Chronic exercise decreases cytokine production in healthy rat skeletal muscle

Skeletal muscle is the source of pro‐ and anti‐inflammatory cytokines, and recently, it has been recognized as an important source of interleukin‐6 (IL‐6). Acute physical exercise is known to induce a pro‐inflammatory cytokine profile in the plasma. However, the effect of chronic physical exercise in the production of pro‐ and anti‐inflammatory cytokines by the skeletal muscle has never been examined. We assessed IL‐6, TNF‐α, IL‐1β and IL‐10 levels in the skeletal muscle of rats submitted to endurance training. Animals were randomly assigned to either a sedentary group (S, n = 7) or an endurance exercise trained group (T, n = 8). Trained rats ran on a treadmill for 5 days week^?1 for 8 weeks (60% VO_2max). Detection of IL‐6, TNF‐α, IL‐1β and IL‐10 protein expression was carried out by ELISA. We found decreased expression of IL‐1β, IL‐6, TNF‐α and IL‐10 (28%, 27%, 32% and 37%, respectively, p < 0.05) in the extensor digital longus (EDL) from T, when compared with S. In the soleus, IL‐1β, TNF‐α and IL‐10 protein levels were similarly decreased (34%, 42% and 50%, respectively, p < 0.05) in T in relation to S, while IL‐6 expression was not affected by the training protocol. In conclusion, exercise training induced decreased cytokine protein expression in the skeletal muscle. These data show that in healthy rats, 8‐week moderate‐intensity aerobic training down regulates skeletal muscle production of cytokines involved in the onset, maintenance and regulation of inflammation, and that the response is heterogeneous according to fibre composition. Copyright © 2009 John Wiley & Sons, Ltd.     

Lipid-heparin infusion suppresses the IL-10 response to trauma in subcutaneous adipose tissue in humans

An imbalance between pro‐ and anti‐inflammatory cytokine productions in adipose tissue is thought to contribute to chronic, systemic, low‐grade inflammation and consequently to an increased risk of cardiovascular complications in obese and type 2 diabetic patients. Nonesterified fatty acids (NEFA), whose serum levels are elevated in such patients, have been shown to interfere with cytokine production in vitro. In order to evaluate the effects of elevated NEFA levels on cytokine production in adipose tissue in vivo we used an 18‐gauge open‐flow microperfusion (OFM) catheter to induce local inflammation in the subcutaneous adipose tissue (SAT) of healthy volunteers and to sample interstitial fluid (IF) specifically from the inflamed tissue. In two crossover studies, nine subjects received either an intravenous lipid‐heparin infusion to elevate circulating NEFA levels or saline over a period of 28 h. The former increased the circulating levels of triglycerides (TGs), NEFA, glucose, and insulin over the study period. NEFA effects on locally induced inflammation were estimated by measuring the levels of a panel adipokines in the OFM probe effluent. Interleukin‐6 (IL‐6), IL‐8, tumor necrosis factor‐α (TNF‐α) and monocyte chemoattractant protein‐1 (MCP‐1) levels increased during the study period but were not affected by lipid‐heparin infusion. In contrast, the level of IL‐10, an anti‐inflammatory cytokine, was significantly reduced during the final hour of lipid‐heparin infusion (saline: 449.2 ± 105.9 vs. lipid‐heparin: 65.4 ± 15.4 pg/ml; P = 0.02). These data provide the first in vivo evidence that elevated NEFA can modulate cytokine production by adipose tissue.     

Induction of cytokine formation by human intestinal bacteria in gut epithelial cell lines

Aims: To investigate the effects of human gut micro‐organisms on cytokine production by human intestinal cell lines. Methods and Results: Quantitative real‐time PCR assays were developed to measure the production of pro‐inflammatory (IL‐1α, IL‐6, IL‐18 and TNFα) and anti‐inflammatory (TGF‐β1, TGF‐β2, TGF‐β3, IL‐4 and IL‐10) cytokines in HT‐29 and Caco‐2 cell lines. They were co‐cultured with a range of mucosal bacteria isolated from ulcerative colitis patients, together with lactobacilli and bifidobacteria obtained from healthy people. HT‐29 cells were also co‐cultured with Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli and Salmonella typhimurium. The majority of commensal bacteria tested suppressed the expression of anti‐inflammatory cytokine mRNA, increased IL‐18, reduced IL‐1α, and with the exception of nonpathogenic E. coli, reduced TNF‐α. All overtly pathogenic species increased both pro‐inflammatory and anti‐inflammatory cytokine mRNA. Conclusion: Commensal and pathogenic species induced fundamentally different cytokine responses in human intestinal epithelial cell lines. Significance and Impact of the Study: Interactions between commensal bacteria tested in this study and the innate immune system were shown to be anti‐inflammatory in nature, in contrast to the pathogenic organisms investigated. These data contribute towards our understanding of how potential probiotic species can be used to suppress the pro‐inflammatory response in inflammatory bowel disease.     

