Effects of Urocortin 2 Gene Transfer on Glucose Disposal in Insulin-Resistant db/db Mice on Metformin.

The study was designed to determine whether urocortin 2 (Ucn2) gene transfer is as safe and effective as metformin in insulin-resistant mice. Four groups of insulin-resistant db/db mice and a nondiabetic group were studied: (1) metformin; (2) Ucn2 gene transfer; (3) metformin + Ucn2 gene transfer; (4) saline; and (5) nondiabetic mice. After completion of the 15-week protocol, glucose disposal was quantified, safety assessed, and gene expression documented. Ucn2 gene transfer was superior to metformin, providing reductions in fasting glucose and glycated hemoglobin and enhanced glucose tolerance. The combination of metformin + Ucn2 gene transfer provided no better glucose control than Ucn2 gene transfer alone and was not associated with hypoglycemia. Metformin alone, Ucn2 gene transfer alone, and metformin + Ucn2 gene transfer together reduced fatty infiltration of the liver. Serum alanine transaminase concentration was elevated in all db/db groups (vs. nondiabetic controls), but the metformin + Ucn2 gene transfer combined group had the lowest alanine transaminase levels. No group differences in fibrosis were detected. In a hepatoma cell line, activation of AMP kinase showed a rank order of combined metformin + Ucn2 peptide > Ucn2 peptide > metformin. We conclude (1) The combination of metformin + Ucn2 gene transfer does not result in hypoglycemia. (2) Ucn2 gene transfer alone provides superior glucose disposal versus metformin alone. (3) The combination of Ucn2 gene transfer and metformin is safe and has additive effects in reducing serum alanine transaminase concentration, activating AMP kinase activity, and increasing Ucn2 expression, but is no more efficacious than Ucn2 gene transfer alone in reducing hyperglycemia. These data indicate that Ucn2 gene transfer is more effective than metformin in the db/db model of insulin resistance and combined treatment with metformin + Ucn2 gene transfer appears to have favorable effects on liver function and Ucn2 expression.

Exploring the Influence of Hepatitis B Immunoglobulin on the Immune Response to the Hepatitis B Vaccine in a Mouse Model.

Background: The extent to which maternal antibodies against the hepatitis B surface antigen (HBsAb) acquired transplacentally affect the immune responses to the hepatitis B vaccine (HBVac) in infants is still uncertain. Objective: To explore the impact of the HBsAb on the immune response to the HBVac in a mouse model. Methods: According to the doses of the HBVac (2, 5 µg) injected, 267 BALB/c mice were divided into two groups. Each group was subdivided into 3 subgroups based on the doses of the hepatitis B immunoglobulin (HBIG) (0, 25, 50 IU) administered. The HBsAb titers were detected 4 weeks after completing the HepB vaccination. Results: Among all the mice, 40 had an HBsAb titer <100 mIU/mL (non- or low-response to the HBVac). The rates of the HBsAb titer <100 mIU/mL in 0, 25 and 50 IU HBIG groups were 1.1%, 23.1%, and 20.7%, respectively. Multivariate logistic regression analysis showed that the risk factors for low- or non-response to the HBVac were injection with the HBIG, low HBVac dose, and hypodermic injection. The mean HBsAb titers (log10) reduced gradually in the 0, 25 and 50 IU HBIG groups (P<0.001). Conclusion: The HBIG administration has negative impacts on the peak level of the HBsAb and the rate of an effective immune response. This implies that the maternal HBsAb acquired transplacentally might inhibit the immune responses to the HBVac in infants.

Urocortin 2 Gene Transfer for Systolic and Diastolic Dysfunction Due to Chronically Increased Left Ventricular Pressure.

