Adiposity is associated with widespread transcriptional changes and downregulation of longevity pathways in aged skeletal muscle.

BACKGROUND: Amongst healthy older people, a number of correlates of impaired skeletal muscle mass and function have been defined. Although the prevalence of obesity is increasing markedly in this age group, information is sparse about the particular impacts of obesity on ageing skeletal muscle or the molecular mechanisms that underlie this and associated disease risk. METHODS: Here, we examined genome-wide transcriptional changes using RNA sequencing in muscle biopsies from 40 older community-dwelling men from the Hertfordshire Sarcopenia Study with regard to obesity (body mass index [BMI] >30 kg/m2 , n = 7), overweight (BMI 25-30, n = 19), normal weight (BMI < 25, n = 14), and per cent and total fat mass. In addition, we used EPIC DNA methylation array data to investigate correlations between DNA methylation and gene expression in aged skeletal muscle tissue and investigated the relationship between genes within altered regulatory pathways and muscle histological parameters. RESULTS: Individuals with obesity demonstrated a prominent modified transcriptional signature in muscle tissue, with a total of 542 differentially expressed genes associated with obesity (false discovery rate ≤0.05), of which 425 genes were upregulated when compared with normal weight. Upregulated genes were enriched in immune response (P = 3.18 × 10-41 ) and inflammation (leucocyte activation, P = 1.47 × 10-41 ; tumour necrosis factor, P = 2.75 × 10-15 ) signalling pathways and downregulated genes enriched in longevity (P = 1.5 × 10-3 ) and AMP-activated protein kinase (AMPK) (P = 4.5 × 10-3 ) signalling pathways. Furthermore, differentially expressed genes in both longevity and AMPK signalling pathways were associated with a change in DNA methylation, with a total of 256 and 360 significant cytosine-phosphate-guanine-gene correlations identified, respectively. Similar changes in the muscle transcriptome were observed with respect to per cent fat mass and total fat mass. Obesity was further associated with a significant increase in type II fast-fibre area (P = 0.026), of which key regulatory genes within both longevity and AMPK pathways were significantly associated. CONCLUSIONS: We provide for the first time a global transcriptomic profile of skeletal muscle in older people with and without obesity, demonstrating modulation of key genes and pathways implicated in the regulation of muscle function, changes in DNA methylation associated with such pathways and associations between genes within the modified pathways implicated in muscle regulation and changes in muscle fibre type.

Epigenome-wide association study of sarcopenia: findings from the Hertfordshire Sarcopenia Study (HSS).

BACKGROUND: Sarcopenia is the age-related loss of muscle mass, strength, and function. Epigenetic processes such as DNA methylation, which integrate both genetic and environmental exposures, have been suggested to contribute to the development of sarcopenia. This study aimed to determine whether differences in the muscle methylome are associated with sarcopenia and its component measures: grip strength, appendicular lean mass index (ALMi), and gait speed. METHODS: Using the Infinium Human MethylationEPIC BeadChip, we measured DNA methylation in vastus lateralis muscle biopsies of 83 male participants (12 with sarcopenia) with a mean (standard deviation) age of 75.7 (3.6) years from the Hertfordshire Sarcopenia Study (HSS) and Hertfordshire Sarcopenia Study extension (HSSe) and examined associations with sarcopenia and its components. Pathway, histone mark, and transcription factor enrichment of the differentially methylated CpGs (dmCpGs) were determined, and sodium bisulfite pyrosequencing was used to validate the sarcopenia-associated dmCpGs. Human primary myoblasts (n = 6) isolated from vastus lateralis muscle biopsies from male individuals from HSSe were treated with the EZH2 inhibitor GSK343 to assess how perturbations in epigenetic processes may impact myoblast differentiation and fusion, measured by PAX7 and MYHC immunocytochemistry, and mitochondrial bioenergetics determined using the Seahorse XF96. RESULTS: Sarcopenia was associated with differential methylation at 176 dmCpGs (false discovery rate ≤ 0.05) and 141 differentially methylated regions (Stouffer ≤ 0.05). The sarcopenia-associated dmCpGs were enriched in genes associated with myotube fusion (P = 1.40E-03), oxidative phosphorylation (P = 2.78E-02), and voltage-gated calcium channels (P = 1.59E-04). ALMi was associated with 71 dmCpGs, grip strength with 49 dmCpGs, and gait speed with 23 dmCpGs (false discovery rate ≤ 0.05). There was significant overlap between the dmCpGs associated with sarcopenia and ALMi (P = 3.4E-35), sarcopenia and gait speed (P = 4.78E-03), and sarcopenia and grip strength (P = 7.55E-06). There was also an over-representation of the sarcopenia, ALMi, grip strength, and gait speed-associated dmCpGs with sites of H3K27 trimethylation (all P ≤ 0.05) and amongst EZH2 target genes (all P ≤ 0.05). Furthermore, treatment of human primary myoblasts with the EZH2 inhibitor GSK343 inhibitor led to an increase in PAX7 expression (P ≤ 0.05), decreased myotube fusion (P = 0.043), and an increase in ATP production (P = 0.008), with alterations in the DNA methylation of genes involved in oxidative phosphorylation and myogenesis. CONCLUSIONS: These findings show that differences in the muscle methylome are associated with sarcopenia and individual measures of muscle mass, strength, and function in older individuals. This suggests that changes in the epigenetic regulation of genes may contribute to impaired muscle function in later life.

