge with OVA. However, administration of the ASOs into mice that had been sensitized and locally challenged with the allergen did not abate the consequent pulmonary inflammatory sequelae. These results suggest that GSLs contribute to the initiation phase of the pathogenesis of airway hyperreactivity and asthma and lowering GSL levels may offer a novel strategy to modulate these manifestations.

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. Moreover, expression of a constitutively active form of hypoxia-inducible factor 1alpha (HIF-1alpha) did not enhance osteoclastogenesis and actually attenuated the differentiation process. DMOG did not affect cell viability or receptor activator of nuclear factor kappaB ligand (RANKL)-dependent phosphorylation of mitogen-activated protein (MAP) kinases. However, RANKL-dependent transcription of tartrate-resistant acid phosphatase (TRAP) was reduced in the presence of DMOG. Additionally, DMOG promoted transcription of the pro-apoptotic mediator B-Nip3. These studies suggest that a hypoxia-responsive factor other than HIF-1alpha is necessary for enhancing the formation of osteoclasts in hypoxic settings.

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. The ob/ob mice were treated for 7 weeks with a specific inhibitor of glucosylceramide synthase, the initial enzyme involved in the synthesis of GSLs. Besides lowering glucose and hemoglobin A1c (HbA1c) levels, drug treatment also significantly reduced the liver/body weight ratio, decreased the accumulation of triglycerides, and improved several markers of liver pathology. Drug treatment reduced liver glucosylceramide (GL1) levels in the ob/ob mouse. Treatment also reduced the expression of several genes associated with hepatic steatosis, including those involved in lipogenesis, gluconeogenesis, and inflammation. In addition, inhibiting GSL synthesis in diet-induced obese mice both prevented the development of steatosis and partially reversed preexisting steatosis. CONCLUSION: These data indicate that inhibiting GSL synthesis ameliorates the liver pathology associated with obesity and diabetes, and may represent a novel strategy for treating fatty liver disease and NASH.

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. Importantly, treatment of immunotolerized Pompe mice with oxime-neo-rhGAA translated to greater improvements in muscle function and strength. Treating older, symptomatic Pompe mice also reduced tissue glycogen levels but provided only modest improvements in motor function. Examination of the muscle pathology suggested that the poor response in the older animals might have been due to a reduced regenerative capacity of the skeletal muscles. These findings lend support to early therapeutic intervention with a targeted enzyme as important considerations in the management of Pompe disease.

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. RESULTS: After the removal of the right femoral artery, the mRNA levels of VEGF, angiopoietin-1 and angiopietin-4 in the calf muscles, as measured by Taqman reverse transcriptase-polymerase chain reaction, were transiently elevated in Zucker lean (ZL) but not Zucker diabetic fatty (ZDF) rats. The angiographic score, as determined by post-mortem angiography, was significantly lower in ZDF animals 35 days after surgery compared to their ZL counterparts. In separate animals, intramuscular injection of Ad2/HIF-1a/VP16 and Ad/2VEGF into the thigh muscles significantly increased the angiographic score and capillary density 21 and 35 days after the injection compared to Ad2/CMVEV (a vector expressing no transgene) or vehicle. After the injection of Ad2/CMVEV or vehicle, the Evans-blue dye content in the thigh muscles was significantly higher in ZDF rats than their ZL counterparts. Ad2/HIF-1 alpha/VP16 but not Ad2/VEGF reduced tissue Evans blue dye content. CONCLUSIONS: The endogenous angiogenic response to ischemia was impaired in ZDF rats, possibly due to down-regulation of angiogenic factors. Ad2/HIF-1 alpha/VP16 enhanced collateral development and reduced vascular leakage in the ischemic hindlimb of ZDF rats indicating that hybrid HIF-1 alpha angiogenic therapy may be efficacious for peripheral vascular disease with a diabetic comorbidity.

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. We showed that either hypoxia or adenovirus-mediated expression of a constitutively stable hybrid form (HIF-1alpha/VP16) suppressed mitochondrial fatty acid metabolism, as indicated by an accumulation of intracellular neutral lipid. Both treatments also reduced the mRNA levels of muscle carnitine palmitoyltransferase I which catalyzes the rate-limiting step in the mitochondrial import of fatty acids for beta-oxidation. Furthermore, adenovirus-mediated expression of HIF-1alpha/VP16 in cardiomyocytes under normoxic conditions also mimicked the reduction in the DNA binding activity of peroxisome proliferator-activated receptor alpha (PPARalpha)/retinoid X receptor (RXR), in the presence or absence of a PPARalpha ligand. These results suggest that HIF-1 may be involved in hypoxia-induced suppression of fatty acid metabolism in cardiomyocytes by reducing the DNA binding activity of PPARalpha/RXR.