We used transverse aortic constriction (TAC) in mice to test the hypothesis that urocortin 2 (Ucn2) gene transfer would increase left ventricular (LV) systolic and diastolic function in the pressure-stressed LV. Three groups were studied: (1) control mice (no TAC); (2) mice that received saline 6 weeks after TAC; and (3) mice that received Ucn2 gene transfer 6 weeks after TAC, using adeno-associated virus 8 encoding murine Ucn2 (AAV8.mUcn2; 2 × 1013 genome copies (gc)/kg, i.v. per mouse). Echocardiography was performed 6 and 12 weeks after TAC. In terminal studies 12 weeks after TAC, rates of LV pressure development and decay and Tau were measured, and LV cardiac myocytes (CMs) were isolated and cytosolic Ca2+ transients and sarcomere shortening rates recorded. Reverse transcription polymerase chain reaction and immunoblotting were used to measure key proteins in LV samples. A CM cell line (HL-1) was used to explore mechanisms. Concentric LV hypertrophy was evident on echocardiography 6 weeks after TAC. Twelve weeks after TAC, LV ejection fraction (EF) was higher in mice that received Ucn2 gene transfer (TAC-saline: 65% ± 3%; TAC-Ucn2: 75% ± 2%; p = 0.01), as was LV peak +dP/dt (1.9-fold increase; p = 0.001) and LV peak -dP/dt (1.7-fold increase; p = 0.017). Tau was more rapid (23% reduction, p = 0.02), indicating improved diastolic function. The peak rates of sarcomere shortening (p = 0.002) and lengthening (p = 0.002) were higher in CMs from TAC-Ucn2 mice, and Tau was reduced (p = 0.001). LV (Ser-16) phosphorylation of phospholamban (PLB) was increased in TAC-Ucn2 mice (p = 0.025), and also was increased in HL-1 cells treated with angiotensin II to induce hypertrophy and incubated with Ucn2 peptide (p = 0.001). Ucn2 gene transfer in TAC-induced heart failure with preserved ejection fraction increased cardiac function in the intact LV and provided corresponding benefits in CMs isolated from study animals, including increased myofilament Ca2+ sensitivity during contraction. The mechanism includes enhanced CM Ca2+ handling associated with increased (Ser-16)-PLB.

Simultaneous Determination of α-Glucosidase Inhibitory Triterpenoids in Psidium guajava Using HPLC-DAD-ELSD and Pressurized Liquid Extraction.

Psidium guajava, a popular food and medicine dual purposes plant cultivated in tropical and subtropical regions, has been widely used as food crop and folk medicine, such as anti-diabetes agent, around the world. Triterpenoids have been considered as the major active ingredients of P. guajava. In the present study, a high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors (HPLC-DAD-ELSD) method was developed for simultaneous determination of nine triterpenoids in P. guajava. Pressurized liquid extraction (PLE) was performed for sample preparation, and the analysis was achieved on a Cosmosil 5C18-MS-II (Nacalai Tesque, Kyoto, Japan) column eluted with gradient 0.1% aqueous formic acid-methanol system. The drift tube temperature of ELSD was set at 40 °C, and nitrogen flow-rate was at 1.6 L/min. All calibration curves for the analytes showed good linear regression (R2 > 0.9992) within test ranges. The established method was validated for intra-day and inter-day precisions (RSDs < 5%) and accuracy (recovery 94.23-106.87%). The validated method was successfully applied to determinate nine triterpenoids in 15 samples from the leave or fruit of P. guajava. In addition, the α-glucosidase inhibition assay showed good α-glucosidase inhibition activity in almost all the determined triterpenoids. The present study suggested that triterpenoids should be the quality control markers for P. guajava and HPLC-DAD-ELSD was an effective tool for the quality control of P. guajava.

Urocortin 2 Gene Transfer Improves Glycemic Control and Reduces Retinopathy and Mortality in Murine Insulin Deficiency.

Type 1 diabetes affects 20 million patients worldwide. Insulin is the primary and commonly the sole therapy for type 1 diabetes. However, only a minority of patients attain the targeted glucose control and reduced adverse events. We tested urocortin 2 gene transfer as single-agent therapy for insulin deficiency using two mouse models. Urocortin 2 gene transfer reduced blood glucose for months after a single intravenous injection, through increased skeletal muscle insulin sensitivity, increased insulin release in response to glucose stimulation, and increased plasma insulin levels before and during euglycemic clamp. The combined increases in both insulin availability and sensitivity resulted in improved glycemic indices-events that were not anticipated in these insulin-deficient models. In addition, urocortin 2 gene transfer reduced ocular manifestations of long-standing insulin deficiency such as vascular leak and improved retinal function. Finally, mortality was reduced by urocortin 2 gene transfer. The mechanisms for these beneficial effects included increased activities of AMP-activated protein kinase and Akt (protein kinase B) in skeletal muscle, increased skeletal muscle glucose uptake, and increased insulin release. These data suggest that urocortin 2 gene transfer may be a viable therapy for new onset type 1 diabetes and might reduce insulin needs in later stage disease.