Folic Acid Induces Intake-Related Changes in the Mammary Tissue Transcriptome of C57BL/6 Mice.

Folic acid (FA) intake has been associated with increased breast cancer risk in some studies. Although underlying mechanisms are unknown, epigenetic modifications that persistently alter transcription have been suggested. We tested the hypothesis that high FA (HFA) intake alters the adult mammary transcriptome in a manner consistent with increased potential for carcinogenesis, detectable beyond the period of intake. C57BL/6 mice were fed control FA (CFA) (1 mg/kg diet) or HFA (5 mg/kg diet) diets for 4 weeks, followed by AIN93M maintenance diet for 4 weeks. Plasma 5-methyltetrahydrofolate, p-aminobenzoylglutamate and unmetabolised FA concentrations were greater (1.62, 1.56, 5.80-fold, respectively) in HFA compared to CFA mice. RNA sequencing of the mammary transcriptome (~20 million reads) showed 222 transcripts (191 upregulated) differentially expressed between groups. Gene Set Enrichment showed upregulated genes significantly enriched in Epithelial Mesenchymal Transition, Myogenesis and Apical Junction and downregulated genes in E2F targets, MYC targets and G2M checkpoint. Cancer was the most altered Disease and Disorder pathway, with Metastasis, Mammary Tumour and Growth of Tumour the most upregulated pathways. ChIP-seq enrichment analysis showed that targets of histone methyltransferase EZH2 were enriched in HFA mice. This study demonstrates HFA intake during adulthood induces mammary transcriptome changes, consistent with greater tumorigenic potential.

Increased tumor localization and reduced immune response to adenoviral vector formulated with the liposome DDAB/DOPE.

UNLABELLED: We aimed to increase the efficiency of adenoviral vectors by limiting adenoviral spread from the target site and reducing unwanted host immune responses to the vector. We complexed adenoviral vectors with DDAB-DOPE liposomes to form adenovirus-liposomal (AL) complexes. AL complexes were delivered by intratumoral injection in an immunocompetent subcutaneous rat tumor model and the immunogenicity of the AL complexes and the expression efficiency in the tumor and other organs was examined. Animals treated with the AL complexes had significantly lower levels of beta-galactosidase expression in systemic tissues compared to animals treated with the naked adenovirus (NA) (P<0.05). The tumor to non-tumor ratio of beta-galactosidase marker expression was significantly higher for the AL complex treated animals. NA induced significantly higher titers of adenoviral-specific antibodies compared to the AL complexes (P<0.05). The AL complexes provided protection (immunoshielding) to the adenovirus from neutralizing antibody. Forty-seven percent more beta-galactosidase expression was detected following intratumoral injection with AL complexes compared to the NA in animals pre-immunized with adenovirus. CONCLUSIONS: Complexing of adenovirus with liposomes provides a simple method to enhance tumor localization of the vector, decrease the immunogenicity of adenovirus, and provide protection of the virus from pre-existing neutralizing antibodies.

