TLR4-dependant pro-inflammatory effects of HMGB1 on human adipocyte.

Chronic low grade inflammation is one of the major metabolic disorders in case of obesity and associated pathologies. By its important secretion function, the role of adipose tissue in this metabolic low grade inflammation is well known. Recently, it was demonstrated that the alarmin high mobility group box protein 1 (HMGB1) is involved in obesity-related pathologies by its increased serum levels in obese compared to normal weight individuals, and by its pro-inflammatory effects. However, the role of HMGB1 on adipocytes inflammation is poorly documented and we propose to investigate this point. Primary culture of human subcutaneous adipocytes were performed from human adipose tissue samples. Cells were treated with recombinant HMGB1 with/without anti-TLR4 antibody and inhibitors of NF-κB and P38 MAPK. Supernatants were collected for IL-6 and MCP-1 ELISA. HMGB1 initiates Toll-like receptor 4 (TLR4)-dependent activation of inflammation through the downstream NF-κB and P38 MAPK signaling pathway to upregulate the secretion of the pro-inflammatory cytokine IL-6. HMGB1 has pro-inflammatory effects on adipocytes. This reinforces the role of TLR4 in adipose tissue inflammation and antagonizing the HMGB1 inflammatory pathway could bring on new therapeutic targets to counteract obesity-associated pathologies.

Effect of Washes and Centrifugation on the Efficacy of Lipofilling With or Without Local Anesthetic.

BACKGROUND: Among the different parameters that influence fat graft survival and lipofilling success, the use of local anesthetic and the way to process the fat before injection have often been pointed out. Likewise, we evaluated different techniques for processing adipose tissue before its injection and analyzed the quality of the grafts. METHODS: Adipose tissue from the same patient was gently harvested from one side of the abdomen after infiltration of a tumescent solution without lidocaine and from the other side of the abdomen using a tumescent solution containing lidocaine 2%. Harvested tissue was prepared with different protocols, from simple decantation to advanced protocols including single or multiple washes and centrifugations. Each type of processed adipose tissue was then injected subcutaneously into immunodeficient mice. Adipose grafts were collected after 1 month and analyzed by histology with a detailed scoring method. RESULTS: After lidocaine use, decantation protocol led to adipose grafts of poor quality with high resorption rate and oil vacuole formation. Larger grafts were obtained after centrifugation, but centrifugation alone resulted in increased fibrosis and necrosis, with or without the use of lidocaine. Finally, multiple washes and centrifugations greatly improved the quality of the lipografts. CONCLUSIONS: Centrifugation alone is not sufficient and must be associated with multiple washes to improve graft quality. This article aims to provide further evidence of lidocaine and washing/centrifugation effects in fat grafting to provide easy tips aimed at ensuring graft efficiency with a long-term clinical outcome.

Autologous Fat Grafting in the Breast: Critical Points and Technique Improvements.

BACKGROUND: Breast augmentation or reconstruction is a major challenge in esthetic and reconstructive surgery. While autologous fat grafting (AFG) provides a natural filler and seems easy to harvest, AFG in breast surgery is still problematic especially due to the high resorption rate associated with megavolume transfer. Despite this pending issue, there is growing interest in this method, which is becoming more and more widespread, as can be seen by the recent increase in the number of clinical studies. This review aims to highlight recent knowledge in the technique of AFG to the breast and recent refined procedures to improve fat viability and long-term success of the graft. METHODS: Clinical publications and trials of AFG to the breast from the past 5 years were examined. Attention was focused on the different AFG steps and the clinical outcomes, in order to highlight the strengths and weaknesses of the available protocols. RESULTS: Recent studies have concentrated on new techniques to improve fat viability and graft intake. However, all of these studies use different protocols at each step of the procedure. Furthermore, results may vary depending on the technique used for fat harvesting and processing. CONCLUSION: This review points out the recent advances in breast AFG techniques and their associated outcomes and complications. The bibliography has been carefully examined to reach a consensus so that recommendations could be made for each step of the technique with the aim of improving graft viability and long-term volume maintenance.

Inflammation triggers high mobility group box 1 (HMGB1) secretion in adipose tissue, a potential link to obesity.

