TNF and TNF receptor expression and insulin sensitivity in human omental and subcutaneous adipose tissue--influence of BMI and adipose distribution

Tumour necrosis factor (TNF)alpha is implicated in the relationship between obesity and insulin resistance/ type 2 diabetes. In an effort to understand this association better we (i) profiled gene expression patterns of TNF, TNFR1 and TNFR2 and (ii) investigated the effects of TNF on glucose uptake in isolated adipocytes and adipose tissue explants from omental and subcutaneous depots from lean, overweight and obese individuals. TNF expression correlated with expression of TNFR2, but not TNFR1, and TNF and TNFR2 expression increased in obesity. TNFR1 expression was higher in omental than in subcutaneous adipocytes. Expression levels of TNF or either receptor did not differ between adipocytes from individuals with central and peripheral obesity. TNF only suppressed glucose uptake in insulin-stimulated subcutaneous tissue and this suppression was only observed in tissue from lean subjects. These data support a relationship between the TNF system and body mass index (BMI), but not fat distribution, and suggest depot specificity of the TNF effect on glucose uptake. Furthermore, adipose tissue from obese subjects already appears insulin 'resistant' and this may be a result of the increased TNF levels.

BDNF and TNF-α polymorphisms in memory

Here, we investigate the genetic basis of human memory in healthy individuals and the potential role of two polymorphisms, previously implicated in memory function. We have explored aspects of retrospective and prospective memory including semantic, short term, working and long-term memory in conjunction with brain derived neurotrophic factor (BDNF) and tumor necrosis factor-alpha (TNF-alpha). The memory scores for healthy individuals in the population were obtained for each memory type and the population was genotyped via restriction fragment length polymorphism for the BDNF rs6265 (Val66Met) SNP and via pyrosequencing for the TNF-alpha rs113325588 SNP. Using univariate ANOVA, a significant association of the BDNF polymorphism with visual and spatial memory retention and a significant association of the TNF-alpha polymorphism was observed with spatial memory retention. In addition, a significant interactive effect between BDNF and TNF-alpha polymorphisms was observed in spatial memory retention. In practice visual memory involves spatial information and the two memory systems work together, however our data demonstrate that individuals with the Val/Val BDNF genotype have poorer visual memory but higher spatial memory retention, indicating a level of interaction between TNF-alpha and BDNF in spatial memory retention. This is the first study to use genetic analysis to determine the interaction between BDNF and TNF-alpha in relation to memory in normal adults and provides important information regarding the effect of genetic determinants and gene interactions on human memory.

Long-term stability of adipose tissue generated from a vascularized pedicled fat flap inside a chamber

Background Numerous studies demonstrate the generation and short-term survival of adipose tissue; however, long-term persistence remains elusive. This study evaluates long-term survival and transferability of de novo adipose constructs based on a ligated vascular pedicle and tissue engineering chamber combination. Methods Defined adipose tissue flaps were implanted into rats in either intact or perforated domed chambers. In half of the groups, the chambers were removed after 10 weeks and the constructs transferred on their vascular pedicle to a new site, where they were observed for a further 10 weeks. In the remaining groups, the tissue construct was observed for 20 weeks inside the chamber. Tissue volume was assessed using magnetic resonance imaging and histologic measures, and constructs were assessed for stability and necrosis. Sections were assessed histologically and for proliferation using Ki-67. Results At 20 weeks, volume analysis revealed an increase in adipose volume from 0.04 ± 0.001 ml at the time of insertion into the chambers to 0.27 ± 0.004 ml in the closed and 0.44 ± 0.014 ml in the perforated chambers. There was an additional increase of approximately 10 to 15 percent in tissue volume in flaps that remained in chambers for 20 weeks, whereas the volume of the transferred tissue not in chambers remained unaltered. Histomorphometric assessment of the tissues documented no signs of hypertrophy, fat necrosis, or atypical changes of the newly generated tissue. Conclusion This study presents a promising new method of generating significant amounts of mature, vascularized, stable, and transferable adipose tissue for permanent autologous soft-tissue replacement.