Study of the proinflammatory role of human differentiated omental adipocytes

Infiltration of monocyte‐derived macrophages into adipose tissue has been associated with tissue and systemic inflammation. It has been suggested that macrophage infiltration affects fat expansion through a paracrine action on adipocyte differentiation. Our working hypothesis is that factors released by monocytes/macrophages may also affect mature adipocyte biology. Human differentiated omental adipocytes were incubated with LPS and conditioned media obtained from human macrophage‐like cell line THP‐1, previously activated or not with LPS. We show that LPS greatly increased the secretion levels of pro‐inflammatory adipokines including IL‐6, IL‐8, GRO, and MCP‐1. Macrophage‐conditioned medium also upregulated IL‐6, IL‐8, GRO, and MCP‐1 mRNA expression and protein levels and led to the novo secretion of ICAM‐1, IL‐1β, IP‐10, MIP‐1α, MIP‐1β, VEGF, and TNFα. Human differentiated adipocytes treated by macrophage‐conditioned medium displayed marked reduction of adipocyte function as assessed by decreased phosphorylation levels of ERK1, ERK2, and p38α and reduced gene expression of lipogenic markers including PPAR‐γ and fatty acid synthase. These data show that macrophage‐secreted factors not only inhibit the formation of mature adipocytes but alter their function, suggesting that human differentiated omental adipocytes might also contribute to systemic chronic low‐grade inflammation associated with human obesity. J. Cell. Biochem. 107: 1107–1117, 2009. © 2009 Wiley‐Liss, Inc.     

In senescence,age‐associated B cells secrete TNFα and inhibit survival of B‐cell precursors*

Aged mice exhibit ~ 5–10‐fold increases in an ordinarily minor CD21/35^? CD23^? mature B‐cell subset termed age‐associated B cells (ABCs). ABCs from old, but not young, mice induce apoptosis in pro‐B cells directly through secretion of TNFα. In addition, aged ABCs, via TNFα, stimulate bone marrow cells to suppress pro‐B‐cell growth. ABC effects can be prevented by the anti‐inflammatory cytokine IL‐10. Notably, CD21/35⁺ CD23⁺ follicular (FO) splenic and FO‐like recirculating bone marrow B cells in both young and aged mice contain a subpopulation that produces IL‐10. Unlike young adult FO B cells, old FO B cells also produce TNFα; however, secretion of IL‐10 within this B‐cell population ameliorates the TNFα‐mediated effects on B‐cell precursors. Loss of B‐cell precursors in the bone marrow of old mice in vivo was significantly associated with increased ABC relative to recirculating FO‐like B cells. Adoptive transfer of aged ABC into RAG‐2 KO recipients resulted in significant losses of pro‐B cells within the bone marrow. These results suggest that alterations in B‐cell composition during old age, in particular, the increase in ABC within the B‐cell compartments, contribute to a pro‐inflammatory environment within the bone marrow. This provides a mechanism of inappropriate B‐cell ‘feedback’ that promotes down‐regulation of B lymphopoiesis in old age.     

Inflammatory profile in X‐linked adrenoleukodystrophy patients: Understanding disease progression