Urocortin 2 Gene Transfer Improves Heart Function in Aged Mice.

Prevalence of left ventricular (LV) systolic and diastolic dysfunction increases with aging. We previously reported that urocortin 2 (Ucn2) gene transfer increases heart function in mice with heart failure with reduced ejection fraction. Here, we test the hypotheses that (1) Ucn2 gene transfer will increase LV function in aged mice and that (2) Ucn2 gene transfer given in early life will prevent age-related LV dysfunction. Nineteen-month-old (treatment study) and 3-month-old (prevention study) mice received Ucn2 gene transfer or saline. LV function was examined 3-4 months (treatment study) or 20 months (prevention study) after Ucn2 gene transfer or saline injection. In both the treatment and prevention strategies, Ucn2 gene transfer increased ejection fraction, reduced LV volume, increased LV peak -dP/dt and peak +dP/dt, and reduced global longitudinal strain. Ucn2 gene transfer-in both treatment and prevention strategies-was associated with higher levels of LV SERCA2a protein, reduced phosphorylation of LV CaMKIIa, and reduced LV α-skeletal actin mRNA expression (reflecting reduced cardiac stress). In conclusion, Ucn2 gene transfer restores normal cardiac function in mice with age-related LV dysfunction and prevents development of LV dysfunction.

Significant alteration of liver metabolites by AAV8.Urocortin 2 gene transfer in mice with insulin resistance.

INTRODUCTION: Urocortin 2 (Ucn2) is a 38-amino acid peptide of the corticotropin-releasing factor family. Intravenous (IV) delivery of an adeno-associated virus vector serotype 8 encoding Ucn2 (AAV8.Ucn2) increases insulin sensitivity and glucose disposal in mice with insulin resistance. OBJECTIVE: To determine the effects of Ucn2 on liver metabolome. METHODS: Six-week-old C57BL6 mice were divided into normal chow (CHOW)-fed and high fat diet (HFD)-fed groups. The animals received saline, AAV8 encoding no gene (AAV8.Empt) or AAV8.Ucn2 (2x1013 genome copy/kg, IV injection). Livers were isolated from CHOW-fed and HFD-fed mice and analyzed by untargeted metabolomics. Group differences were statistically analyzed. RESULTS: In CHOW-fed mice, AAV8.Ucn2 gene transfer (vs. saline) altered the metabolites in glycolysis, pentose phosphate, glycogen synthesis, glycogenolysis, and choline-folate-methionine signaling pathways. In addition, AAV8.Ucn2 gene transfer increased amino acids and peptides, which were associated with reduced protein synthesis. In insulin resistant (HFD-induced) mice, HFD (vs CHOW) altered 448 (112 increased and 336 decreased) metabolites and AAV8.Ucn2 altered 239 metabolites (124 increased and 115 reduced) in multiple pathways. There are 61 metabolites in 5 super pathways showed interactions between diet and AAV8.Ucn2 treatment. Among them, AAV8.Ucn2 gene transfer reversed HFD effects on 13 metabolites. Finally, plasma Ucn2 effects were determined using a 3-group comparison of HFD-fed mice that received AAV8.Ucn2, AAV.Empt or saline, where 18 metabolites that altered by HFD (15 increased and 3 decreased), but restored levels to that seen in CHOW-fed mice by increased plasma Ucn2. CONCLUSIONS: Metabolomics study revealed that AAV8.Ucn2 gene transfer, through increased plasma Ucn2, provided counter-HFD effects in restoring hepatic metabolites to normal levels, which could be the underlying mechanisms for Ucn2 effects on increasing glucose disposal and reducing insulin assistance.

Four new corynanthe-type alkaloids from the roots of Alstonia scholaris.

Four new corynanthe-type alkaloids, meloslines C-F (1-4), together with four known ones (5-8) were isolated from the roots of Alstonia scholaris. Their structures including absolute configurations were elucidated by extensive spectroscopic analysis and electronic circular dichroism (ECD) calculation. Compounds 1 and 2 exhibited potent vasorelaxant activity on endothelium-intact renal arteries precontracted with KCl.

