Vascular Regeneration in Ischemic Hindlimb by Adeno-Associated Virus Expressing Conditionally Silenced Vascular Endothelial Growth Factor.

BACKGROUND: Critical limb ischemia (CLI) is the extreme manifestation of peripheral artery disease, a major unmet clinical need for which lower limb amputation is the only option for many patients. After 2 decades in development, therapeutic angiogenesis has been tested clinically via intramuscular delivery of proangiogenic proteins, genes, and stem cells. Efficacy has been modest to absent, and the largest phase 3 trial of gene therapy for CLI reported a worsening trend of plasmid fibroblast growth factor. In all clinical trials to date, gene therapy has used unregulated vectors with limited duration of expression. Only unregulated extended expression vectors such as adeno-associated virus (AAV) and lentivirus have been tested in preclinical models. METHODS AND RESULTS: We present preclinical results of ischemia (hypoxia)-regulated conditionally silenced (CS) AAV-human vascular endothelial growth factor (hVEGF) gene delivery that shows efficacy and safety in a setting where other strategies fail. In a BALB/c mouse model of CLI, we show that gene therapy with AAV-CS-hVEGF, but not unregulated AAV or plasmid, vectors conferred limb salvage, protection from necrosis, and vascular regeneration when delivered via intramuscular or intra-arterial routes. All vector treatments conferred increased capillary density, but organized longitudinal arteries were selectively generated by AAV-CS-hVEGF. AAV-CS-hVEGF therapy reversibly activated angiogenic and vasculogenic genes, including Notch, SDF1, Angiopoietin, and Ephrin-B2. Reoxygenation extinguished VEGF expression and inactivated the program with no apparent adverse side effects. CONCLUSIONS: Restriction of angiogenic growth factor expression to regions of ischemia supports the safe and stable reperfusion of hindlimbs in a clinically relevant murine model of CLI.

High protein/fish oil diet prevents hepatic steatosis in NONcNZO10 mice; association with diet/genetics-regulated micro-RNAs.

OBJECTIVE: NONcNZO10 (NZ10) mice are predisposed to obesity and develop type 2 diabetes (T2D) and hepatic steatosis even when maintained on a control diet (CD) of 6% fat. Studies were designed to determine whether this extreme susceptibility phenotype could be alleviated by diet and if so the molecular targets of diet. METHODS: NZ10 and SWR/J (SWR) control mice were fed a CD or a test diet of high protein and fish oil (HPO) for 19 weeks and then analyzed for steatosis, blood chemistry, hepatic gene and micro-RNA expression. RESULTS: HPO diet prevented steatosis, significantly increased serum adiponectin and reduced serum cholesterol and triglycerides only in NZ10 mice. The HPO diet repressed hepatic expression of fatty acid metabolic regulators including PPAR-γ, sterol regulatory element-binding protein-c1, peroxisome proliferator-activated receptor gamma co-activator-1, fatty acid synthase, fatty acid binding protein-4, and apolipoprotein A4 genes only in NZ10 mice. Also repressed by a HPO diet were adiponectinR2 receptor, leptin-R, PPAR-α, pyruvate dehydrogenase kinase isoforms 2 and 4, AKT2 and GSK3ß. Micro-RNA (miR) arrays identified miRs that were diet and/or genetics regulated. QRTPCR confirmed increased expression of miR-205 and suppression of a series of miRs including miRs-411, 155, 335 and 21 in the NZ10-HPO group, each of which are implicated in the progression of diabetes and/or steatosis. Evidence is presented that miR-205 co-regulates with PPARγ and may regulate fibrosis and EMT during the progression of steatosis in the livers of NZ10-CD mice. The dietary responses of miR-205 are tissue-specific with opposite effects in adipose and liver. CONCLUSION: The results confirm that a HPO diet overrides the genetic susceptibility of NZ10 mice and this correlates with the suppression of key genes and perhaps micro-RNAs involved in hyperglycemia, dyslipidemia and inflammation including master PPAR regulators, adiponectin and leptin receptors.

Role of Micro RNA-205 in Promoting Visceral Adiposity of NZ10 Mice with Polygenic Susceptibility for Type 2 Diabetes.