X‐linked adrenoleukodystrophy (X‐ALD) is an inherited disease characterized by progressive inflammatory demyelization in the brain, adrenal insufficiency, and an abnormal accumulation of very long chain fatty acids (VLCFA) in tissue and body fluids. Considering that inflammation might be involved in pathophysiology of X‐ALD, we aimed to investigate pro‐ and anti‐inflammatory cytokines in plasma from three different male phenotypes (CCER, AMN, and asymptomatic individuals). Our results showed that asymptomatic patients presented increased levels of pro‐inflammatory cytokines IL‐1β, IL‐2, IL‐8, and TNF‐α and the last one was also higher in AMN phenotype. Besides, asymptomatic patients presented higher levels of anti‐inflammatory cytokines IL‐4 and IL‐10. AMN patients presented higher levels of IL‐2, IL‐5, and IL‐4. We might hypothesize that inflammation in X‐ALD is related to plasmatic VLCFA concentration, since there were positive correlations between C26:0 plasmatic levels and pro‐inflammatory cytokines in asymptomatic and AMN patients and negative correlation between anti‐inflammatory cytokine and C24:0/C22:0 ratio in AMN patients. The present work yields experimental evidence that there is an inflammatory imbalance associated Th1, (IL‐2, IL‐6, and IFN‐γ), Th2 (IL‐4 and IL‐10), and macrophages response (TNF‐α and IL‐1β) in the periphery of asymptomatic and AMN patients, and there is correlation between VLCFA plasmatic levels and inflammatory mediators in X‐ALD. Furthermore, we might also speculate that the increase of plasmatic cytokines in asymptomatic patients could be considered an early biomarker of brain damage and maybe also a predictor of disease progression.     

Variants of the IL‐10 gene associate with muscle strength in elderly from rural Africa: a candidate gene study

Recently, it has been shown that the capacity of the innate immune system to produce cytokines relates to skeletal muscle mass and strength in older persons. The interleukin‐10 (IL‐10) gene regulates the production capacities of IL‐10 and tumour necrosis factor‐α (TNF‐α). In rural Ghana, IL‐10 gene variants associated with different production capacities of IL‐10 and TNF‐α are enriched compared with Caucasian populations. In this setting, we explored the association between these gene variants and muscle strength. Among 554 Ghanaians aged 50 years and older, we determined 20 single nucleotide polymorphisms in the IL‐10 gene, production capacities of IL‐10 and TNF‐α in whole blood upon stimulation with lipopolysaccharide (LPS) and handgrip strength as a proxy for skeletal muscle strength. We distinguished pro‐inflammatory haplotypes associated with low IL‐10 production capacity and anti‐inflammatory haplotypes with high IL‐10 production capacity. We found that distinct haplotypes of the IL‐10 gene associated with handgrip strength. A pro‐inflammatory haplotype with a population frequency of 43.2% was associated with higher handgrip strength (P = 0.015). An anti‐inflammatory haplotype with a population frequency of 7.9% was associated with lower handgrip strength (P = 0.006). In conclusion, variants of the IL‐10 gene contributing to a pro‐inflammatory cytokine response associate with higher muscle strength, whereas those with anti‐inflammatory response associate with lower muscle strength. Future research needs to elucidate whether these effects of variation in the IL‐10 gene are exerted directly through its role in the repair of muscle tissue or indirectly through its role in the defence against infectious diseases.     

IL‐35 recombinant protein reverses inflammatory bowel disease and psoriasis through regulation of inflammatory cytokines and immune cells

Interleukin‐35 (IL‐35), a member of the IL‐12 family, functions as a new anti‐inflammatory factor involved in arthritis, psoriasis, inflammatory bowel disease (IBD) and other immune diseases. Although IL‐35 can significantly prevent the development of inflammation in many diseases, there have been no early studies accounting for the role of IL‐35 recombinant protein in IBD and psoriasis. In this study, we assessed the therapeutic potential of IL‐35 recombinant protein in three well‐known mouse models: the dextransulfate sodium (DSS)‐induced colitis mouse model, the keratin14 (K14)‐vascular endothelial growth factor A (VEGF‐A)‐transgenic (Tg) psoriasis mouse model and the imiquimod (IMQ)‐induced psoriasis mouse model. Our results indicated that IL‐35 recombinant protein can slow down the pathologic process in DSS‐induced acute colitis mouse model by decreasing the infiltrations of macrophages, CD4⁺T and CD8⁺T cells and by promoting the infiltration of Treg cells. Further analysis demonstrated that IL‐35 recombinant protein may regulate inflammation through promoting the secretion of IL‐10 and inhibiting the expression of pro‐inflammatory cytokines such as IL‐6, TNF‐α and IL‐17 in acute colitis model. In addition, lower dose of IL‐35 recombinant protein could achieve long‐term treatment effects as TNF‐α monoclonal antibody did in the psoriasis mouse. In summary, the remarkable therapeutic effects of IL‐35 recombinant protein in acute colitis and psoriasis mouse models indicated that IL‐35 recombinant protein had a variety of anti‐inflammatory effects and was expected to become an effective candidate drug for the treatment of inflammatory diseases.     