Cardiac-Directed Expression of Adenylyl Cyclase Catalytic Domain (C1C2) Attenuates Deleterious Effects of Pressure Overload.

A fusion protein (C1C2) constructed by fusing the intracellular C1 and C2 segments of adenylyl cyclase type 6 (AC6) retains beneficial effects of AC6 expression, without increasing cyclic adenosine monophosphate generation. The effects of cardiac-directed C1C2 expression in pressure overload is unknown. Left ventricular (LV) pressure overload was induced by transverse aortic constriction (TAC) in C1C2 mice and in transgene negative (TG-) mice. Four weeks after TAC, LV systolic function and diastolic function were measured, and Ca2+ handling was assessed. Four weeks after TAC, TG- animals showed reduced LV peak +dP/dt. LV peak +dP/dt in C1C2 mice was statistically indistinguishable from that of normal mice and was higher than that seen in TG- mice 4 weeks after TAC (p = 0.02), despite similar and substantial cardiac hypertrophy. In addition to higher LV peak +dP/dt in vivo, cardiac myocytes from C1C2 mice showed shorter time-to-peak Ca2+ transient amplitude (p = 0.002) and a reduced time constant of cytosolic Ca2+ decline (Tau; p = 0.003). Sarcomere shortening fraction (p < 0.03) and the rate of sarcomere shortening (p < 0.02) increased in C1C2 cardiac myocytes. Myofilament sensitivity to Ca2+ was increased in systole (p = 0.02) and diastole (p = 0.04) in C1C2 myocytes. These findings indicate enhanced Ca2+ handling associated with C1C2 expression. Favorable effects on Ca2+ handling and LV function were associated with increased LV SERCA2a protein content (p = 0.015) and reduced LV fibrosis (p = 0.008). Cardiac-directed C1C2 expression improves Ca2+ handling and increases LV contractile function in pressure overload. These data provide a rationale for further exploration of C1C2 gene transfer as a potential treatment for heart failure.

Urocortin 2 Gene Transfer Reduces the Adverse Effects of a Western Diet on Cardiac Function in Mice.

Diabetes mellitus is associated with increased risk of heart failure. It has been previously demonstrated in mice that a single injection of adeno-associated virus 8 encoding urocortin 2 (AAV8.UCn2) increases glucose disposal in models of insulin resistance and improves the function of the failing heart. The present study tested the hypothesis that UCn2 gene transfer would reduce diabetes-related left ventricular (LV) dysfunction. Eight-week-old C57BL6 male mice were fed a Western diet (WD; 45% fat, 35% carbohydrate) for 40 weeks. At week 30, they received saline or AAV8.UCn2 (2 × 1013 genome copies/kg) via intravenous injection. Ten weeks after gene transfer, fasting blood glucose, glucose tolerance, and cardiac function were measured via echocardiography and in vivo measurement of LV contractile function, and the results were compared to those of mice fed normal chow (NC; 10% fat; 70% carbohydrate). The contents of key LV signaling proteins were also measured to probe mechanisms. WD increased 12 h fasting glucose (WD: 190 ± 11 mg/dL, n = 8; NC: 105 ± 12 mg/dL, n = 7; p = 0.0004). WD tended to reduce LV peak +dP/dt (p = 0.08) and LV peak -dP/dt (p = 0.05). LV ejection fraction was unchanged. Among WD-fed mice, UCn2 gene transfer reduced 12 h fasting glucose (WD-UCn2: 149 ± 6 mg/dL, n = 8; WD-Saline: 190 ± 11 mg/dL, n = 8; p = 0.012), increased LV peak +dP/dt (p < 0.001) and LV peak -dP/dt (p = 0.013), and reduced Tau (p < 0.02), indicating beneficial effects on systolic and diastolic LV function. In addition, among WD-fed mice, UCn2 gene transfer increased LV ejection fraction (p < 0.005) and the velocity of circumferential fiber shortening (p = 0.0005). Finally, a reduction was seen in fatty infiltration of the liver in WD-fed mice that had received UCn2 gene transfer. LV samples from WD-UCn2 mice showed increased phosphorylation of the protein kinase A catalytic domain (p = 0.03). In conclusion, UCn2 gene transfer increased LV systolic and diastolic function and reduced blood glucose in mice with diabetes-related LV dysfunction, indicating that UCn2 gene transfer may be of potential therapeutic benefit.

