Oxidative stress mediates depot-specific functional differences of human adipose-derived stem cells.

BACKGROUND: Visceral (VS) fat depot is known to have defective adipogenic functions compared to subcutaneous (SC) fat, but its mechanism of origin is unclear. OBJECTIVE: We tested our hypothesis that the degree of oxidative stress in adipose-derived stem cells (ASCs) from these depots may account for this difference. METHODS: ASCs were isolated from VS (omental region) and SC (abdominal region) fat depots of human subjects undergoing bariatric surgery. ASCs from VS and SC fat were investigated for their cellular characteristics in reactive oxygen species (ROS), metabolism, gene expression, proliferation, senescence, migration, and adipocyte differentiation. ASCs were also treated with antioxidant ascorbic acid (vitamin C). RESULTS: We found that human VS-derived ASCs exhibit excessive oxidative stress characterized by high reactive oxygen species (ROS), compared to SC-derived ASCs. Gene expression analyses indicate that the VS-ASCs exhibit higher levels of genes involved in pro-oxidant and pro-inflammatory pathways and lower levels of genes in antioxidant and anti-inflammatory pathways. VS-ASCs have impaired cellular functions compared to SC-ASCs, such as slower proliferation, early senescence, less migratory activity, and poor adipogenic capability in vitro. Treatment with ascorbic acid decreased ROS levels drastically in VS-ASCs. Ascorbic acid treatment substantially improved proliferation, senescence, migration, and adipogenic capacities of compromised ASCs caused by high ROS. CONCLUSIONS: This finding suggests the fat depot-specific differences of cellular defects originating from stem cell population. Considering clinical potentials of human ASCs for cell therapies, this also offers a possible strategy for improving their therapeutic qualities through antioxidants.

Adipose tissue macrophages do not affect atherosclerosis development in mice.

BACKGROUND AND AIMS: Obese individuals have a higher risk of developing atherosclerosis, possibly driven by adipose tissue (AT) inflammation. We recently showed that AT macrophages (ATMs), which accumulate in the expanding obese AT, produce mediators causing immune cell recruitment from the bone marrow. In the current study, we evaluated whether ATMs are directly involved in atherosclerotic plaque development. METHODS: Lean ldlr-/- acceptor mice received visceral AT (vAT) from lean, obese, or ATM-depleted obese ldlr-/- mice. Acceptor mice were fed high cholesterol diet (HCD) for 4 weeks before and 8 weeks after AT transplantation to induce atherosclerosis. Atherosclerotic plaque development was studied 8 weeks after transplantation. RESULTS: Transplanting donor vAT from obese mice increased circulating triglycerides and B-cells, but decreased Ly6c- monocytes. Plasma cholesterol, Ly6c+ monocytes, T-cells, NK-cells and eosinophils were unaffected. Depleting ATMs from obese AT using clodronate liposomes prior to vAT transplantation prevented the increase in triglycerides and B-cells and decrease in Ly6c- monocytes, but did increase eosinophils. Circulating Cxcl1 was reduced by obese AT transplantation and Ifn-γ tended to be increased while Tnf and Il-1ß were unaffected. ATM-depleted obese AT transplantation also reduced Cxcl1, but increased circulating Tnf levels. However, obese AT transplantation with or without depletion of ATMs did not influence atherosclerotic plaque size, phenotype, or stability. CONCLUSIONS: ATMs from obese vAT do not affect atherosclerotic plaque development or phenotype.

Adipose tissue-derived stromal cells are sources of cancer-associated fibroblasts and enhance tumor progression by dense collagen matrix.

Although recent studies revealed that adipose tissue accelerates pancreatic tumor progression with excessive extracellular matrix, key players for desmoplasia in the adipose microenvironment remains unknown. Here, we investigated the roles of adipose tissue-derived stromal cells (ASCs) in desmoplastic lesions and tumor progression by in vitro and in vivo experiments. In a three-dimensional (3-D) organotypic fat invasion model using visceral fat from CAG-EGFP mice, GFP-positive fibroblastic cells infiltrated toward cancer cells. When tumor cells were inoculated into transplanted visceral fat pads in vivo, tumor weights and stromal components were enhanced compared to subcutaneous and orthotopic tumor cells inoculated without fat pads. Expression of αSMA in established human ASCs was lower compared to cancer associated fibroblasts, and the 3-D collagen matrices produced by ASCs cultured in cancer cell-conditioned medium changed from loose to dense structures that affected the motility of cancer cells. Microarray analyses revealed upregulation of S100A4 in ASCs, while S100A4-positive stromal cells were observed at extrapancreatic invasion sites of human pancreatic cancer. The present findings indicate that ASCs are recruited to extrapancreatic invasion sites and produce dense collagen matrices that lead to enhanced tumor progression. Both inhibition of ASCs recruitment and activation could lead to a novel antistromal therapy.