Chemical Composition of Bawei Longzuan Granule and Its Anti‐Arthritic Activity on Collagen‐Induced Arthritis in Rats by Inhibiting Inflammatory Responses

Bawei Longzuan granule (BLG) is a representative Zhuang medicine preparation. The present work aims to characterize the chemical constituents of BLG and evaluate its anti‐arthritic activity. The major chemical constituents of BLG were tentatively identified by ultra‐performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry (UPLC‐Q‐TOF/MS), which revealed the presence of some alkaloids (e. g., magnoflorine, sinomenine and nitidine) and flavonoids (e. g., hesperidin, diosmin and sinensetin) that may be partly responsible for the anti‐arthritic effect of BLG. In addition, the collagen‐induced arthritis (CIA) model in rats was induced by intradermal injection of bovine collagen‐II in complete Freund's adjuvant at the base of tail. The CIA rats received oral administration of BLG (1.25, 2.5 and 5 g/kg) for 30 days. Then, various indicators were determined to evaluate its anti‐arthritic activity, including paw swelling, arthritic score, body weight, knee joint pathology, thymus index and spleen index. Additionally, the serum levels of tumor necrosis factor (TNF)‐α, interferon (IFN)‐γ, interleukin (IL)‐1β, IL‐6, IL‐4 and IL‐10 were measured to determine the underlying mechanisms. The results showed that BLG efficiently ameliorated the severity of arthritis in CIA rats by decreasing paw swelling and arthritis score and improving the histological lesions of knee joint. Moreover, the serum levels of several pro‐inflammatory cytokines (i. e., IL‐1β, TNF‐α, IL‐6 and IFN‐γ) were downregulated, whereas two anti‐inflammatory factors (i. e., IL‐4 and IL‐10) were upregulated after BLG administration. These results indicated that BLG possessed promising therapeutic effect on collagen‐induced arthritis by inhibiting inflammatory responses. BLG can be used as a complementary or alternative traditional medicine to treat rheumatoid arthritis.     

Effect of peroxisome proliferator-activated receptor-alpha ligands in the interaction between adipocytes and macrophages in obese adipose tissue

Objective: This study was designed to examine the effect of peroxisome proliferator‐activated receptor‐α (PPAR‐α) ligands on the inflammatory changes induced by the interaction between adipocytes and macrophages in obese adipose tissue. Methods and Procedures: PPAR‐α ligands (Wy‐14,643 and fenofibrate) were added to 3T3‐L1 adipocytes, RAW264 macrophages, or co‐culture of 3T3‐L1 adipocytes and RAW264 macrophages in vitro, and monocyte chemoattractant protein‐1 (MCP‐1) and tumor necrosis factor‐α (TNF‐α) mRNA expression and secretion were examined. PPAR‐α ligands were administered to genetically obese ob/ob mice for 2 weeks. Moreover, the effect of PPAR‐α ligands was also evaluated in the adipose tissue explants and peritoneal macrophages obtained from PPAR‐α‐deficient mice. Results: In the co‐culture of 3T3‐L1 adipocytes and RAW264 macrophages, PPAR‐α ligands reduced MCP‐1 and TNF‐α mRNA expression and secretion in vitro relative to vehicle‐treated group. The anti‐inflammatory effect of Wy‐14,643 was observed in adipocytes treated with macrophage‐conditioned media or mouse recombinant TNF‐α and in macrophages treated with adipocyte‐conditioned media or palmitate. Systemic administration of PPAR‐α ligands inhibited the inflammatory changes in adipose tissue from ob/ob mice. Wy‐14,643 also exerted an anti‐inflammatory effect in the adipose tissue explants but not in peritoneal macrophages obtained from PPAR‐α‐deficient mice. Discussion: This study provides evidence for the anti‐inflammatory effect of PPAR‐α ligands in the interaction between adipocytes and macrophages in obese adipose tissue, thereby improving the dysregulation of adipocytokine production and obesity‐related metabolic syndrome.     

Mesenteric adipose tissue B lymphocytes promote local and hepatic inflammation in non‐alcoholic fatty liver disease mice

Mesenteric adipose tissue (MAT) inflammation is associated with non‐alcoholic fatty liver disease (NAFLD), and immune cells play pivotal roles in the inflammation of adipose tissue. Here, we investigated the roles of MAT B lymphocytes in NAFLD. Mice fed with high‐fat diet (HFD) and normal diet (ND) were killed in time gradients (4, 8 and 12 weeks). Compared with ND‐fed mice, intra‐hepatic CD45⁺CD19⁺ B lymphocytes increased after 4 weeks (P < 0.01) of HFD feeding, and lasted until the 12th week, infiltrated earlier than CD45⁺CD3⁺ T lymphocytes and CD45⁺F4/80⁺ macrophages. The mRNA expression of tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and monocyte chemotactic protein (MCP)‐1 decreased in MAT of B^null HFD‐fed mice compared to that in wild‐type HFD‐fed mice, along with lesser macrophages. Mesenteric adipose tissue B cells from HFD‐fed mice promoted macrophage differentiation to type‐Ι macrophages and expression of pro‐inflammatory cytokines in vitro. Macrophages pre‐treated with MAT B cells from HFD‐fed mice showed elevated mRNA expression of IL‐6 and TNF‐α and declined IL‐10 levels in adipocytes compared to ND MAT B cell pre‐treated macrophages. Besides, internal near‐infrared scanning and external transwell assay showed that HFD MAT B cells migrated to the liver more than ND MAT B cells. High‐fat diet MAT B cells induced higher MCP‐1 and lower IL‐10 expression in primary hepatocytes compared to ND MAT B cells in co‐culture experiment. These data indicate that B lymphocytes infiltrate early in MAT during the development of NAFLD, which may not only promote MAT inflammation by regulating macrophages but also migrate to the liver and induce hepatocytes inflammation.     

Murine macrophage inflammatory cytokine production and immune activation in response to Vibrio parahaemolyticus infection

Vibrio parahaemolyticus is the most common cause of bacterial, seafood‐related illness in the USA. Currently, there is a dearth of published reports regarding immunity to infection with this pathogen. Here, production of both pro‐ and anti‐inflammatory cytokines by V. parahaemolyticus‐infected RAW 264.7 murine macrophages was studied. It was determined that this infection results in increased concentrations of IL‐1α, IL‐6, TNF‐α and IL‐10. Additionally, decreases in cell surface TLR2 and TLR4 and increases in T‐cell co‐stimulatory molecules CD40 and CD86 were discovered. The data presented here begin to identify the immune variables required to eliminate V. parahaemolyticus from infected host tissues.     

The Effect of the Interleukin‐1 Cytokine Family Members IL‐1F6 and IL‐1F8 on Adipocyte Differentiation

Obesity is characterized by chronic low‐grade inflammation originating from expanding adipose tissue. In the present study, we examined the adipogenic expression levels of IL‐1F6 and IL‐1F8, both members of the IL‐1 family of cytokines, and their effects on adipose tissue gene expression. Although IL‐1F6 is primarily present in adipose tissue resident macrophages and induced by inflammation, IL‐1F8 is absent. IL‐1F6, but not IL‐1F8, reduces adipocyte differentiation, as shown by a significant decrease in PPARγ gene expression. Finally, both IL‐1F6 and IL‐1F8 are able to induce inflammatory gene expression in mature adipocytes. In conclusion, we demonstrate for the first time that IL‐1F6 is present in adipose tissue and that IL‐1F6 and IL‐1F8 are involved in the regulation of adipose tissue gene expression. Importantly, IL‐1F6 inhibits PPARγ expression which may lead to reduced adipocyte differentiation suggesting metabolic effects of this cytokine.     

Macrophage-induced expression and release of matrix metalloproteinase 1 and 3 by human preadipocytes is mediated by IL-1β via activation of MAPK signaling

Obesity is associated with a chronic low‐grade inflammation and increased macrophage infiltration in adipose tissue. Matrix metalloproteinases (MMPs) are involved in adipose tissue remodeling and inflammatory responses in obesity. This study investigated whether macrophage‐derived factors modulate expression and secretion of MMP1 and MMP3 in human preadipocytes. The potential mediators and signaling pathways were also explored. MMP1 and MMP3 were primarily expressed and secreted by preadipocytes and dramatically reduced post‐differentiation. Preadipocytes were incubated with RPMI 1640 medium (control) or THP‐1 macrophage‐conditioned (MC) medium (25% and 100%) for 24 h. MC medium markedly increased mRNA levels of MMP1 (up to 122‐fold) and MMP3 (up to 59‐fold), as well as protein release of MMP1 (up to 378‐fold) and MMP3 (up to 10‐fold) in a dose‐dependent manner. Treatment with IL‐1β or TNFα, the major products of macrophages, also induced MMP1 and MMP3 secretion by preadipocytes. Neutralizing IL‐1β abolished the induction of MMP1 and MMP3 in preadipocytes by MC medium while the effects of TNFα neutralization were modest. Furthermore, MC medium or IL‐1β led to the phosphorylation of p38, ERK and JNK MAPKs. Inhibition of p38, ERK and JNK reversed the stimulatory effects of MC or IL‐1β on MMP1 and MMP3 production. MC medium and IL‐1β also activated NF‐κB p65 whereas reduced IκBα protein expression in preadipocytes. These results suggest that macrophage accumulation in adipose tissue has a central role in stimulating MMP1 and MMP3 production by preadipocytes, and this is partially mediated by IL‐1β via activation of the MAPK and NF‐κB signaling pathways. J. Cell. Physiol. 226: 2869–2880, 2011. © 2011 Wiley‐Liss, Inc.     

Calcium and 1,25-dihydroxyvitamin D3 regulation of adipokine expression

Objective: Obesity is associated with elevated oxidative stress and low‐grade systemic inflammation. We have demonstrated recently that 1α,25‐(OH)₂‐D₃ promotes reactive oxygen species production in cultured adipocytes, whereas suppression of 1α,25‐(OH)₂‐D₃ by increasing dietary calcium down‐regulates diet‐induced oxidative stress in aP2‐agouti transgenic mice. However, whether the anti‐obesity effect of dietary calcium plays a role in regulation of obesity‐associated inflammation is not clear. Research Methods and Procedures: We investigated the role of dietary calcium in the regulation of inflammatory cytokine production in aP2‐agouti transgenic mice fed low‐ and high‐calcium obesigenic diets and in the modulation of cytokine production by 1α,25‐(OH)₂‐D₃ in cultured murine and human adipocytes. Results: The high‐calcium diet inhibited the expression of pro‐inflammatory factors tumor necrosis factor α and interleukin (IL)‐6 by 64% and 51%, respectively (p < 0.001), in visceral fat, stimulated the expression of the anti‐inflammatory factors IL‐15 and adiponectin by 52% (p = 0.001) and 54% (p = 0.025), respectively, in visceral fat, and induced a 2‐fold increase in IL‐15 expression in soleus muscle (p = 0.01) compared with litter mate controls on a low‐calcium diet. 1α,25‐(OH)₂‐D₃ also markedly stimulated the expression of tumor necrosis factor α (p < 0.001) and IL‐6 (p = 0.016) in differentiated 3T3‐L1 adipocytes and increased IL‐6 (p = 0.004) and IL‐8 (p < 0.001) production in differentiated human adipocytes. These effects were blocked by calcium channel antagonism with nifedipine. Discussion: These data demonstrate that 1α,25‐(OH)₂‐D₃ favors inflammatory cytokine expression and inhibits anti‐inflammatory cytokine expression; accordingly, suppression of 1α,25‐(OH)₂‐D₃ by dietary calcium inhibits adipocyte‐derived inflammation associated with obesity.     

Adipose stromal cell and sarpogrelate orchestrate the recovery of inflammation‐induced angiogenesis in aged hindlimb ischemic mice