Zymosan-induced inflammation stimulates neo-adipogenesis

Objective: To investigate the potential of inflammation to induce new adipose tissue formation in the in vivo environment. Methods and results: Using an established model of in vivo adipogenesis, a silicone chamber containing a Matrigel and fibroblast growth factor 2 (1 μg/ml) matrix was implanted into each groin of an adult male C57Bl6 mouse and vascularized with the inferior epigastric vessels. Sterile inflammation was induced in one of the two chambers by suspending Zymosan-A (ZA) (200-0.02 μg/ml) in the matrix at implantation. Adipose tissue formation was assessed at 6, 8, 12 and 24 weeks. ZA induced significant adipogenesis in an inverse dose-dependent manner (P<0.001). At 6 weeks adipose tissue formation was greatest with the lowest concentrations of ZA and least with the highest. Adipogenesis occurred both locally in the chamber containing ZA and in the ZA-free chamber in the contralateral groin of the same animal. ZA induced a systemic inflammatory response characterized by elevated serum tumour necrosis factor-α levels at early time points. Aminoguanidine (40 μg/ml) inhibited the adipogenic response to ZA-induced inflammation. Adipose tissue formed in response to ZA remained stable for 24 weeks, even when exposed to the normal tissue environment. Conclusions: These results demonstrate that inflammation can drive neo-adipogenesis in vivo. This suggests the existence of a positive feedback mechanism in obesity, whereby the state of chronic, low-grade inflammation, characteristic of the condition, may promote further adipogenesis. The mobilization and recruitment of a circulating population of adipose precursor cells is likely to be implicated in this mechanism.

Spontaneous large volume adipose tissue generation from a vascularized pedicled fat flap inside a chamber space

A novel method of spontaneous generation of new adipose tissue from an existing fat flap is described. A defined volume of fat flap based on the superficial inferior epigastric vascular pedicle in the rat was elevated and inset into a hollow plastic chamber implanted subcutaneously in the groin of the rat. The chamber walls were either perforated or solid and the chambers either contained poly(D,L-lactic-co-glycolic acid) (PLGA) sponge matrix or not. The contents were analyzed after being in situ for 6 weeks. The total volume of the flap tissue in all groups except the control groups, where the flap was not inserted into the chambers, increased significantly, especially in the perforated chambers (0.08 ± 0.007 mL baseline compared to 1.2 ± 0.08 mL in the intact ones). Volume analysis of individual component tissues within the flaps revealed that the adipocyte volume increased and was at a maximum in the chambers without PLGA, where it expanded from 0.04 ± 0.003 mL at insertion to 0.5 ± 0.08 mL (1250% increase) in the perforated chambers and to 0.16 ± 0.03 mL (400% increase) in the intact chambers. Addition of PLGA scaffolds resulted in less fat growth. Histomorphometric analysis rather than simple hypertrophy documented an increased number of adipocytes. The new tissue was highly vascularized and no fat necrosis or atypical changes were observed.

Adipose tissue induction in vivo

Engineering adipogenic tissue in vivo requires the concomitant induction of angiogenesis to generate a stable long-term three-dimensional construct. Histiocon-ductive tissue engineering strategies have been used. The disadvantage of using biodegradable scaffolds is a delayed angiogenic induction resulting in ischemic necrosis of the central cell population in the scaffold. We evaluated an histioinductive approach for adipose tissue engineering by combining essential key components for adipogenic induction: (1) a precursor cell source; (2) a vascular pedicle; (3) a supportive matrix, and; (4) a chamber to preserve space for the new tissue to develop. We observed concomitant adipogenic and angiogenic induction after 6 weeks in three-dimensional adipose tissue constructs.

The effect of the histological properties of tumors on transfection efficiency of electrically assisted gene delivery to solid tumors in mice

Uniform DNA distribution in tumors is a prerequisite step for high transfection efficiency in solid tumors. To improve the transfection efficiency of electrically assisted gene delivery to solid tumors in vivo, we explored how tumor histological properties affected transfection efficiency. In four different tumor types (B16F1, EAT, SA-1 and LPB), proteoglycan and collagen content was morphometrically analyzed, and cell size and cell density were determined in paraffin-embedded tumor sections under a transmission microscope. To demonstrate the influence of the histological properties of solid tumors on electrically assisted gene delivery, the correlation between histological properties and transfection efficiency with regard to the time interval between DNA injection and electroporation was determined. Our data demonstrate that soft tumors with larger spherical cells, low proteoglycan and collagen content, and low cell density are more effectively transfected (B16F1 and EAT) than rigid tumors with high proteoglycan and collagen content, small spindle-shaped cells and high cell density (LPB and SA-1). Furthermore, an optimal time interval for increased transfection exists only in soft tumors, this being in the range of 5-15 min. Therefore, knowledge about the histology of tumors is important in planning electrogene therapy with respect to the time interval between DNA injection and electroporation.