BACKGROUND: Low grade inflammation is one of the major metabolic disorders in case of obesity due to variable secretion of adipose derived cytokines called adipokines. Recently the nuclear protein HMGB1 was identified as an inflammatory alarmin in obesity associated diseases. However HMGB1 role in adipose tissue inflammation is not yet studied. OBJECTIVES: The aim of this study was to prove the expression of HMGB1 in human adipose tissue and to assess the levels of expression between normo-weight and obese individuals. Furthermore we determined which type of cells within adipose tissue is involved in HMGB1 production under inflammatory signal. METHODS: Western-blot was performed on protein lysates from human normo-weight and obese adipose tissue to study the differential HMGB1 expression. Human normo-weight adipose tissue, adipose-derived stromal cells (ASCs) and adipocytes were cultured and stimulated with LPS to induce inflammation. HMGB1, IL-6 and MCP-1 secretion and gene expression were quantified by ELISA and Q-PCR respectively, as well as cell death by LDH assay. HMGB1 translocation during inflammation was tracked down by immunofluorescence in ASCs. RESULTS: HMGB1 was expressed 2-fold more in adipose tissue from obese compared to normo-weight individuals. LPS led to an up-regulation in HMGB1 secretion and gene expression in ASCs, while no change was noticed in adipocytes. Moreover, this HMGB1 release was not attributable to any cell death. In LPS-stimulated ASCs, HMGB1 translocation from nucleus to cytoplasm was detectable at 12h and the nuclear HMGB1 was completely drained out after 24h of treatment. CONCLUSION: The expression level studies between adipose tissue from normo-weight and obese individuals together with in vitro results strongly suggest that adipose tissue secretes HMGB1 in response to inflammatory signals which characterized obesity.

Effect of centrifugation and washing on adipose graft viability: a new method to improve graft efficiency.

BACKGROUND: Adipose tissue grafting is a promising method in the field of surgical filling. We studied the effect of centrifugation on fat grafts, and we propose an optimised protocol for the improvement of adipose tissue viability. METHODS: Adipose tissue was subjected to different centrifugations, and the volumes of interstitial liquid and oil released were measured to choose the optimal condition. Tissue from this condition was then compared to tissue obtained from two traditional techniques: strong centrifugation (commonly 3 min at 3000 rpm/900 g), and decantation, by injecting into immunodeficient mice. The cytokine interleukin-6 (IL-6) and chemokine monocyte chemotactic protein-1 (MCP-1) were assayed 24 h post-injection, and after 1 month of grafting the state of the lipografts was evaluated through macroscopic and histological analysis, with oil gap area measurement. RESULTS: Strong centrifugation (900 g, 1800 g) is deleterious for adipose tissue because it leads to until threefold more adipocyte death compared to low centrifugation (100 g, 400 g). In addition, mice injected with strong centrifuged and non-centrifuged adipose tissue have higher rates of blood IL-6 and MCP-1, compared to those grafted with soft centrifuged fat. Moreover, extensive lipid vacuoles were detectable on histological sections of the non-centrifuged lipografts, whereas lipografts from soft centrifugation contain a higher amount of connective tissue containing collagen fibres. CONCLUSION: It is necessary to wash and centrifuge adipose tissue before reinjection in order to remove infiltration liquid and associated toxic molecules, which in the long term are deleterious for the graft. However, strong centrifugation is not recommended since it leads very quickly to greater adipocyte death. Thus, soft centrifugation (400 g/1 min), preceded by washings, seems to be the most appropriate protocol for the reinjection of adipose tissue.

New insights into lidocaine and adrenaline effects on human adipose stem cells.

BACKGROUND: Adipose stem cells have gained great interest in plastic and reconstructive surgery with their ability to improve engraftment after fat transfer for soft tissue filling. It is therefore essential to know the effect of the drugs commonly used during the lipoaspiration procedure, such as lidocaine and adrenaline. Indeed, these drugs are infiltrated at the fat donor site for local anesthesia and for reduction of bleeding. This study analyzed the effects of these drugs on the viability of adipose-derived stem cells and on their inflammatory status. METHODS: Adipose-derived stem cells from lipoaspirates were grown in culture before being treated with different clinical doses of lidocaine at different times of exposure (1-24 h), and with adrenaline (1 µg/mL). Cytotoxicity was measured by lactate dehydrogenase assay and by flow cytometry with annexin V/propidium iodide staining. In parallel, the secretion of the proinflammatory cytokines tumor necrosis factor-alpha (TNFα), interleukin-6 (IL-6), and monocyte chemotactic protein-1 (MCP-1) was tested by enzyme-linked immunoassay. RESULTS: Lidocaine affected cell viability after 24 h, even when the cells were exposed for only 1 or 2 h. Apoptosis was not involved in lidocaine cytotoxicity. Regarding inflammation, no TNFα was produced, and lidocaine decreased the levels of IL-6 and MCP-1 in a dose-dependent manner. In contrast, adrenaline did not influence cell viability or cytokine secretions. CONCLUSIONS: Adipose tissue should be handled appropriately to remove lidocaine and adrenaline, with such procedures as washing and centrifugation. This study provides new insights into the use of lidocaine and adrenaline for fat transfer or stem cell isolation from lipoaspirates. LEVEL OF EVIDENCE II: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .