Cultured Adipose Tissue Research Articles

The expression and regulation of bone-acting cytokines in human peripheral adipose tissue in organ culture

The expression and regulation of bone-acting cytokines in human peripheral adipose tissue in organ culture

Clinical and Experimental Study of Autologous Fat Grafting After Processing by Centrifugation and Serum Lavage

This clinical and experimental study compared adipose tissue transplant behavior after two different techniques of purifying: centrifugation at 3400 rpm for 3 min and serum lavage without centrifugation. Clinical evaluation was performed under standardized conditions for lipofilling on a series of 51 female patients, intentionally selected to have similar characteristics and assigned to two groups based on the method of processing. Experimentally, a culture system in diffusion chambers with vitaline membranes was designed to mimic the behavior and to study the morphology of the adipose tissue used for autografting. Survival, structure, and proliferation of the adipose cells in vitro were examined by classical histologic H&E staining and immunohistochemistry for leptin and cyclin D1. The main differences encountered experimentally were the presence of a greater amount of preadipocytes in the noncentrifuged adipose tissue cultures and more distinctly expressed cell proliferation. The postoperative clinical results favored of the serum lavage purifying technique. Our data suggest that with transplantation of noncentrifuged adipose tissue more active preadipocytes are applied which could possibly lead to better potential chances of survival and even de novo development of fat.

Isolation and Characterization of Mouse Mesenchymal Stem Cells

Objective Mesenchymal stem cells (MSCs) have been studied in regenerative medicine because of their unique immunologic characteristics. However, before clinical application in humans, animal models are needed to confirm their safety and efficacy. To date, appropriate methods and sources to obtain mouse MSCs have not been identified. Therefore, we investigated MSCs isolated from 3 strains of mice and 3 sources for the development of MSCs in a mouse model. Materials and Methods Male BALB/c, C3H and C57BL/6 mice were used to isolate MSCs from various tissues including bone marrow (BM), compact bone, and adipose tissue. The MSCs were maintained in StemXVivo medium. Immunophenotypes of the MSCs were analyzed by FACS and their growth potential estimated by the number of colony-forming unit fibroblasts. Results All MSCs that were isolated from BM, compact bone, and adipose tissue showed plastic-adherent, fibroblastic-like morphologic characteristics regardless of the mouse strain or cell source. However, culture of BM MSCs was less successful than the other tissue types. The FACS phenotype analysis revealed that the MSCs were positive for CD29, CD44, CD105, and Sca-1, but negative for CD34, TER-119, CD45, and CD11b. According to the results of the characterization, the adipose tissue MSCs showed higher growth potential than did other MSCs. Conclusion The results of this study showed that culture of adipose tissue and compact bone-MSCs was easier than BM MSCs. Based on the results of immunophenotype and growth potential, C57BL/6 AT-MSCs might be a suitable source to establish a mouse model of MSCs.

Adipose tissue as a source of nicotinamide N-methyltransferase and homocysteine

Nicotinamide N-methyltransferase (NNMT) catalyses the conversion of nicotinamide to 1-methylnicotinamide and plays an important role in hepatic detoxification reactions. Here we show that, in addition to the liver, 3T3-L1 adipocytes as well as human and murine adipose tissue explants express high amounts of enzymatically active NNMT. NNMT mRNA levels and enzyme activity increased in 3T3-L1 cells in a differentiation-dependent manner. Homocysteine, the atherogenic product of the NNMT-catalyzed reaction, was secreted from 3T3-L1 cells or adipose tissue cultures. Homocysteine release increased during 3T3-L1 differentiation and was reduced when adipose tissue was treated with the NNMT inhibitor 1-methylnicotinamide. Nicotinic acid (NA), a widely used drug to lower elevated plasma lipid levels, induced NNMT enzyme activity in white adipose tissue of mice. In tissue culture nicotinamide treatment led to an increase in adipose tissue homocysteine secretion. These data support the concept that adipose tissue NNMT contributes to the increased plasma homocysteine levels in patients treated with NA.