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. Expression profile analysis revealed that the brain natriuretic peptide (BNP) gene was significantly up-regulated in response to HIF-1alpha/VP16 in human fetal cardiac cells. Real-time reverse transcription-polymerase chain reaction analyses confirmed transcriptional activation of the BNP gene by HIF-1alpha/VP16 in human but not rat cardiac cells. Because hypoxia itself did not increase human BNP gene expression in these analyses, the mechanism of the HIF-1alpha/VP16 effect was determined. Analyses of promoter deletion mutants suggested that the cis-acting sequence in the human BNP promoter mediating activation by HIF-1alpha/VP16 was a putative HIF-1 responsive element (HRE) located at -466. An SV40 basal promoter-luciferase plasmid containing a minimal BNP HRE was up-regulated by HIF-1alpha/VP16, whereas a similar construct carrying a mutation within the HIF-1 binding site was not. Mutation of an E-box motif within the BNP HRE reduced HIF-1alpha/VP16-mediated transcriptional activation by 50%. Gel-shift analyses showed that both the native HIF-1alpha and HIF-1alpha/VP16 are able to bind to a probe containing the HIF-1 binding site. These experiments demonstrate the existence of a functional HRE in the BNP promoter and further define the scope and mechanism of action of Ad2/HIF-1alpha/VP16.

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. six synthetic ligands on to each enzyme. The resulting modified enzyme [neo-rhGAA (modified recombinant human GAA harbouring synthetic oligosaccharide ligands)] displayed near-normal specific activity and significantly increased affinity for the CI-MPR. However, binding to the mannose receptor was unaffected despite the introduction of additional mannose residues in neo-rhGAA. Uptake studies using L6 myoblasts showed neo-rhGAA was internalized approx. 20-fold more efficiently than the unmodified enzyme. Administration of neo-rhGAA into Pompe mice also resulted in greater clearance of glycogen from all the affected muscles when compared with the unmodified rhGAA. Comparable reductions in tissue glycogen levels in the Pompe mice were realized using an approx. 8-fold lower dose of neo-rhGAA in the heart and diaphragm and an approx. 4-fold lower dose in the skeletal muscles. Treatment of older Pompe mice, which are more refractory to enzyme therapy, with 40 mg/kg neo-rhGAA resulted in near-complete clearance of glycogen from all the affected muscles as opposed to only partial correction with the unmodified rhGAA. These results demonstrate that remodelling the carbohydrate of rhGAA to improve its affinity for the CI-MPR represents a feasible approach to enhance the efficacy of enzyme replacement therapy for Pompe disease.

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. Transduction of the external jugular vein with Ad2/betaARKct (5E9, 5E10, or 5E11 particles per vein) did not result in systemic toxicity, as measured by clinical and pathological assessments. Ad2/betaARKct significantly reduced neointimal hyperplasia in the graft/vein anastomosis. It also improved the graft patency rate and angiographic score, as measured histologically and angiographically, compared with vehicle or empty viral vector controls. CONCLUSIONS: Our results suggest that local administration of adenoviral vectors encoding betaARKct into the jugular vein represents a viable strategy to treat AV graft hemodialysis vascular access 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. Cultured neonatal rat cardiomyocytes were subjected to simulated ischemia-reperfusion with or without preinfection with recombinant adenoviral vectors [Ad2/HIF-1alpha/herpes simplex virus protein VP16 and Ad2/HIF-1alpha/nuclear factor-kappaB (NF-kappaB)]. Cellular viability and mRNA levels of several cardioprotective genes were measured. We demonstrated that infection with Ad2/HIF-1alpha/VP16 and Ad2/HIF-1alpha/NF-kappaB mimicked the upregulation of the mRNA levels of vascular endothelial growth factor (VEGF), Glut-1, Glut-4, HSP70, and inducible NO synthase (iNOS) and the protection of cultured neonatal rat cardiomyocytes by late-phase preconditioning against simulated ischemia-reperfusion. The same dose of a control viral vector expressing no transgene had no effect. Preconditioning also elevated HIF-1alpha protein levels. These results suggest that adenovirus-mediated expression of HIF-1alpha/VP16 or HIF-1alpha/NF-kappaB, a constitutively stable hybrid transcriptional factor, protected cultured neonatal cardiomyocytes against simulated ischemia-reperfusion injury by inducing multiple protective genes.