Gene electrotransfer into murine skeletal muscle: A systematic analysis of parameters for long-term gene expression

Skeletal muscle is an attractive target tissue for delivery of therapeutic genes, since it is well vascularized, easily accessible, and has a high capacity for protein synthesis. For efficient transfection in skeletal muscle, several protocols have been described, including delivery of low voltage electric pulses and a combination of high and low voltage electric pulses. The aim of this study was to determine the influence of different parameters of electrotransfection on short-term and long-term transfection efficiency in murine skeletal muscle, and to evaluate histological changes in the treated tissue. Different parameters of electric pulses, different time lags between plasmid DNA injection and application of electric pulses, and different doses of plasmid DNA were tested for electrotransfection of tibialis cranialis muscle of C57BI/6 mice using DNA plasmid encoding green fluorescent protein (GFP). Transfection efficiency was assessed on frozen tissue sections one week after electrotransfection using a fluorescence microscope and also noninvasively, followed by an in vivo imaging system using a fluorescence stereo microscope over a period of several months. Histological changes in muscle were evaluated immediately or several months after electrotransfection by determining infiltration of inflammatory mononuclear cells and presence of necrotic muscle fibers. The most efficient electrotransfection into skeletal muscle of C57BI/6 mice in our experiments was achieved when one high voltage (HV) and four low voltage (LV) electric pulses were applied 5 seconds after the injection of 30 μg of plasmid DNA. This protocol resulted in the highest short-term as well as long-term transfection. The fluorescence intensity of the transfected area declined after 2-3 weeks, but GFP fluorescence was still detectable 18 months after electrotransfection. Extensive inflammatory mononuclear cell infiltration was observed immediately after the electrotransfection procedure using the described parameters, but no necrosis or late tissue damage was observed. This study showed that electric pulse parameters, time lag between the injection of DNA and application of electric pulses, and dose of plasmid DNA affected the duration of transgene expression in murine skeletal muscle. Therefore, transgene expression in muscle can be controlled by appropriate selection of electrotransfection protocol.

Atmospheric gas plasma–induced ROS production activates TNF-ASK1 pathway for the induction of melanoma cancer cell apoptosis

Atmospheric gas plasmas (AGPs) are able to selectively induce apoptosis in cancer cells, offering a promising alternative to conventional therapies that have unwanted side effects such as drug resistance and toxicity. However, the mechanism of AGP-induced cancer cell death is unknown. In this study, AGP is shown to up-regulate intracellular reactive oxygen species (ROS) levels and induce apoptosis in melanoma but not normal melanocyte cells. By screening genes involved in apoptosis, we identify tumor necrosis factor (TNF)-family members as the most differentially expressed cellular genes upon AGP treatment of melanoma cells. TNF receptor 1 (TNFR1) antagonist-neutralizing antibody specifically inhibits AGP-induced apoptosis signal, regulating apoptosis signal-regulating kinase 1 (ASK1) activity and subsequent ASK1-dependent apoptosis. Treatment of cells with intracellular ROS scavenger N-acetyl-l-cysteine also inhibits AGP-induced activation of ASK1, as well as apoptosis. Moreover, depletion of intracellular ASK1 reduces the level of AGP-induced oxidative stress and apoptosis. The evidence for TNF-signaling dependence of ASK1-mediated apoptosis suggests possible mechanisms for AGP activation and regulation of apoptosis-signaling pathways in tumor cells.

A linear-elastic model of anisotropic tumour growth

This paper examines the effect of anisotropic growth on the evolution of mechanical stresses in a linear-elastic model of a growing, avascular tumour. This represents an important improvement on previous linear-elastic models of tissue growth since it has been shown recently that spatially-varying isotropic growth of linear-elastic tissues does not afford the necessary stress-relaxation for a steady-state stress distribution upon reaching a nutrient-regulated equilibrium size. Time-dependent numerical solutions are developed using a Lax-Wendroff scheme, which show the evolution of the tissue stress distributions over a period of growth until a steady-state is reached. These results are compared with the steady-state solutions predicted by the model equations, and key parameters influencing these steady-state distributions are identified. Recommendations for further extensions and applications of this model are proposed.