SCOPE: To characterize diet-dependent miRNA profiles and their targets in the visceral adipose of mice with polygenic susceptibility to type 2 diabetes. METHODS AND RESULTS: Six-week NONcNZO10/LtJ (NZ10) and control SWR/J mice were subjected to high protein-fish oil or control diets for 19 weeks and micro-RNA microarray analyses were implemented on visceral adipose RNA. We found that 27 miRNAs were significantly induced and 10 significantly repressed in the VA of obese NZ10 mice compared with controls. 12 selected regulated miRNAs were confirmed by RT-PCR based on the microarray data and we demonstrated that the expression of these miRNAs remained unaltered in the VA of control SWR mice. To assess the possible functional roles of miRNAs in adipogenesis, we also analyzed their expression in 3T3-L1 cells during growth and differentiation. This revealed that suppression of miRNA-205 alone correlated selectively with increased cell proliferation and lipid formation of adipocytes. CONCLUSION: Diet and genetics control the expression of obesity-regulated miRNAs in the visceral adipose of NZ10 mice.

Influence of diet on visceral adipose remodeling in NONcNZO10 mice with polygenic susceptibility for type 2 diabetes.

Visceral adipose tissue (VAT) is a source of inflammatory cytokines that in obese subjects may contribute to low-level systemic inflammation and development of metabolic syndrome. Expansion of VAT involves adipocyte hyperplasia and hypertrophy and requires breakdown of the extracellular matrix and increased vascular outgrowth. To investigate changes of gene expression associated with VAT expansion and the role of combined genetics and diet, we implemented gene microarray analyses of VAT in NONcNZO10 (NZ10) and control SWR/J mice subjected to control chow (CD) or a diet of high protein and fish oil (HPO). NZ10 mice on CD showed increased body weight, hyperglycemia, and hyperinsulinemia at 25 weeks whereas those on HPO diet retained normal insulin levels and were normoglycemic. Two-way ANOVA revealed a significant interaction between diet and strain on blood glucose, serum insulin, and percent fat but not for body weight. Microarray heat maps revealed a remarkable combined effect of genetics and diet on genes that regulate extracellular matrix as well as angiogenic genes. Real time-PCR (RT-PCR) confirmed markedly increased expression of matrix metalloproteinases (MMPs) 2, 3, 11, and 12, vascular endothelial growth factor-A and C (VEGF-A and C), Von Willebrand Factor, and peroxisome proliferator-activated receptor-γ (PPAR-γ) selectively in the NZ10/CD group. MMP7 was significantly decreased. Protein levels of MMP2, 3, and 9 were significantly increased in the VA of NZ10 mice fed CD while those of MMP7 were downregulated. Microarrays also revealed diet-dependent two to fourfold increased expression of all four tissue inhibitor of metalloproteinases (TIMP) isoforms in NZ10 mice. Two-way ANOVA confirmed strongly interactive roles of diet and genetics on fat deposition and progression of type 2 diabetes in this polygenic mouse model.

Impaired CXCR4 expression and cell engraftment of bone marrow-derived cells from aged atherogenic mice.

OBJECTIVES: Reduced numbers and activity of circulating progenitor cells are associated with aging and have been linked with coronary artery disease. To determine the impact of aging and atherosclerotic disease on the chemotaxic activity of bone marrow derived cells (BMCs), we examined CXCR4 surface expression on BMCs from aged and atherosclerotic mice. METHODS: CXCR4 expression and cellular mobility were compared between BMCs of young (6-week old) ApoE null mice (ApoE(-/-)) and aged ApoE(-/-) mice that had been fed with a high-fat, high-cholesterol diet for 6-months. RESULTS: Age and atherosclerosis correlated with significantly lower surface expression of CXCR4 that was less inducible by calcium. The impaired calcium response was associated with defective calcium influx and was partially recovered by treatment with the calcium ionophore ionomycin. ApoE(-/-) mice fed high fat diet for 6-months had defective CXCR4 expression and SDF-1 regulation that is equivalent to that of 24-month old wild type mice. BMCs from aged, atherogenic ApoE(-/-) mice also displayed defective homing to SDF-1, and the animals had lower serum and bone marrow levels of SDF-1. CONCLUSION: Evolution of atherosclerosis in ApoE(-/-) mice is paralleled by progressive loss of mobility of BMCs with reductions of CXCR4 expression, and reduced levels of SDF-1 in both serum and bone marrow. These changes mute the homing capability of BMCs and may contribute to the progression of atherosclerosis in this model.