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. Twenty-eight days after gene transfer, angiographic and histological assessments were performed. RESULTS: Angiographic and histological assessments indicate significant (p < 0.05) inhibition of iliac artery neointima formation and lumen stenosis by Ad2/betaARKct. Our studies demonstrate that an inhibitory effect of Ad2/betaARKct on neointima formation is achievable using a catheter-based vector delivery system and that the inhibition of neointima formation translates into a gain in the vessel minimal luminal diameter. The extent of inhibition (35%) was comparable to that observed with adenoviral-mediated expression of thymidine kinase plus ganciclovir treatment, a cytotoxic gene therapy approach for restenosis. CONCLUSIONS: These results suggest that adenoviral-mediated gene transfer of betaARKct is a clinically viable cytostatic gene therapy strategy for the treatment of restenosis.

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. In this study, we compared the mRNA levels of angiopoietins in human kidney proximal tubule epithelial (RPTE) and RCC4 and RCC786-0 VHL- cells. In RPTE cells, angiopoietin-4 (Ang-4) expression was selectively induced by hypoxia or by expression of a hybrid form of HIF-1alpha. Under normoxic conditions, the mRNA levels of Ang-4 were higher in RCC4 and RCC786-0 VHL- than RPTE cells. Angiopoietin-1 expression was detectable in RCC4 and RCC786-0 VHL- cells but not RPTE cells. In RCC786-0 VHL+ cells, which were stably transfected with a wild-type copy of VHL, the mRNA levels of VEGF and Ang-4 were suppressed and the hypoxic response was restored. We also demonstrated that stimulation of endothelial tube formation by conditioned medium harvested from RCC4 cells was inhibited by a soluble Tie-2 receptor. These results suggest that the angiopoietin/Tie-2 system may participate in the angiogenic response to hypoxia in renal tissues and in tumor angiogenesis in renal carcinoma.

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. Shifts in this balance could account for the accumulation of vascular smooth muscle cells in response to arterial injury, a major feature of vascular intimal hyperplasia. Intimal hyperplasia occurs in more than a third of patients receiving percutaneous transluminal balloon angioplasty. Stenting with or without coating significantly reduces the incidence rate of angiographic restenosis and that of target vessel revascularization. However, "in-stent" intimal hyperplasia/restenosis remains a challenge for clinical cardiologists. Although both the cell types and mechanisms that contribute to intimal hyperplasia in response to vascular injury remain controversial, vascular smooth muscle cell migration and proliferation appear to play an important role in the process. In animal models, cytotoxic and cytostatic gene therapy strategies targeted at the vascular smooth muscle cells have shown therapeutic potential for the treatment of vascular intimal hyperplasia. However, Fas ligand-based gene therapy appears to offer several advantages. In this review article, we will discuss the mode of FasL/Fas signaling in vascular smooth muscle cells and its therapeutic implications. We will also compare the relative merits of FasL with other cytotoxic and cytostatic gene therapy approaches for the treatment of intimal hyperplasia.

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. In primary human endothelial cells, hypoxia, desferrioxamine, or infection with Ad2/HIF-1alpha/VP16, an adenoviral vector encoding a constitutively stable hybrid form of HIF-1alpha, increased the mRNA and protein levels of VEGF, angiopoietin-2 (Ang-2), and angiopoietin-4 (Ang-4). Infection with Ad2/CMVEV (a control vector expressing no transgene) had no effect. Angiopoietin-1 (Ang-1) expression was not detected in human endothelial cells. Ang-4 was also induced by hypoxia or Ad2/HIF-1alpha/VP16 in human cardiac cells, whereas Ang-1 expression remained unchanged. Recombinant Ang-4 protein protected endothelial cells against serum starvation-induced apoptosis and increased cultured endothelial cell migration and tube formation. Ad2/HIF-1alpha/VP16 stimulated endothelial cell proliferation and tube formation. Hypoxia- or Ad2/HIF-1alpha/VP16-induced tube formation was significantly reduced by a Tie-2 inhibitor. These results suggest that HIF-1 mediates the angiogenic response to hypoxia by upregulating the expression of multiple angiogenic factors. Ang-4 can function similarly as Ang-1 and substitute for Ang-1 to participate in hypoxia-induced angiogenesis. Activation of the angiopoietin/Tie-2 system may play a role in the ability of HIF-1 to induce hypervascularity without excessive permeability.

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. Direct injection of Ad2/CMVp35 into the left ventricular wall inhibited myocardial caspase 3 activity and apoptosis and improved left ventricular performance in rats treated with doxorubicin, whereas the same dose of Ad2/CMV beta gal encoding beta-galactosidase had no effect. These results suggest that adenovirus-mediated expression of p35 protects cardiomyocytes against doxorubicin cardiotoxicity, possibly by inhibiting caspase activity and by reducing cellular ROS levels. Localized delivery of gene transfer vectors expressing an antiapoptotic protein such as p35 to the myocardium may represent a therapeutic approach to alleviate doxorubicin-induced cardiomyopathy.