Adipose-Derived Exosomes Exert Proatherogenic Effects by Regulating Macrophage Foam Cell Formation and Polarization.

BACKGROUND: Obesity is causally associated with atherosclerosis, and adipose tissue (AT)-derived exosomes may be implicated in the metabolic complications of obesity. However, the precise role of AT-exosomes in atherogenesis remains unclear. We herein aimed to assess the effect of AT-exosomes on macrophage foam cell formation and polarization and subsequent atherosclerosis development. METHODS AND RESULTS: Four types of exosomes isolated from the supernatants of ex vivo subcutaneous AT and visceral AT (VAT) explants that were derived from wild-type mice and high-fat diet (HFD)-induced obese mice were effectively taken up by RAW264.7 macrophages. Both treatment with wild-type VAT exosomes and HFD-VAT exosomes, but not subcutaneous AT exosomes, markedly facilitated macrophage foam cell generation through the downregulation of ATP-binding cassette transporter (ABCA1 and ABCG1)-mediated cholesterol efflux. Decreased expression of liver X receptor-α was also observed. Among the 4 types of exosomes, only HFD-VAT exosomes significantly induced M1 phenotype transition and proinflammatory cytokine (tumor necrosis factor α and interleukin 6) secretion in RAW264.7 macrophages, which was accompanied by increased phosphorylation of NF-κB-p65 but not the cellular expression of NF-κB-p65 or IκB-α. Furthermore, systematic intravenous injection of HFD-VAT exosomes profoundly exacerbated atherosclerosis in hyperlipidemic apolipoprotein E-deficient mice, as indicated by the M1 marker (CD16/32 and inducible nitric oxide synthase)-positive areas and the Oil Red O/Sudan IV-stained area, without affecting the plasma lipid profile and body weight. CONCLUSIONS: This study demonstrated a proatherosclerotic role for HFD-VAT exosomes, which is exerted by regulating macrophage foam cell formation and polarization, indicating a novel link between AT and atherosclerosis in the context of obesity.

Assessment of the Cancer Risk of the Fat-Grafted Breast in a Murine Model.

Background: The results of experimental studies indicate that grafting of autologous adipose tissue may induce tumorigenesis at the recipient site, but clinical results do not support a carcinogenic effect of fat grafting to the breast. Objectives: The authors assessed cancer risk following transplantation of autologous fat into murine mammary tissue. Methods: In this animal study, mammary tissues from 54 breasts of 9 female rats were either grafted with autologous subcutaneous fat, grafted with autologous omental fat, or unmanipulated. Tissues were harvested and processed for histologic and immunohistochemical analyses, and the mRNA expression levels of specific genes were determined. Results: No atypia or changes in lobular structures were observed in lipofilled breasts compared with controls. The numbers of ductal cell layers and terminal ductal units were similar for lipofilled and control breasts. Macrophage concentrations also were similar for the 3 groups. The localization and magnitude of plasminogen activator inhibitor 1 were similar for lipofilled and unmanipulated breast tissue. The percentages of cells expressing Ki67 or estrogen receptor (ER) and the ER/Ki67 balance were similar for the 3 groups. Gene expression was not altered in lipofilled breasts, compared with controls. Conclusions: No theoretical risk of cancer was detected in the microenvironment of the lipofilled rat breast.

Peritoneal free autologous fat graft for the control of pulmonary air leaks in emphysematous rat lungs.