Time course of high-fat diet-induced reductions in adipose tissue mitochondrial proteins: potential mechanisms and the relationship to glucose intolerance

Increasing evidence suggests that reduced adipose tissue mitochondrial content is associated with the pathogenesis of type 2 diabetes. These investigations have utilized severely insulin-resistant rodent models. Thus, it is difficult to ascertain the potential mechanisms that initiate these changes and whether reductions in adipose mitochondria are an initiating event in the development of impaired glucose homeostasis. Thus, we sought to determine the time course of high-fat diet-induced reductions of mitochondrial content in epididymal adipose tissue in relation to changes in purported mediators of mitochondrial biogenesis and the development of impaired glucose homeostasis. Male Wistar rats were fed a high-fat diet ( approximately 59% of kcals from fat) for 2, 4, or 6 wk. Six weeks of high-fat feeding resulted in reductions in CORE I, COX IV, cytochrome c, HSP60, relative mtDNA copy number, and PGC-1alpha expression. These changes were not associated with decreases in eNOS and AMPK or increases in markers of oxidative stress. Interestingly, ex vivo treatment of adipose tissue cultures with palmitate led to decreases in PGC-1alpha expression and COX IV and CORE I protein content as observed in vivo. Thus, the high-fat diet-induced reductions in adipose tissue mitochondrial proteins may be mediated by increases in plasma fatty acids. Importantly, reductions in adipose tissue mitochondrial content occurred after the development of impaired glucose homeostasis. Thus, reductions in adipose tissue mitochondrial proteins are most likely not a causal event in the development of impaired glucose homeostasis.

Effects of curcumin on secretion of adiponectin and interleukin-6 in human adipose tissues: an in vitro study

To investigate the effects of different concentrations of curcumin on secretion of adiponectin (APN) and interleukin-6 (IL-6) in human adipose tissues cultivated in vitro. Seven male patients with kidney stones were admitted. Abdominal subcutaneous adipose tissue and perirenal adipose tissue were collected from the operating-patients, and were cultivated with different concentrations of curcumin (10 and 100 microg/ml) in vitro. The contents of APN and IL-6 in the culture medium of adipose tissue cells were measured by enzyme-linked immunosorbent assay (ELISA) after they were cultured for 6 and 24 hours. Compared with the blank control group, the content of APN in the adipose tissue culture medium was increased by 100 microg/ml curcumin (P<0.05) after 6-hour culture, and the content of IL-6 was significantly decreased by 100 microg/ml curcumin after 6- and 24-hour culture (P<0.05). 100 microg/ml curcumin can increase APN secretion and decrease IL-6 secretion in human adipose tissues cultivated in vitro.

Effects of trans-10, cis-12 conjugated linoleic acid on gene expression and lipid metabolism of adipose tissue of growing pigs

The objective of the present study was to determine the effects of trans-10, cis-12 conjugated linoleic acid (CLA) in adipose tissue explant cultures of growing pigs on the following responses: lipogenesis (measured as rate of (14)C-labeled glucose incorporation over a subsequent 2-h incubation in the presence or absence of insulin), lipolysis (release of non-esterified fatty acid over a 2-h incubation in the presence or absence of isoproterenol), activities of lipogenic enzymes, and mRNA abundance of fatty acid synthase (FAS). Adipose tissue explants from nine growing pigs (78 +/- 3 kg) were cultured in 199 medium with insulin, dexamethasone and antibiotics for 4, 12, 24, and 48 h. The treatments were 1) control: 100 microM polyvinyl alcohol (PVA); 2) pGH: 100 ng/mL porcine growth hormone (pGH) plus 100 microM PVA; 3) CLA200: 200 microM trans-10, cis-12 CLA; 4) CLA50: 50 microM trans-10, cis-12 CLA, and 5) LA: 200 microM linoleic acid. Fatty acids were added along with PVA (2:1), respectively, for 24 h. Explants were collected after each culture period and assayed for lipogenesis. Transcripts of FAS mRNA were quantified by real-time RT-PCR after 24 and 48 h. Lipolysis and activities of FAS, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and NADP-malate dehydrogenase were determined after 48 h. As expected, glucose incorporation was decreased (P < 0.05) in response to pGH treatment (positive control). LA had no effect on any parameter evaluated. Treatment with trans-10, cis-12 CLA decreased FAS activity (P < 0.05), but NADPH-generating enzymes were unaffected by treatments. Consistent with reduction in FAS activity, both lipid synthesis and FAS mRNA abundance were reduced with chronic CLA treatment, pGH increased baseline and stimulated lipolysis (P < 0.05) after 48 h of culture, while CLA treatment had no effect on non-esterified fatty acid release. Results of this study showed that trans-10, cis-12 CLA alters lipogenesis but has no effect on lipolysis in cultures of pig adipose tissue.