An essential role for diet in exercise-mediated protection against dyslipidemia, inflammation and atherosclerosis in ApoE⁻/⁻ mice.

BACKGROUND: Diet and exercise promote cardiovascular health but their relative contributions to atherosclerosis are not fully known. The transition from a sedentary to active lifestyle requires increased caloric intake to achieve energy balance. Using atherosclerosis-prone ApoE-null mice we sought to determine whether the benefits of exercise for arterial disease are dependent on the food source of the additional calories. METHODS AND RESULTS: Mice were fed a high-fat diet (HF) for 4.5 months to initiate atherosclerosis after which time half were continued on HF while the other half were switched to a high protein/fish oil diet (HP). Half of each group underwent voluntary running. Food intake, running distance, body weight, lipids, inflammation markers, and atherosclerotic plaque were quantified. Two-way ANOVA tests were used to assess differences and interactions between groups. Exercised mice ran approximately 6-km per day with no difference between groups. Both groups increased food intake during exercise and there was a significant main effect of exercise F((1,44) = 9.86, p<0.01) without interaction. Diet or exercise produced significant independent effects on body weight (diet: F(1,52) = 6.85, p = 0.012; exercise: F(1,52) = 9.52, p<0.01) with no significant interaction. The combination of HP diet and exercise produced a greater decrease in total cholesterol (F(1, 46) = 7.9, p<0.01) and LDL (F(1, 46) = 7.33, p<0.01) with a large effect on the size of the interaction. HP diet and exercise independently reduced TGL and VLDL (p<0.05 and 0.001 respectively). Interleukin 6 and C-reactive protein were highest in the HF-sedentary group and were significantly reduced by exercise only in this group. Plaque accumulation in the aortic arch, a marker of cardiovascular events was reduced by the HP diet and the effect was significantly potentiated by exercise only in this group resulting in significant plaque regression (F1, 49 = 4.77, p<0.05). CONCLUSION: In this model exercise is beneficial to combat dyslipidemia and protect from atherosclerosis only when combined with diet.

c-Jun N-terminal kinase (JNK-1) confers protection against brief but not extended ischemia during acute myocardial infarction.

Brief periods of ischemia do not damage the heart and can actually protect against reperfusion injury caused by extended ischemia. It is not known what causes the transition from protection to irreversible damage as ischemia progresses. c-Jun N-terminal kinase-1 (JNK-1) is a stress-regulated kinase that is activated by reactive oxygen and thought to promote injury during severe acute myocardial infarction. Because some reports suggest that JNK-1 can also be protective, we hypothesized that the function of JNK-1 depends on the metabolic state of the heart at the time of reperfusion, a condition that changes progressively with duration of ischemia. Mice treated with JNK-1 inhibitors or transgenic mice wherein the JNK-1 gene was ablated were subjected to 5 or 20 min of ischemia followed by reperfusion. When JNK-1 was inactive, ischemia of only 5 min duration caused massive apoptosis, infarction, and negative remodeling that was equivalent to or greater than extended ischemia. Conversely, when ischemia was extended JNK-1 inactivation was protective. Mechanisms of the JNK-1 switch in function were investigated in vivo and in cultured cardiac myocytes. In vitro there was a comparable switch in the function of JNK-1 from protective when ATP levels were maintained during hypoxia to injurious when reoxygenation followed glucose and ATP depletion. Both apoptotic and necrotic death pathways were affected and responded reciprocally to JNK-1 inhibitors. JNK-1 differentially regulated Akt phosphorylation of the regulatory sites Ser-473 and Thr-450 and the catalytic Thr-308 site in vivo. The studies define a novel role for JNK-1 as a conditional survival kinase that protects the heart against brief but not protracted ischemia.