BACKGROUND: Persistent pulmonary air leak is the most frequent complication after lung resection, resulting in an increase in postoperative morbidity and mortality. We evaluated the viability, integration, and efficacy of a free peritoneal fat graft as a method for controlling air leak in normal and emphysematous rat lungs. METHODS: Sixty Wistar rats were divided into two groups: elastase-produced lung emphysema (n=30) and control (normal) lungs (n=30). Pulmonary air leak was produced by puncture of the right lower lobe, and aerostasis was attempted by means of intrapulmonary injection of autologous free peritoneal fat graft. Rats in each group (n=6) were randomly allocated to subgroups and were sacrificed at 7, 14, 21, 30, and 60 days. Then, lungs were removed for histology, morphometry, vessel identification and counting, and immunohistochemistry for caspase 3, vascular endothelial growth factor, and factor VIII. RESULTS: Tissue integration of the free fat grafts was found in all animals in both groups. Vessels stained with India ink inside the fat grafts were present at all assessment periods in both groups. Vascular endothelial growth factor expression was significantly higher in all periods in the emphysema group compared with normal lungs (p<0.001). There was a significant increase in caspase 3 expression in the emphysema group at 7, 21, 30, and 60 days (p<0.001). Factor VIII showed a significant increase (p<0.001) at 30 and 60 days in emphysematous lungs. CONCLUSIONS: The use of free peritoneal fat graft was able to control the air leaks in normal and emphysematous rat lungs, with persisting graft viability for as long as 60 days after implantation.

Endoscopic abdominoplasty providing a perforator fat flap for treatment of hemi-facial microsomia.

A patient with a history of an extended unilateral hemifacial cleft desired the restoration of the buccal fat on the hollow cheeked side and also wished to reduce an abdominal bulge at the same time. The amount of tissue volume needed exceeded the possibilities of free autologous fat grafting, lipofilling and allogenic implants. Therefore a free fat flap with microvascular anastomoses harvested as part of a minimal invasive abdominoplastic procedure using an incision well hidden within the bikini zone was the most attractive surgical option. This is the first case reported, where the standard free deep inferior epigastric artery (DIEA) perforator flap was utilised.

Transplantation of non-visceral fat to the visceral cavity improves glucose tolerance in mice: investigation of hepatic lipids and insulin sensitivity.

AIMS/HYPOTHESIS: Intra-abdominal transplantation of non-visceral adipose tissue in rodents, simulating increased abdominal fat in obesity, paradoxically improves glucose tolerance and insulin sensitivity. We hypothesised that this improvement is due to transplant-induced enhanced uptake of fatty acids by adipose tissue, thus reducing fatty acid flux into, and triacylglycerol storage in, the liver. METHODS: In Experiment 1, mice were sham-operated or received heterologous epididymal white adipose tissue (WAT; EWAT) or visceral WAT (VWAT) transplantation to the portal and splanchnic circulation regions in the visceral cavity. In Experiment 2, inguinal WAT (IWAT) or EWAT was removed and subsequently transplanted to the visceral cavity of the same mouse (autotransplant). IWAT and EWAT autotransplants were repeated in Experiment 3 and compared with heterotransplants. RESULTS: Heterotransplantation of VWAT did not alter glucose tolerance, whereas auto- or hetero-transplantation of EWAT or IWAT significantly improved glucose tolerance. Transplantation-induced improvements in glucose tolerance 4 weeks after surgery coincided with decreased liver triacylglycerol, decreased portal plasma lipids and increased hepatic insulin sensitivity. By 8 weeks, these changes were apparent only in mice with autotransplantation. Heterologous EWAT transplantation-induced glucose improvement persisted without altered liver metabolism. CONCLUSIONS/INTERPRETATION: Increases in visceral fat, via transplantation of visceral or non-visceral adipose tissue, is not a major risk factor for glucose intolerance. In fact, there are dynamic metabolic improvements following transplantation that include decreased portal lipids and improved liver metabolism, but these improvements are transient under certain circumstances.

Removal of intra-abdominal visceral adipose tissue improves glucose tolerance in rats: role of hepatic triglyceride storage.

Epidemiological studies have demonstrated a strong link between increased visceral fat and metabolic syndrome. In rodents, removal of intra-abdominal but non-visceral fat improves insulin sensitivity and glucose homeostasis, though previous studies make an imprecise comparison to human physiology because actual visceral fat was not removed. We hypothesize that nutrient release from visceral adipose tissue may have greater consequences on metabolic regulation than nutrient release from non-visceral adipose depots since the latter drains into systemic but not portal circulation. To assess this we surgically decreased visceral white adipose tissue (~0.5 g VWATx) and compared the effects to removal of non-visceral epididymal fat (~4 g; EWATx), combination removal of visceral and non-visceral fat (~4.5 g; EWATx/VWATx) and sham-operated controls, in chow-fed rats. At 8 weeks after surgery, only the groups with visceral fat removed had a significantly improved glucose tolerance, although 8 times more fat was removed in EWATx compared with VWATx. This suggests that mechanisms controlling glucose metabolism are relatively more sensitive to reductions in visceral adipose tissue mass. Groups with visceral fat removed also had significantly decreased hepatic lipoprotein lipase (LPL) and triglyceride content compared with controls, while carnitine palmitoyltransferase (CPT-1A) was decreased in all fat-removal groups. In a preliminary experiment, we assessed the opposite hypothesis; i.e., we transplanted excess visceral fat from a donor rat to the visceral cavity (omentum and mesentery), which drains into the hepatic portal vein, of a recipient rat but observed no major metabolic effect. Overall, our results indicate surgical removal of intra-abdominal fat improves glucose tolerance through mechanism that may be mediated by reductions in liver triglyceride.