Trans-10, cis-12 conjugated linoleic acid activates the integrated stress response pathway in adipocytes

Trans-10, cis-12 conjugated linoleic acid (t10c12 CLA) causes fat loss in mouse white adipose tissue (WAT) and adipocytes in culture. The early transcriptome changes in treated WAT and 3T3-L1 adipocytes were analyzed using high-density microarrays to better characterize the signaling pathways responding to t10c12 CLA. Gene expression responses between 4 and 24 h after treatment showed a common set of early gene expression changes indicative of an integrated stress response (ISR). The responses of 3T3-L1 preadipocytes treated with t10c12 CLA or adipocytes treated with the cis-9, trans-11 isomer of CLA did not show the ISR, indicating the effect is specific to adipocytes responding to t10c12 CLA. Western blot analysis found increased phosphorylation of eIF2 alpha and increased production of ATF4 confirming at least part of the response to t10c12 CLA is mediated through the ISR pathway. Immunofluorescence microscopy found that the cell type expressing ATF3, an indicator of the ISR, was early stage adipocytes containing oil droplets but lacking the abundant levels of fatty acid binding protein-4 (FABP4) (AP2) found in mature adipocytes. Our data suggests that the ISR precedes and is possibly the cause of the later induction of proinflammatory cytokines observed in t10c12 CLA treated adipocytes. The release of proinflammatory cytokines may explain how the ISR in early stage adipocytes causes lipid loss in mature adipocytes.

Sequential Regulation of Diacylglycerol Acyltransferase 2 Expression by CAAT/Enhancer-binding Protein β (C/EBPβ) and C/EBPα during Adipogenesis

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triacylglycerol (TG) synthesis. Despite the existence of an alternative acyltransferase (DGAT1), mice lacking DGAT2 have a severe deficiency of TG in adipose tissue, indicating a nonredundant role for this enzyme in adipocyte TG synthesis. We have studied the regulation of DGAT2 expression during adipogenesis. In both isolated murine preadipocytes and 3T3-L1 cells the temporal pattern of DGAT2 expression closely mimicked that of genes whose expression is regulated by CAAT/enhancer-binding protein beta (C/EBPbeta). Inhibition of C/EBPbeta expression in differentiating preadipocytes reduced DGAT2 expression, and electrophoretic mobility shift assay and chromatin immunoprecipitation experiments identified a promoter element in the DGAT2 gene that is likely to mediate this effect. The importance of C/EBPbeta in adipocyte expression of DGAT2 was confirmed by the finding of reduced DGAT2 expression in the adipose tissue of C/EBPbeta-null animals. However, DGAT2 expression is maintained at high levels during the later stages of adipogenesis, when C/EBPbeta levels decline. We show that, at these later stages of differentiation, C/EBPalpha is capable of substituting for C/EBPbeta at the same promoter element. These observations provide novel insight into the transcriptional regulation of DGAT2 expression. Moreover, they further refine the complex and serial roles of the C/EBP family of transcription factors in inducing and maintaining the metabolic properties of mature adipocytes.

Profiling of adipokines secreted from human subcutaneous adipose tissue in response to PPAR agonists

The role of PPARs in the regulation of human adipose tissue secretome has received little attention despite its potential importance in the therapeutic actions of PPAR agonists. Here, we have investigated the effect of selective PPARγ, PPARα, and PPARβ/δ agonists on the production of adipokines by human subcutaneous adipose tissue. Antibody arrays were used to measure secreted factors in media from cultured adipose tissue explants. Sixteen proteins were produced in significant amounts. Activation of PPARs regulated the production of five proteins. Treatments with the three PPAR agonists decreased the secretion of leptin and interleukin-6. PPARα and β/δ agonists markedly enhanced hepatocyte growth factor secretion whereas PPARβ/δ down-regulated angiogenin and up-regulated TIMP-1 release. Hepatocyte growth factor, interleukin-6, and TIMP-1 are chiefly expressed in cells from the stromal vascular fraction whereas angiogenin is expressed in both adipocytes and cells from the stromal vascular fraction. Our data show that PPAR agonists modulate secretion of bioactive molecules from the different cell types composing human adipose tissue.