Hyperinsulinemia down-regulates TLR4 expression in the mammalian heart

Toll-like receptors (TLR) are key regulators of innate immune and inflammatory responses and their activation is linked to impaired glucose metabolism during metabolic disease. Determination of whether TLR4 signaling can be activated in the heart by insulin may shed light on the pathogenesis of diabetic cardiomyopathy, a process that is often complicated by obesity and insulin resistance. The aim of the current study was to determine if supraphysiological insulin concentrations alter the expression of TLR4, markers of TLR4 signaling and glucose transporters (GLUTs) in the heart. Firstly, the effect of insulin on TLR4 protein expression was investigated in vitro in isolated rat cardiac myocytes. Secondly, protein expression of TLR4, the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) suppressor of cytokine signaling 3 (SOCS3) and GLUTs (1, 4, 8, 12) were examined in the equine ventricular myocardium following a prolonged, euglycemic, hyperinsulinemic clamp. Down-regulation of TLR4 protein content in rat cardiac myocytes was observed after incubation with a supraphysiologic concentration of insulin as well as in the equine myocardium after prolonged insulin infusion. Further, cardiac TLR4 expression was negatively correlated with serum insulin concentration. Markers of cardiac TLR4 signaling and GLUT expression were not affected by hyperinsulinemia and concomitant TLR4 down-regulation. Since TLRs are major determinants of the inflammatory response, our findings suggest that insulin infusion exerts an anti-inflammatory effect in the hearts of non-obese individuals. Understanding the regulation of cardiac TLR4 signaling during metabolic dysfunction will facilitate improved management of cardiac sequela to metabolic syndrome and diabetes.

MAS NMR measurements of intact articular bovine cartilage

Articular cartilage (AC), an avascular connective tissue lining articulating surfaces of the long bones, comprises extracellular biopolymers. In functionally compromised states such as osteoarthritis, thinned or lost AC causes reduced mobility and increased health-care costs. Understanding of the characteristics responsible for the load bearing efficiency of AC and the factors leading to its degradation are incomplete. DTI shows the structural alignment of collagen in AC [1] and T2 relaxation measurements suggest that the average director of reorientational motion of water molecules depends on the degree of alignment of collagen in AC [2]. Information on the nature of the chemical interactions involved in functional AC is lacking. The need for AC structural integrity makes solid state NMR an ideal tool to study this tissue. We examined the contribution of water in different functional ‘compartments’ using 1H-MAS, 13C-MAS and 13C-CPMAS NMR of bovine patellar cartilage incubated in D2O. 1H-MAS spectra signal intensity was reduced due to H/D exchange without a measureable redistribution of relative signal intensity. Chemical shift anisotropy was estimated by lineshape analysis of multiple peaks in the 1H-MAS spinning sidebands. These asymmetrical sidebands suggested the presence of multiple water species in AC. Therefore, water was added in small aliquots to D2O saturated AC and the influence of H2O and D2O on organic components was studied with 13C-MAS-NMR and 13C-CPMAS-NMR. Signal intensity in 13C-MAS spectra showed no change in relative signal intensity throughout the spectrum. In 13C-CPMAS spectra, displacement of water by D2O resulted in a loss of signal in the aliphatic region due to a reduction in proton availability for cross-polarization. These results complement dehydration studies of cartilage using osmotic manipulation [3] and demonstrate components of cartilage that are in contact with mobile water.

Solid-state MAS NMR measurements of intact articular bovine cartilage

Articular cartilage (AC), an avascular connective tissue lining articulating surfaces of the long bones, comprises extracellular biopolymers. In functionally compromised states such as osteoarthritis, thinned or lost AC causes reduced mobility and increased health-care costs. Understanding of the characteristics responsible for the load bearing efficiency of AC and the factors leading to its degradation are incomplete. DTI shows the structural alignment of collagen in AC [1] and T2 relaxation measurements suggest that the average director of reorientational motion of water molecules depends on the degree of alignment of collagen in AC [2]. Information on the nature of the chemical interactions involved in functional AC is lacking. The need for AC structural integrity makes solid state NMR an ideal tool to study this tissue. We examined the contribution of water in different functional ‘compartments’ using 1H-MAS, 13C-MAS and 13C-CPMAS NMR of bovine patellar cartilage incubated in D2O. 1H-MAS spectra signal intensity was reduced due to H/D exchange without a measureable redistribution of relative signal intensity. Chemical shift anisotropy was estimated by lineshape analysis of multiple peaks in the 1H-MAS spinning sidebands. These asymmetrical sidebands suggested the presence of multiple water species in AC. Therefore, water was added in small aliquots to D2O saturated AC and the influence of H2O and D2O on organic components was studied with 13C-MAS-NMR and 13C-CPMAS-NMR. Signal intensity in 13C-MAS spectra showed no change in relative signal intensity throughout the spectrum. In 13C-CPMAS spectra, displacement of water by D2O resulted in a loss of signal in the aliphatic region due to a reduction in proton availability for cross-polarization. These results complement dehydration studies of cartilage using osmotic manipulation [3] and demonstrate components of cartilage that are in contact with mobile water.