Modification of liposomal concentration in liposome/adenoviral complexes allows significant protection of adenoviral vectors from neutralising antibody, in vitro.

Adenoviral vectors have been commonly used in gene therapy protocols, however the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced which limits further administration. This study examines the efficacy of complexing liposomes to adenovirus for the protection of the adenovirus from neutralising antibodies in an in vitro setting. Dimethyldioctadecylammonium bromide (DDAB)-dioleoyl-l-phosphatidylethanolamine (DOPE) liposomes were bound at varying concentrations to adenovirus to form AL complexes and tested these complexes' ability to prevent adenoviral neutralisation. It is shown that by increasing the concentration of liposomes in the adenoviral-liposome (AL) complexes we can increase the level of immuno-shielding afforded the adenovirus. It is also shown that the increase in liposomal concentration may lead to drawbacks such as increased cytotoxicity and reductions in expression levels.

Microsphere-liposome complexes protect adenoviral vectors from neutralising antibody without losses in transfection efficiency, in-vitro.

Adenoviral vectors have been commonly used in gene therapy protocols but the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced, which limits further administration. This study examines the effectiveness of a novel combination of microspheres and liposomes for the shielding of adenovirus from neutralising antibodies in an in-vitro setting. We show that liposomes are effective in the protection of adenovirus from neutralising antibody and that the conjugation of these complexes to microspheres augments the level of protection. This study further reveals that previously neutralised adenovirus may still be transported into the cell via liposome-cell interactions and is still capable of expressing its genes, making this vector an effective tool for circumvention of the humoral immune response. We also looked at possible side effects of using the complexes, namely increases in cytotoxicity and reductions in transfection efficiency. Our results showed that varying the liposome:adenovirus ratio can reduce the cytotoxicity of the vector as well as increase the transfection efficiency. In addition, in cell lines that are adenoviral competent, transfection efficiencies on par with uncomplexed adenoviral vectors were achievable with the combination vector.

In-vitro evaluation of ion-exchange microspheres for the sustained release of liposomal-adenoviral conjugates.

This study looks at the development of a novel combination vector consisting of adenovirus conjugated to liposomes (AL complexes) bound to cation-exchanging microspheres (MAL complexes). With adenovirus having a net negative charge and the liposomes a net positive charge it was possible to modify the net charge of the AL complexes by varying the concentrations of adenovirus to liposomes. The modification of the net charge resulted in altered binding and release characteristics. Of the complexes tested, the 5:1 and 2:1 ratio AL complexes were able to be efficiently bound by the microspheres and exhibited sustained release over 24 h. The 1:1 and 1:2 AL complexes, however, bound poorly to the microspheres and were rapidly released. In addition the MAL complexes also were able to reduce the toxicity of the AL complexes, which was seen with the 10:1 ratio. The AL complexes showed considerably more toxicity alone than in combination with microspheres, highlighting a potential benefit of this vector.

Pre-treatment with a non-therapeutic dose of cisplatin increases solid tumour response to liposomal-p53 gene therapy- An in vivo study.

Successful liposomal-mediated gene therapy is often limited by poor transfection efficiencies. One method previously shown to increase the efficiency of liposomal gene delivery is through the administration of a non-therapeutic dose of the chemotherapeutic drug cisplatin prior to lipofection. The currents study aims to utilise this method to deliver lipoplexes containing the p53 tumour suppressor gene with the aim of increasing therapeutic effect of the p53 gene on a solid tumour in vivo. Rats, implanted with solid salivary adenocarcinomas, were pre-treated with a low dose of cisplatin seven days prior to liposomal mediated p53 treatment. Following treatment with p53, tumour growth, p53 expression and levels of apoptosis were examined and compared to animals treated with p53 without cisplatin pre-treatment and a saline control. Tumours that had been pre-treated with cisplatin prior to p53-lipofection were significantly smaller than both the saline control and the non-cisplatin treated tumours. Saline treated tumours increased in size by an average of 164% over a 96-hour period compared to 64% and 101% for the cisplatin and non-cisplatin p53-liposome treated tumours. The cisplatin pre-treated tumours resulted in significantly higher levels of apoptosis surrounding the treatment site and exhibited prolonged p53 expression when compared to the non-cisplatin pre-treated tumours. The results suggest that the use of cisplatin to pre-sensitise tumours to lipofection has significant benefits when used in conjunction with p53.