Acute and selective regulation of glyceroneogenesis and cytosolic phosphoenolpyruvate carboxykinase in adipose tissue by thiazolidinediones in type 2 diabetes

Regulation of glyceroneogenesis and its key enzyme cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) plays a major role in the control of fatty acid release from adipose tissue. Here we investigate the effect of rosiglitazone on the expression of genes involved in fatty acid metabolism and the resulting metabolic consequences. Rosiglitazone was administered to Zucker fa/fa rats for 4 days and to 24 diabetic patients for 12 weeks, then mRNA expression for the genes encoding PEPCK-C, mitochondrial PEPCK, adipocyte lipid-binding protein, glycerol kinase, lipoprotein lipase and glycerol-3-phosphate dehydrogenase was examined in s.c. adipose tissue by real-time RT-PCR. Glyceroneogenesis was determined using [1-(14)C]pyruvate incorporation into lipids. Cultured adipose tissue explants from overweight women undergoing plastic surgery were incubated with rosiglitazone for various times before mRNA determination and analysis of PEPCK-C protein, activity and glyceroneogenesis. Rosiglitazone administration to rats induced the expression of the gene encoding PEPCK-C mRNA (PCK1) and PEPCK-C activity in adipose tissue with a resulting 2.5-fold increase in glyceroneogenesis. This was accompanied by an improvement in dyslipidaemia as demonstrated by the decrease in plasma NEFAs and triacylglycerol. In rosiglitazone-treated diabetic patients, PCK1 mRNA was raised 2.5-fold in s.c. adipose tissue. Rosiglitazone treatment of adipose tissue explants from overweight women caused a selective augmentation in PCK1 mRNA which reached a maximum of 9-fold at 14 h, while mRNA for other genes remained unaffected. Experiments with inhibitors showed a direct and transcription-only effect, which was followed by an increase in PEPCK-C protein, enzyme activity and glyceroneogenesis. These results favour adipocyte glyceroneogenesis as the initial thiazolidinedione-responsive pathway leading to improvement in dyslipidaemia.

Fractional Factorial Design for Optimization of the SELDI Protocol for Human Adipose Tissue Culture Media

The early factors inducing insulin resistance are not known. Therefore, we are interested in studying the secretome of the human visceral adipose tissue as a potential source of unknown peptides and proteins inducing insulin resistance. Surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry is a high-throughput proteomics technology to generate peptide and protein profiles (MS spectra). To obtain good quality and reproducible data from SELDI-TOF, many factors in the sample pretreatment and SELDI protocol should be optimized. To identify the optimal combination of factors resulting in the best and the most reproducible spectra, we designed an experiment where factors were varied systematically according to a fractional factorial design. In this study, seven protein chip preparation protocol factors were tested in 32 experiments. The main effects of these factors and their interactions contributing to the best quality spectra were identified by ANOVA. To assess the reproducibility, in a subsequent experiment the eight protocols generating the highest quality spectra were applied to samples in quadruplicates on different chips. This approach resulted in the development of an improved chip protocol, yielding higher quality peaks and more reproducible spectra.

Hyperphosphorylation of MEF2A in primary adipocytes correlates with downregulation of human GLUT4 gene promoter activity

GLUT4 promoter activity is regulated by hormonal, metabolic, and tissue-specific controls. This complicates the study of GLUT4 gene transcription, as no cell culture model adequately recapitulates these extracellular regulators. While investigating cultured primary adipocytes as a model system for GLUT4 transcription, we observed that GLUT4 mRNA was specifically and rapidly downregulated upon tissue dispersal. Downregulation of GLUT4 mRNA was mediated in part by loss of regulatory control by the trans-acting factors that control GLUT4 transcriptional activity [the myocyte enhancer factor 2 (MEF2) transcription factor family and the GLUT4 enhancer factor] and their cognate DNA binding sites in transgenic mice. The differences in GLUT4 transcription when whole adipose tissue and cell culture model systems are compared can be correlated to a posttranslational phosphorylation of the transcription factor MEF2A. The difference in the MEF2A phosphorylation state in whole tissue vs. isolated cells may provide a further basis for the development of an in vitro system that could recapitulate fully regulated GLUT4 promoter activity. Development of an in vitro system to reconstitute GLUT4 transcriptional regulation will further efforts to discern the molecular mechanisms that underlie GLUT4 expression.