Human amnion epithelial cell transplantation abrogates lung fibrosis and augments repair

Rationale: Chronic lung disease characterized by loss of lung tissue,inflammation, and fibrosis represents a major global health burden. Cellular therapies that could restore pneumocytes and reduce inflammation and fibrosis would be a major advance in management. Objectives: To determine whether human amnion epithelial cells (hAECs), isolated from term placenta and having stem cell–like and antiinflammatory properties, could adopt an alveolar epithelial phenotype and repair a murine model of bleomycin-induced lung injury. Methods: Primary hAECs were cultured in small airway growth medium to determine whether the cells could adopt an alveolar epithelial phenotype. Undifferentiated primary hAECs were also injected parenterally into SCID mice after bleomycin-induced lung injury and analyzed for production of surfactant protein (SP)-A, SP-B, SP-C, and SP-D. Mouse lungs were also analyzed for inflammation and collagen deposition. Measurements and Main Results: hAECs grown in small airway growth medium developed an alveolar epithelial phenotype with lamellar body formation, production of SPs A–D, and SP-D secretion. Although hAECs injected into mice lacked SPs, hAECs recovered from mouse lungs 2 weeks posttransplantation produced SPs. hAECs remained engrafted over the 4-week test period. hAEC administration reduced inflammation in association with decreased monocyte chemoattractant protein-1, tumor necrosis factor-a, IL-1 and -6, and profibrotic transforming growth factor-b in mouse lungs. In addition,lung collagen content was significantly reduced by hAEC treatment as a possible consequence of increased degradation by matrix metalloproteinase-2 and down-regulation of the tissue inhibitors f matrix metalloproteinase-1 and 2. Conclusions: hAECs offer promise as a cellular therapy for alveolar restitution and to reduce lung inflammation and fibrosis.

Weight-related differences in glucose metabolism and free fatty acid production in two South African population groups

OBJECTIVE The effects of free fatty acids (FFA), leptin, tumour necrosis factor (TNF) alpha and body fat distribution on in vivo oxidation of a glucose load were studied in two South African ethnic groups. DESIGN AND MEASUREMENTS Anthropometric and various metabolic indices were measured at fasting and during a 7h oral glucose tolerance test (OGTT). Body composition was measured using bioelectrical impedance analysis and subcutaneous and visceral fat mass was assessed using a five- and two-level CT-scan respectively. Glucose oxidation was evaluated by measuring the ratio of (13)CO(2) to (12)CO(2) in breath following ingestion of 1-(13)C-labelled glucose. SUBJECTS Ten lean black women (LBW), ten obese black women (OBW), nine lean white women (LWW) and nine obese white women (OWW) were investigated after an overnight fast. RESULTS Visceral fat levels were significantly higher (P < 0.01) in obese white than black women, despite similar body mass indexes (BMIs). There were no ethnic differences in glucose oxidation however; in the lean subjects of both ethnic groups the area under the curve (AUC) was higher than in obese subjects (P < 0.05 for both) and was found to correlate negatively with weight (r = -0.69, P < 0.01) after correcting for age. Basal TNF alpha concentrations were similar in all groups. Percentage suppression of FFAs at 30 min of the OCTT was 24 +/- 12% in OWW and - 38 +/- 23% (P < 0.05) in OBW, ie the 30 min FFA level was higher than the fasting level in the latter group. AUC for FFAs during the late postprandial period (120 - 420 min) was significantly higher in OWW than OBW (P < 0.01) and LWW (P < 0.01) and correlated positively with visceral fat mass independent of age (r = 0.78, P < 0.05) in the OWW only. Leptin levels were higher (P < 0.01) both at fasting and during the course of the OCTT in obese women from both ethnic groups compared to the lean women. CONCLUSIONS Glucose oxidation is reduced in obese subjects of both ethnic groups; inter- and intra-ethnic differences were observed in visceral fat mass and FFA production and it is possible that such differences may play a role in the differing prevalences of obesity-related disorders that have been reported in these two populations.