Involvement of GDF-9, leptin, and IGF1 receptors associated with adipose tissue transplantation on fertility restoration in obese anovulatory mice.

The aim was to analyze the effect of adipose tissue transplantation on growth differentiation factor-9 (GDF-9), insulin growth factor 1 receptor (IGF1R), and leptin receptor (LEPR) protein expression in ovaries of obese anovulatory mice. Leptin-deficient female (ob/ob) and wild-type mice were divided into untreated ob/ob mice and gonadal white adipose tissue transplanted ob/ob mice, with evaluation after 7, 15, and 45 days and compared to control wild-type mice. The corporal weight and glycemia levels increased in the obese group concomitant with polymicrocyst formation and abundant estrone, mimicking anovulatory disease. In the treated group after 45 days, glycemia, weight, ovarian size, and number of follicles were decreased and corpora lutea were decreased. The analysis of GDF-9 revealed that, whereas control ovaries presented follicular localization, the obese ovary lacked this protein. On the other hand, obese ovaries showed elevated expression of IGF1R that was normalized after the transplantation. Finally, LEPR was reduced in obese ovaries, and adipose tissue transplantation was efficient in returning it to normal levels. In conclusion, the adipose tissue transplantation, especially after 45 days, seems to stimulate ovulation, supported by the fact that several proteins involved in ovulation returned to basal levels.

The portal theory supported by venous drainage-selective fat transplantation.

OBJECTIVE: The "portal hypothesis" proposes that the liver is directly exposed to free fatty acids and cytokines increasingly released from visceral fat tissue into the portal vein of obese subjects, thus rendering visceral fat accumulation particularly hazardous for the development of hepatic insulin resistance and type 2 diabetes. In the present study, we used a fat transplantation paradigm to (artificially) increase intra-abdominal fat mass to test the hypothesis that venous drainage of fat tissue determines its impact on glucose homeostasis. RESEARCH DESIGN AND METHODS: Epididymal fat pads of C57Bl6/J donor mice were transplanted into littermates, either to the parietal peritoneum (caval/systemic venous drainage) or, by using a novel approach, to the mesenterium, which confers portal venous drainage. RESULTS: Only mice receiving the portal drained fat transplant developed impaired glucose tolerance and hepatic insulin resistance. mRNA expression of proinflammatory cytokines was increased in both portally and systemically transplanted fat pads. However, portal vein (but not systemic) plasma levels of interleukin (IL)-6 were elevated only in mice receiving a portal fat transplant. Intriguingly, mice receiving portal drained transplants from IL-6 knockout mice showed normal glucose tolerance. CONCLUSIONS: These results demonstrate that the metabolic fate of intra-abdominal fat tissue transplantation is determined by the delivery of inflammatory cytokines to the liver specifically via the portal system, providing direct evidence in support of the portal hypothesis.

Transplantation or removal of intra-abdominal adipose tissue prevents age-induced glucose insensitivity.

Increases in intra-abdominal fat, a common feature associated with aging, is an established risk factor for insulin resistance, diabetes and the metabolic syndrome. To examine the direct contribution of intra-abdominal fat in the pathophysiology of insulin resistance we altered fat volume via removal or transplantation in a naturally occurring age-induced moderate model of obesity and insulin resistance. This was accomplished by bilateral removal of epididymal white adipose tissue (Lipx) or transplantation of donor fat into the intra-abdominal side of the peritoneal cavity of 28-week old rats. Control animals received sham surgery. Glucose tolerance was evaluated at baseline and 4 and 8weeks post-surgery in all groups, and fasting insulin and leptin were additionally measured in 28-week old rats. In addition, fasted and fed triglyceride, cholesterol and fatty acid concentrations were measured. Before surgery 28-week old rats weighed more and were glucose intolerant compared with 8-week old controls. Both Lipx and transplantation significantly prevented age-induced decreases in glucose tolerance, with Lipx causing improvement at 4weeks which declined by 8weeks; and with a significant transplantation improvement at 8weeks only. Lipx significantly increased insulin secretion 15min after a bolus injection of 0.75mg/kg dextrose at 4 and 8weeks compared with controls, while transplantation caused a significant ( approximately 220%) increase in fasted leptin level at 4weeks only. Taken together, these data suggest that surgical removal or addition of intra-abdominal fat prevents age-induced insulin resistance by different mechanisms and is a suitable model to investigate naturally occurring obesity.