Adipose Triglyceride Lipase and Hormone-sensitive Lipase Are the Major Enzymes in Adipose Tissue Triacylglycerol Catabolism

The mobilization of free fatty acids from adipose triacylglycerol (TG) stores requires the activities of triacylglycerol lipases. In this study, we demonstrate that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major enzymes contributing to TG breakdown in in vitro assays and in organ cultures of murine white adipose tissue (WAT). To differentiate between ATGL- and HSL-specific activities in cytosolic preparations of WAT and to determine the relative contribution of these TG hydrolases to the lipolytic catabolism of fat, mutant mouse models lacking ATGL or HSL and a mono-specific, small molecule inhibitor for HSL (76-0079) were used. We show that 76-0079 had no effect on TG catabolism in HSL-deficient WAT but, in contrast, essentially abolished free fatty acid mobilization in ATGL-deficient fat. CGI-58, a recently identified coactivator of ATGL, stimulates TG hydrolase activity in wild-type and HSL-deficient WAT but not in ATGL-deficient WAT, suggesting that ATGL is the sole target for CGI-58-mediated activation of adipose lipolysis. Together, ATGL and HSL are responsible for more than 95% of the TG hydrolase activity present in murine WAT. Additional known or unknown lipases appear to play only a quantitatively minor role in fat cell lipolysis.

Acute-Phase Serum Amyloid A: An Inflammatory Adipokine and Potential Link between Obesity and Its Metabolic Complications

BackgroundObesity is associated with low-grade chronic inflammation, and serum markers of inflammation are independent risk factors for cardiovascular disease (CVD). However, the molecular and cellular mechanisms that link obesity to chronic inflammation and CVD are poorly understood.Methods and FindingsAcute-phase serum amyloid A (A-SAA) mRNA levels, and A-SAA adipose secretion and serum levels were measured in obese and nonobese individuals, obese participants who underwent weight-loss, and persons treated with the insulin sensitizer rosiglitazone. Inflammation-eliciting activity of A-SAA was investigated in human adipose stromal vascular cells, coronary vascular endothelial cells and a murine monocyte cell line. We demonstrate that A-SAA was highly and selectively expressed in human adipocytes. Moreover, A-SAA mRNA levels and A-SAA secretion from adipose tissue were significantly correlated with body mass index ( r = 0.47; p = 0.028 and r = 0.80; p = 0.0002, respectively). Serum A-SAA levels decreased significantly after weight loss in obese participants ( p = 0.006), as well as in those treated with rosiglitazone ( p = 0.033). The magnitude of the improvement in insulin sensitivity after weight loss was significantly correlated with decreases in serum A-SAA ( r = −0.74; p = 0.034). SAA treatment of vascular endothelial cells and monocytes markedly increased the production of inflammatory cytokines, e.g., interleukin (IL)-6, IL-8, tumor necrosis factor alpha, and monocyte chemoattractant protein-1. In addition, SAA increased basal lipolysis in adipose tissue culture by 47%. ConclusionsA-SAA is a proinflammatory and lipolytic adipokine in humans. The increased expression of A-SAA by adipocytes in obesity suggests that it may play a critical role in local and systemic inflammation and free fatty acid production and could be a direct link between obesity and its comorbidities, such as insulin resistance and atherosclerosis. Accordingly, improvements in systemic inflammation and insulin resistance with weight loss and rosiglitazone therapy may in part be mediated by decreases in adipocyte A-SAA production.

Adipose Tissue Proadipogenic Redox Changes in Obesity

The role of inflammation and oxidative stress in the development of obesity and associated metabolic disorders is under debate. We investigated the redox metabolism in a non-diabetic obesity model, i.e. 11-week-old obese Zucker rats. Antioxidant enzyme activities, lipophilic antioxidant (alpha-tocopherol, coenzymes Q) and hydrophilic antioxidant (glutathione, vitamin C) contents and their redox state (% oxidized form), were studied in inguinal white fat and compared with blood and liver. The adipose tissues of obese animals showed a specific higher content of hydrophilic molecules in a lower redox state than those of lean animals, which were associated with lower lipophilic molecule content and lipid peroxidation. Conversely and as expected, glutathione content decreased and its redox state increased in adipose tissues of rats subjected to lipopolysaccharide-induced systemic oxidative stress. In these in vivo models, oxidative stress and obesity thus had opposite effects on adipose tissue redox state. Moreover, the increase in glutathione content and the decrease of its redox state by antioxidant treatment promoted in vitro the accumulation of triglycerides in preadipocytes. Taken together and contrary to the emergent view, our results suggest that obesity is associated with an intracellular reduced redox state that promotes on its own the development of a deleterious proadipogenic process.

