Background and Objective Severe thermal injury is associated with extreme and prolonged inflammatory and hypermetabolic responses, resulting in significant catabolism that delays recovery or even leads to multiple organ failure and death. Burned patients exhibit many symptoms of stress-induced diabetes, including hyperglycemia, hyperinsulinemia, and hyperlipidemia. Recently, the NLRP3 inflammasome has received much attention as the sensor of endogenous “danger signals” and mediator of “sterile inflammation” in type II diabetes. Therefore, we investigated whether the NLRP3 inflammasome is activated in the adipose tissue of burned patients, as we hypothesize that, similar to the scenario observed in chronic diabetes, the cytokines produced by the inflammasome mediate insulin resistance and metabolic dysfunction. Subjects We enrolled 76 patients with burn sizes ranging from 1% to 70% total body surface area (TBSA). Severely burned patients all exhibited burn-induced insulin resistance and hyperglycemia. Measurements and Main Results We examined the adipose tissue of control and burned patients and found, via flow cytometry and gene expression studies, increased infiltration of leukocytes - especially macrophages - and evidence of inflammasome priming and activation. Furthermore, we observed increased levels of IL-1β in the plasma of burned patients when compared to controls. Conclusions In summary, our study is the first to show activation of the inflammasome in burned humans, and our results provide impetus for further investigation of the role of the inflammasome in burn-induced hypermetabolism and, potentially, developing novel therapies targeting this protein complex for the treatment of stress-induced diabetes.
Considerable effort has been directed toward deriving endothelial cells (ECs) from adipose‐derived mesenchymal stem cells (ASCs) since 2004, when it was first suggested that ECs and adipocytes share a common progenitor. While the capacity of ASCs to express endothelial markers has been repeatedly demonstrated, none constitute conclusive evidence of an endothelial phenotype as all reported markers have been detected in other, non‐endothelial cell types. In this study, quantitative phenotypic comparisons to representative EC controls were used to determine the extent of endothelial differentiation being achieved with ASCs. ASCs were harvested from human subcutaneous abdominal white adipose tissue, and their endothelial differentiation was induced using well‐established biochemical stimuli. Reverse transcription quantitative real‐time polymerase chain reaction and parallel reaction monitoring mass spectrometry were used to quantify their expression of endothelial genes and corresponding proteins, respectively. Flow cytometry was used to quantitatively assess their uptake of acetylated low‐density lipoprotein (AcLDL). Human umbilical vein, coronary artery, and dermal microvascular ECs were used as positive controls to reflect the phenotypic heterogeneity between ECs derived from different vascular beds. Biochemically conditioned ASCs were found to upregulate their expression of endothelial genes and proteins, as well as AcLDL uptake, but their abundance remained orders of magnitude lower than that observed in the EC controls despite their global proteomic heterogeneity. The findings of this investigation demonstrate the strikingly limited extent of endothelial differentiation being achieved with ASCs using well‐established biochemical stimuli, and underscore the importance of quantitative phenotypic comparisons to representative primary cell controls in studies of differentiation. Stem Cells Translational Medicine 2019;8:35–45
Endothelial cells are among the fundamental building blocks for vascular tissue engineering. However, a clinically viable source of endothelium has continued to elude the field. Here, we demonstrate the feasibility of sourcing autologous endothelium from human fat – an abundant and uniquely dispensable tissue that can be readily harvested with minimally invasive procedures. We investigate the challenges underlying the overgrowth of human adipose tissue-derived microvascular endothelial cells by stromal cells to facilitate the development of a reliable method for their acquisition. Magnet-assisted cell sorting strategies are established to mitigate the non-specific uptake of immunomagnetic microparticles, enabling the enrichment of endothelial cells to purities that prevent their overgrowth by stromal cells. This work delineates a reliable method for acquiring human adipose tissue-derived microvascular endothelial cells in large quantities with high purities that can be readily applied in future vascular tissue engineering applications.
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