Urocortin 3 Gene Transfer Increases Function of the Failing Murine Heart.

Peptide infusions of peptides the corticotropin releasing factor family, including urocortin 2, stresscopin, and urocortin 3 (UCn3), have favorable acute effects in clinical heart failure (HF), but their short half-lives make them unsuitable for chronic therapy. This study asked whether UCn3 gene transfer, which provides sustained elevation of plasma UCn3 levels, increases the function of the failing heart. HF was induced by transmural left ventricular (LV) cryoinjury in mice. LV function was assessed 3 weeks later by echocardiography. Those with ejection fractions (EF) <40% received intravenous saline or intravenous adeno-associated virus type-8 encoding murine UCn3 (AAV8.mUCn3; 1.9 × 1013 genome copies/kg). Five weeks after randomization, repeat echocardiography, assessment of LV function (+dP/dt, -dP/dt), and quantification of Ca2+ transients and sarcomere shortening in isolated cardiac myocytes were conducted, and assessment of LV Ca2+ handling and stress proteins was performed. Three weeks after myocardial infarction, prior to treatment, EFs were reduced (mean 31%, from 63% in sham-operated animals). Mice randomized to receive UCn3 gene transfer showed increased plasma UCn3 (from 0.1 ± 0.01 ng/mL in the saline group to 5.6 ± 1.1 ng/mL; n = 12 each group; p < 0.0001). Compared to mice that received saline, UCn3 gene transfer was associated with higher values for EF (p = 0.0006); LV +dP/dt (p < 0.0001), and LV -dP/dt (p < 0.0001). Cardiac myocytes from mice that received UCn3 gene transfer showed higher peak Ca2+ transients (p = 0.0005), lower time constant of cytosolic Ca2+ decline (tau, p < 0.0001), and higher rates of sarcomere shortening (+dL/dt, p = 0.03) and lengthening (-dL/dt, p = 0.04). LV samples from mice that received UCn3 gene transfer contained higher levels of SERCA2a (p = 0.0004 vs. HF) and increased amounts of phosphorylated troponin I (p = 0.04 vs. HF). UCn3 gene transfer is associated with improved Ca2+ handling and LV function in mice with HF and reduced EF.

Effects of Urocortin 2 Versus Urocortin 3 Gene Transfer on Left Ventricular Function and Glucose Disposal.

UCn2 and UCn3 peptides have recently been infused to treat patients with heart failure (HF) but are limited by their short half-lives. A 1-time intravenous injection of virus vectors encoding UCn2 or UCn3 provided sustained increases in plasma concentrations of the peptides. This was associated with increases in both systolic and diastolic left ventricular (LV) function, mediated by increased LV SERCA2a expression and Ca2+ handling. UCn2, but not UCn3, gene transfer reduced fasting glucose and increased glucose disposal. These findings support UCn2 and UCn3 gene transfer as potential treatments for HF and indicate that UCn2 may be an optimal selection in patients with diabetes and HF.

Overexpression of Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains induces cluster of differentiation 59-mediated apoptosis in Jurkat cells.

Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains (CBP/PAG) is a membrane-bound adaptor protein that downregulates the activation of Src family kinases present in lipid rafts. To elucidate the role of CBP/PAG in human T cell activation, a cell line overexpressing CBP/PAG was constructed and the function of CBP/PAG in Jurkat cells was examined. The present study revealed that increased CBP/PAG expression in T cells significantly enhanced their apoptosis and reduced cellular activation and proliferation. Overexpression of CBP/PAG suppressed the growth of Jurkat cells by recruiting c-Src and its negative regulator, C-terminal Src kinase (CSK), to lipid rafts. The negative regulation of CBP/PAG was enhanced in the presence of anti-cluster of differentiation (CD)59 monoclonal antibodies. In addition, a significant association was revealed between the location of CBP/PAG and CD59, which were co-expressed in the same region of the cell membrane, implicating a potential overlap of the elicited signaling pathways. These results indicate that CBP/PAG functions as a negative regulator of cell signal transduction and suggest that CD59 may strengthen the role of negative feedback regulation.

A role for GPI-CD59 in promoting T-cell signal transduction via LAT.

Cluster of differentiation 59 (CD59) is a glycosylphosphatidylinositol-anchored protein. Cross-linking of CD59 with specific monoclonal antibodies can cause a series of intracellular signal transduction events. However, the underlying molecular mechanisms are poorly understood. Linker for activation of T-cells (LAT) is a crucial adaptor protein in T-cell signaling, and its phosphorylation and palmitoylation are essential for its localization and function. In a previous study by the present authors, it was demonstrated that CD59 may be responsible for LAT palmitoylation, thereby regulating T-cell signal transduction. The present study detected the co-localization of LAT and CD59 in lipid rafts by transfecting Jurkat cells with lentivirus vectors carrying the LAT-enhanced green fluorescent protein fusion protein. In addition, LAT and CD59 were shown to have a synergistic effect on the proliferation of Jurkat cells. The results also indicated that CD59 may transfer the palmitate group from phosphatidylinositol to LAT to form LAT palmitate, which then localizes to lipid rafts to regulate T-cell activation. The results of the present study provided novel insights into the role of CD59 in T-cell signal transduction.

Cardiac-directed expression of a catalytically inactive adenylyl cyclase 6 protects the heart from sustained ß-adrenergic stimulation.

OBJECTIVES: Increased expression of adenylyl cyclase type 6 (AC6) has beneficial effects on the heart through cyclic adenosine monophosphate (cAMP)-dependent and cAMP-independent pathways. We previously generated a catalytically inactive mutant of AC6 (AC6mut) that has an attenuated response to ß-adrenergic receptor stimulation, and, consequently, exhibits reduced myocardial cAMP generation. In the current study we test the hypothesis that cardiac-directed expression of AC6mut would protect the heart from sustained ß-adrenergic receptor stimulation, a condition frequently encountered in patients with heart failure. METHODS AND RESULTS: AC6mut mice and transgene negative siblings received osmotic mini-pumps to provide continuous isoproterenol infusion for seven days. Isoproterenol infusion caused deleterious effects that were attenuated by cardiac-directed AC6mut expression. Both groups showed reduced left ventricular (LV) ejection fraction, but the reduction was less in AC6mut mice (p = 0.047). In addition, AC6mut mice showed superior left ventricular function, manifested by higher values for LV peak +dP/dt (p = 0.03), LV peak -dP/dt (p = 0.008), end-systolic pressure-volume relationship (p = 0.003) and cardiac output (p<0.03). LV samples of AC6mut mice had more sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) protein (p<0.01), which likely contributed to better LV function. AC6mut mice had lower rates of cardiac myocyte apoptosis (p = 0.016), reduced caspase 3/7 activity (p = 0.012) and increased B-cell lymphoma 2 (Bcl2) expression (p = 0.0001). CONCLUSION: Mice with cardiac-directed AC6mut expression weathered the deleterious effects of continuous isoproterenol infusion better than control mice, indicating cardiac protection.

One-time injection of AAV8 encoding urocortin 2 provides long-term resolution of insulin resistance.

Using mice rendered insulin resistant with high fat diets (HFD), we examined blood glucose levels and insulin resistance after i.v. delivery of an adeno-associated virus type 8 encoding murine urocortin 2 (AAV8.UCn2). A single i.v. injection of AAV8.UCn2-normalized blood glucose and glucose disposal within weeks, an effect that lasted for months. Hyperinsulinemic-euglycemic clamps showed reduced plasma insulin, increased glucose disposal rates, and increased insulin sensitivity following UCn2 gene transfer. Mice with corticotropin-releasing hormone type 2-receptor deletion that were rendered insulin resistant by HFD showed no improvement in glucose disposal after UCn2 gene transfer, indicating that the effect requires UCn2's cognate receptor. We also demonstrated increased glucose disposal after UCn2 gene transfer in db/db mice, a second model of insulin resistance. UCn2 gene transfer reduced fatty infiltration of the liver in both models of insulin resistance. UCn2 increases Glut4 translocation to the plasma membrane in skeletal myotubes in a manner quantitatively similar to insulin, indicating a mechanism through which UCn2 operates to increase insulin sensitivity. UCn2 gene transfer, in a dose-dependent manner, is insulin sensitizing and effective for months after a single injection. These findings suggest a potential long-term therapy for clinical type-2 diabetes.

Intracoronary Gene Transfer of Adenylyl Cyclase 6 in Patients With Heart Failure: A Randomized Clinical Trial.

IMPORTANCE: Gene transfer has rarely been tested in randomized clinical trials. OBJECTIVE: To evaluate the safety and efficacy of intracoronary delivery of adenovirus 5 encoding adenylyl cyclase 6 (Ad5.hAC6) in heart failure. DESIGN, SETTING, AND PARTICIPANTS: A randomized, double-blind, placebo-controlled, phase 2 clinical trial was conducted in US medical centers (randomization occurred from July 19, 2010, to October 30, 2014). Participants 18 to 80 years with symptomatic heart failure (ischemic and nonischemic) and an ejection fraction (EF) of 40% or less were screened; 86 individuals were enrolled, and 56 were randomized. Data analysis was of the intention-to-treat population. Participants underwent exercise testing and measurement of left ventricular EF (echocardiography) and then cardiac catheterization, where left ventricular pressure development (+dP/dt) and decline (-dP/dt) were recorded. Participants were randomized (3:1 ratio) to receive 1 of 5 doses of intracoronary Ad5.hAC6 or placebo. Participants underwent a second catheterization 4 weeks later for measurement of dP/dt. Exercise testing and EF were assessed 4 and 12 weeks after randomization. INTERVENTIONS: Intracoronary administration of Ad5.hAC6 (3.2 × 109 to 1012 virus particles) or placebo. MAIN OUTCOMES AND MEASURES: Primary end points included exercise duration and EF before and 4 and 12 weeks after randomization and peak rates of +dP/dt and -dP/dt before and 4 weeks after randomization. Fourteen placebo participants were compared (intention to treat) with 24 Ad5.hAC6 participants receiving the highest 2 doses (D4 + 5). RESULTS: Fifty-six individuals were randomized and monitored for up to 1 year. Forty-two participants (75%) received Ad5.hAC6 (mean [SE] age, 63 [1] years; EF, 30% [1%]), and 14 individuals (25%) received placebo (age, 62 [1] years; EF, 30% [2%]). Exercise duration showed no significant group differences (4 weeks, P = .27; 12 weeks, P = .47, respectively). The D4 + 5 participants had increased EF at 4 weeks (+6.0 [1.7] EF units; n = 21; P < .004), but not 12 weeks (+3.0 [2.4] EF units; n = 21; P = .16). Placebo participants showed no increase in EF at 4 weeks or 12 weeks. Exercise duration showed no between-group differences (4-week change from baseline: placebo, 27 [36] seconds; D4 + 5, 44 [25] seconds; P = .27; 12-week change from baseline: placebo, 44 [28] seconds; D4 + 5, 58 [29 seconds, P = .47). AC6 gene transfer increased basal left ventricular peak -dP/dt (4-week change from baseline: placebo, +93 [51] mm Hg/s; D4 + 5, -39 [33] mm Hg/s; placebo [n = 21]; P < .03); AC6 did not increase arrhythmias. The admission rate for patients with heart failure was 9.5% (4 of 42) in the AC6 group and 28.6% (4 of 14) in the placebo group (relative risk, 0.33 [95% CI, 0.08-1.36]; P = .10). CONCLUSIONS AND RELEVANCE: AC6 gene transfer safely increased LV function beyond standard heart failure therapy, attainable with one-time administration. Larger trials are warranted. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00787059.

Cardiac-Directed Expression of Adenylyl Cyclase Catalytic Domain Reverses Cardiac Dysfunction Caused by Sustained Beta-Adrenergic Receptor Stimulation.