Dehydroepiandrosterone and human adipose tissue

The aim of this study was to investigate whether DHEA alters the proliferation and differentiation of human sc and visceral adipose cells in primary cultures. Sc and omental adipose tissue was obtained from 10 female donors aged 36+/-3.6 yr with a body mass index (BMI) of 33+/-3.21 kg/m2. Stromal vascular cells were isolated and cultured using modified procedures described by Entenmann and Hauner. For the proliferation assay, stromal-vascular cells from sc and visceral adipose tissue cultures were fed with proliferation media containing 0, 25 or 100 microM DHEA for 3 days. At the end of this treatment period, two type cultures were prepared for determining their metabolic activity using the sulforhodamine B staining procedure. The metabolic activity of proliferating human visceral adipose tissue was higher than sc adipose tissue. The activity of proliferating human visceral tissue cultures decreased more than the sc tissue as the level of DHEA in the cultures was increased. These data suggest that DHEA predominantly influences the proliferation and differentiation of human omental adipose tissue.

Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction

Mesenchymal stem cells are multipotent cells that can differentiate into cardiomyocytes and vascular endothelial cells. Here we show, using cell sheet technology, that monolayered mesenchymal stem cells have multipotent and self-propagating properties after transplantation into infarcted rat hearts. We cultured adipose tissue-derived mesenchymal stem cells characterized by flow cytometry using temperature-responsive culture dishes. Four weeks after coronary ligation, we transplanted the monolayered mesenchymal stem cells onto the scarred myocardium. After transplantation, the engrafted sheet gradually grew to form a thick stratum that included newly formed vessels, undifferentiated cells and few cardiomyocytes. The mesenchymal stem cell sheet also acted through paracrine pathways to trigger angiogenesis. Unlike a fibroblast cell sheet, the monolayered mesenchymal stem cells reversed wall thinning in the scar area and improved cardiac function in rats with myocardial infarction. Thus, transplantation of monolayered mesenchymal stem cells may be a new therapeutic strategy for cardiac tissue regeneration.

Effect of growth hormone on fatty acid synthase gene expression in porcine adipose tissue cultures

We describe an efficient in vitro assay to test growth hormone effects on mRNA levels and fatty acid synthase (FAS, EC. 2.3.1.85) activity. Swine adipose tissue explants were long-term cultured in medium containing growth hormone and FAS mRNA levels and enzyme activity were measured. We quantified FAS transcripts by competitive reverse transcriptase PCR (RT-PCR) using total RNA from cultured adipose tissue explants and RT-PCR standard-curves were constructed using a cloned 307 bp segment of native FAS cDNA and a shorter fragment from which a 64 bp (competitor, 243 bp) internal sequence had been deleted. A known amount of competitor was added to each PCR as an internal control and µ-actin transcripts were also measured to correct for differences in total RNA extraction and reverse transcription efficiency. In cultures with added growth hormone FAS mRNA levels decreased 70% (p < 0.01) and FAS enzyme activity decreased 22% (p < 0.05). These in vitro growth hormone effects were consistent with those observed in vivo, showing that in vitro adipose tissue culture combined with RT-PCR is a useful and accurate tool for studying growth hormone modulation of adipose tissue metabolism. This technique allowed the diagnosis of lower levels of FAS mRNA in the presence of growth hormone and these low levels were associated with decreased FAS activity in the adipose tissue explants.

Novel Autologous Cell Therapy in Ischemic Limb Disease Through Growth Factor Secretion by Cultured Adipose Tissue–Derived Stromal Cells

The delivery of autologous progenitor cells into ischemic tissue of patients is emerging as a novel therapeutic option. Here, we report the potential impact of cultured adipose tissue-derived cells (ADSC) on angiogenic cell therapy. ADSC were isolated from C57Bl/6 mouse inguinal adipose tissue and showed high expression of ScaI and CD44, but not c-kit, Lin, CD34, CD45, CD11b, and CD31, compatible with that of mesenchymal stem cells from bone marrow. In coculture conditions with ADSC and human aortic endothelial cells (ECs) under treatment with growth factors, ADSC significantly increased EC viability, migration and tube formation mainly through secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). At 4 weeks after transplantation of ADSC into the ischemic mouse hindlimb, the angiogenic scores were improved in the ADSC-treated group, which were evaluated with blood flow by laser Doppler imaging (LDI) and capillary density by immunostaining with anti-CD31 antibody. However, injected ADSC did not correspond to CD31, von Willebrand factor, and alpha-smooth muscle actin-positive cells in ischemic tissue. These adipose tissue-derived cells demonstrated potential as angiogenic cell therapy for ischemic disease, which appears to be mainly achieved by their ability to secrete angiogenic growth factors.