Modified microplex vector enhances transfection of cells in culture while maintaining tumour-selective gene delivery in-vivo.

A non-commercial liposome (dimethyl dioctadecyl ammonium bromide:dioleoyl phosphatidylethanolamine) was compared with a commercial variety (Lipofectamine) for transfection of cultured rat adenocarcinoma cells and in an in-vivo kidney tumour model. Transfection of the cells in culture and in tumours in-vivo was variable with both types of liposomes. A high-dose microplex (lipoplex-microsphere) vector enhanced liposome-mediated transfection of cells in culture. When these high-dose microplexes were tested in-vivo, they were better than both microspherical and liposomal delivery modes in terms of transgene expression levels and the tumour-to-normal tissue ratio of gene delivery. Microplexes have been demonstrated to be capable of not only selective delivery of plasmids to solid tumours, but also of increasing transfection in cell culture, a finding that may be used in ex-vivo transfection studies. It is hypothesized that microspheres anchored the combination vector closer to the cultured cells, allowing attached liposomes to gain easier access into cells. In-vivo, microspheres permitted the microplexes to selectively deliver their genetic payload within the tumour tissue, from where the action of cationic liposomes on cellular membranes facilitated increased access of plasmids into the cytosol of target cells.

A model for evaluating selective delivery of plasmid DNA to tumours via the vasculature.

A comparative study of plasmid DNA delivery in a newly established rat renal solid tumour model was undertaken. Free plasmid, plasmids bound to microspheres, and plasmids complexed with liposomes were selectively delivered to tumours via arterial catheterisation. Forty-eight hours post delivery, tumour to normal kidney tissue chloramphenicol acetyltransferase expression ratios were as follows: free (1.8:1), microspherical (3.9:1), and liposomal (1.2:1). Microspheres were able to selectively deliver the plasmids to tumours, whereas cationic liposomes distributed the plasmids to both kidney parenchymal and tumour cells. This tumour model has the potential of screening delivery vehicles as well as therapeutic agents for the capacity of selective delivery to tumours via the vasculature.

Liposomes containing cationic dimethyl dioctadecyl ammonium bromide: formulation, quality control, and lipofection efficiency.

This article describes a novel, simple, and relatively inexpensive method to prepare cationic liposomes using an ethanol injection/pressure extrusion method. The study also demonstrated that binding erythrosine dye to cationic liposomes results in a shift of the absorption maximum of the dye from 528 nm to 549 nm at pH 4.25, allowing quantification and visualization of these vesicles. In addition, a relatively simple Ficoll-based gradient centrifugation method for separation of lipoplexes from unbound molecules is presented. Laboratory-formulated dimethyl dioctadecyl ammonium bromide (DDAB) containing liposomes were just as efficient in complexing nucleic acids as commercially available types, and binding increased as the positive to neutral lipid ratio was increased. Transfection efficiency of the DDAB-containing liposomes increased as the ratio of cationic to neutral lipid was increased from 1:1 to 4:1 with either PtdChol or DOPE as the neutral lipid. A concomitant increase in cytotoxicity of CSU-SA1 cancer cells was noted as the ratio of positive to neutral lipid of the liposomes was increased. Nevertheless, our present study showed that the 2:1 liposome is a good choice since it delivers functional plasmids at a comparable rate to commercial liposome formulations, has similar toxicities to the less harmful commercial liposomes, and is at least 1000-fold more economical to prepare inhouse, a major factor to be considered in preclinical and clinical studies with these carriers.