Monocyte chemoattractant protein-1 deficiency protects against visceral fat-induced atherosclerosis.

OBJECTIVE: To determine the role of monocyte chemoattractant protein-1 (Mcp-1) on the progression of visceral fat-induced atherosclerosis. METHODS AND RESULTS: Visceral fat inflammation was induced by transplantation of perigonadal fat. To determine whether recipient Mcp-1 status affected atherosclerosis induced by inflammatory fat, apolipoprotein E-deficient (ApoE(-/-)) and ApoE(-/-) and Mcp-1-deficient (Mcp-1(-/-)) mice underwent visceral fat transplantation. Intravital microscopy was used to study leukocyte-endothelial interactions. To study the primary tissue source of circulating Mcp-1, both fat and bone marrow transplantation experiments were used. Transplantation of visceral fat increased atherosclerosis in ApoE(-/-) mice but had no effect on atherosclerosis in ApoE(-/-),Mcp-1(-/-) mice. Intravital microscopy revealed increased leukocyte attachment to the endothelium in ApoE(-/-) mice compared with ApoE(-/-),Mcp-1(-/-) mice after receiving visceral fat transplants. Transplantation of visceral fat increased plasma Mcp-1, although donor adipocytes were not the source of circulating Mcp-1 because no Mcp-1 was detected in plasma from ApoE(-/-),Mcp-1(-/-) mice transplanted with Wt fat, indicating that recipient Mcp-1-producing cells were affecting the atherogenic response to the fat transplantation. Consistently, transplantation of Mcp-1(-/-) fat to ApoE(-/-) mice did not lead to atheroprotection in recipient mice. Bone marrow transplantation between Wt and Mcp-1(-/-) mice indicated that the primary tissue source of circulating Mcp-1 was the endothelium. CONCLUSIONS: Recipient Mcp-1 deficiency protects against atherosclerosis induced by transplanted visceral adipose tissue.

Visceral adipose tissue inflammation accelerates atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Fat inflammation may play an important role in comorbidities associated with obesity such as atherosclerosis. METHODS AND RESULTS: To first establish feasibility of fat transplantation, epididymal fat pads were harvested from wild-type C57BL/6J mice and transplanted into leptin-deficient (Lep(ob/ob)) mice. Fat transplantation produced physiological leptin levels and prevented obesity and infertility in Lep(ob/ob) mice. However, the transplanted fat depots were associated with chronically increased macrophage infiltration with characteristics identical to those observed in fat harvested from obese animals. The inflammation in transplanted adipose depots was regulated by the same factors that have been implicated in endogenous fat inflammation such as monocyte chemoattractant protein-1. To determine whether this inflamed adipose depot could affect vascular disease in mice, epididymal fat depots were transplanted into atherosclerosis-prone apolipoprotein E-deficient ApoE(-/-) mice. Plasma from ApoE(-/-) mice receiving fat transplants contained increased leptin, resistin, and monocyte chemoattractant protein-1 compared with plasma from sham-operated ApoE(-/-) mice. Furthermore, mice transplanted with visceral fat developed significantly more atherosclerosis compared with sham-operated animals, whereas transplants with subcutaneous fat did not affect atherosclerosis despite a similar degree of fat inflammation. Treatment of transplanted ApoE(-/-) mice with pioglitazone decreased macrophage content of the transplanted visceral fat pad and reduced plasma monocyte chemoattractant protein-1. Importantly, pioglitazone also reduced atherosclerosis triggered by inflammatory visceral fat but had no protective effect on atherosclerosis in the absence of the visceral fat transplantation. CONCLUSIONS: Our results indicate that visceral adipose-related inflammation accelerates atherosclerosis in mice. Drugs such as thiazolidinediones might be a useful strategy to specifically attenuate the vascular disease induced by visceral inflammatory fat.