OBJECTIVE: To test the hypothesis that cardiac-directed expression of the cytoplasmic domains of adenylyl cyclase-6 (AC6) would have beneficial effects on the heart. BACKGROUND: Eliminating the two transmembrane domains of AC6 yields a protein with an intact catalytic domain that is disengaged from membrane-associated ß-adrenergic receptor stimulation, but with enhanced propensity for intracellular interactions. METHODS: We constructed a peptide of the C1 and C2 segments of AC6 (C1C2), expressed C1C2 in an adenovirus vector and generated transgenic lines with cardiac-directed C1C2 expression, which underwent sustained isoproterenol (Iso) infusion. RESULTS: Gene transfer of C1C2 in cardiac myocytes showed reduced cAMP generation in response to Iso-stimulation. C1C2 transgenic mice had normal left ventricular (LV) structure and function. LV samples from C1C2 mice showed diminished Iso-stimulated cAMP generation but normal LV contractile responses, suggesting a compensatory mechanism. Cardiac myocytes from C1C2 mice showed increased Iso-stimulated Ca2+ release and reduced time to peak Ca2+ release. After 7 days Iso infusion, control mice tended to show reduced LV function, but C1C2 mice showed increases in both LV peak +dP/dt and peak -dP/dt indicating enhanced LV systolic and diastolic function. LV from C1C2 mice showed a 2.6-fold increase in SERCA2a protein, and cardiac myocytes showed increased Ca2+ release, reduced time to peak Ca2+ release and reduced Tau. CONCLUSIONS: In C1C2 mice, sustained isoproterenol infusion increases rather than decreases LV function. Reduced cAMP generation and resistance to catecholamine cardiomyopathy are attractive features of this novel AC-related protein.

Study of the synergistic effects of all-transretinoic acid and C-phycocyanin on the growth and apoptosis of A549 cells.

In the present study, we investigated the effects of the combination of all-transretinoic acid (ATRA) and natural nontoxic C-phycocyanin (C-PC) on the growth of A549 lung cancer cells in vitro and in vivo. Furthermore, the anticancer mechanism of the drug combination was revealed. Results showed both C-PC and ATRA could inhibit the growth of A549 cells in vivo. The combination of ATRA+C-PC could yield a higher inhibition rate. C-PC exerted a major effect on the proliferation of human embryo lung cells, but ATRA at a high concentration exerted an inhibitory effect. In addition, ATRA+C-PC could decrease the CDK4 mRNA level, but upregulated caspase-3 protein expression and induced cell apoptosis. A mouse model with tumor was constructed by a subcutaneous injection to the left axilla of nu nude (NU/NU) mice. Compared with the control group, the tumor weight was decreased in the single-drug treatment group and was the lowest in the combination group. C-PC+ATRA could upregulate tumor necrosis factor levels and downregulate Bcl-2 expression and the cyclin D1 gene in the tumor. C-PC could promote T cells' activities and spleen weight, but a single use of ATRA exerted an opposite effect. The dosage of ATRA could be reduced when combined with C-PC to reduce the toxic side-effects. In summary, the antitumor effects of the C-PC+ATRA combination were more significant than a single drug in vivo and in vitro.

Intravenous AAV8 Encoding Urocortin-2 Increases Function of the Failing Heart in Mice.

Urocortin-2 (UCn2) peptide infusion increases cardiac function in patients with heart failure, but chronic peptide infusion is cumbersome, is costly, and provides only short-term benefits. Gene transfer would circumvent these shortcomings. We previously showed that a single intravenous (IV) injection of AAV8.UCn2 increases plasma UCn2 and left ventricular (LV) systolic and diastolic function for at least 7 months in normal mice. Here we test the hypothesis that IV delivery of AAV8.UCn2 increases function of the failing heart. Myocardial infarction (MI, by coronary ligation) was used to induce heart failure, which was assessed by echocardiography 3 weeks after MI. Mice with LV ejection fraction (EF) <25% received IV delivery of AAV8.UCn2 (5×10(11) gc) or saline, and 5 weeks later echocardiography showed increased LV EF in mice that received UCn2 gene transfer (p=0.01). In vivo physiological studies showed a 2-fold increase in peak rate of LV pressure development (LV +dP/dt; p<0.0001) and a 1.6-fold increase in peak rate of LV pressure decay (LV -dP/dt; p=0.0007), indicating increased LV systolic and diastolic function in treated mice. UCn2 gene transfer was associated with increased peak systolic Ca(2+) transient amplitude and rate of Ca(2+) decline and increased SERCA2a expression. In addition, UCn2 gene transfer reduced Thr286 phosphorylation of Cam kinase II, and increased expression of cardiac myosin light chain kinase, findings that would be anticipated to increase function of the failing heart. We conclude that a single IV injection of AAV8.UCn2 increases function of the failing heart. The simplicity of IV injection of a vector encoding a gene with beneficial paracrine effects to increase cardiac function is an attractive potential clinical strategy.