Aging population displays a much higher risk of peripheral arterial disease (PAD) possibly due to the higher susceptibility, poor prognosis, and fewer therapeutic options. This study was designed to examine the impact of combined multipotent adipose‐derived stromal cells (mADSCs) and sarpogrelate treatment on aging hindlimb ischemia and the mechanism of action involved. mADSCs (1.0 × 10⁷) constitutively expressing enhanced green fluorescent protein (eGFP) or firefly luciferase (Fluc) reporter were engrafted into the hindlimb of aged Vegfr2‐luc transgenic or FVB/N mice subjected to unilateral femoral artery occlusion, followed by a further administration of sarpogrelate. Multimodality molecular imaging was employed to noninvasively evaluate mADSCs' survival and therapeutic efficacy against aging hindlimb ischemia. Aged Tg(Vegfr2‐luc) mice exhibited decreased inflammatory response, and downregulation of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor‐2 (VEGFR2) compared with young ones following hindlimb ischemia induction, resulting in angiogenesis insufficiency and decompensation for ischemia recovery. Engrafted mADSCs augmented inflammation‐induced angiogenesis to yield pro‐angiogenic/anti‐apoptotic effects partly via the VEGF/VEGFR2/mTOR/STAT3 pathway. Nonetheless, mADSCs displayed limited survival and efficacy following transplantation. Sarpogrelate treatment with mADSCs further upregulated mammalian target of rapamycin (mTOR)/STAT3 signal and modulated pro‐/anti‐inflammatory markers including IL‐1β/TNF‐α/IFN‐γ and IL‐6/IL‐10, which ultimately facilitated mADSCs' survival and therapeutic benefit in vivo. Sarpogrelate prevented mADSCs from hypoxia/reoxygenation‐induced cell death via a mTOR/STAT3‐dependent pathway in vitro. This study demonstrated a role of in vivo kinetics of VEGFR2 as a biomarker to evaluate cell‐derived therapeutic angiogenesis in aging. mADSCs and sarpogrelate synergistically restored impaired angiogenesis and inflammation modulatory capacity in aged hindlimb ischemic mice, indicating its therapeutic promise for PAD in the elderly.     

Reversal of Inflammation‐Induced Impairment of Glucose Uptake in Adipocytes by Direct Effect of CB1 Antagonism on Adipose Tissue Macrophages

Macrophage infiltration into adipose tissue (AT‐MP) is thought to induce insulin resistance and diabetes in obesity. Here, we investigated the effect of the antiobesity drug SR141716 (a CB1 antagonist) on macrophage‐mediated inhibition of insulin signaling in adipocytes. THP1 macrophages (THP1) were stimulated in vitro with lipopolysaccharide (LPS) and SR141716 or vehicle. The resulting conditioned medium (CM) was analyzed and incubated on human adipocytes. CM from LPS‐stimulated THP1 inhibited insulin‐induced AKT phosphorylation in adipocytes, in contrast to CM from nonactivated THP1. Moreover, it contained higher concentrations of tumor necrosis factor‐α (TNFα) and lower levels of the anti‐inflammatory cytokine IL‐10. SR141716 reduced TNFα production and increased IL‐10 secretion, resulting in a rescue of insulin signaling in adipocytes. To confirm these findings in vivo, AT‐MP CM from cafeteria diet‐fed or Zucker diabetic fatty (ZDF) rats that had received SR141716 for 3 weeks were isolated, analyzed, and incubated with adipocytes. Cafeteria diet induced macrophage‐mediated inhibition of insulin signaling in adipocytes. Interestingly, SR141716 rescued insulin‐induced glucose uptake in adipocytes. Finally, AT‐MP CM from obese ZDF rats inhibited insulin‐stimulated glucose uptake in adipocytes in contrast to AT‐MP CM from lean ZDF rats. After treatment with SR141716, AT‐MP CM rescued insulin‐induced glucose uptake in adipocytes. In summary, our data indicate that CB1 receptor antagonism in macrophages modified their cytokine production and improved the insulin responsiveness of adipocytes that had been incubated with macrophage CM. Thus, SR141716 ameliorated adipose tissue insulin resistance by direct action on AT‐MP demonstrating a novel peripheral mode of action of CB1 antagonism.     

Macrophages modulate the viability and growth of human mesenchymal stem cells

Following myocardial infarction, tissue repair is mediated by the recruitment of monocytes and their subsequent differentiation into macrophages. Recent findings have revealed the dynamic changes in the presence of polarized macrophages with pro‐inflammatory (M1) and anti‐inflammatory (M2) properties during the early (acute) and late (chronic) stages of cardiac ischemia. Mesenchymal stem cells (MSCs) delivered into the injured myocardium as reparative cells are subjected to the effects of polarized macrophages and the inflammatory milieu. The present study investigated how cytokines and polarized macrophages associated with pro‐inflammatory (M1) and anti‐inflammatory (M2) responses affect the survival of MSCs. Human MSCs were studied using an in vitro platform with individual and combined M1 and M2 cytokines: IL‐1β, IL‐6, TNF‐α, and IFN‐γ (for M1), and IL‐10, TGF‐β1, TGF‐β3, and VEGF (for M2). In addition, polarization molecules (M1: LPS and IFN‐γ; M2: IL‐4 and IL‐13) and common chemokines (SDF‐1 and MCP‐1) found during inflammation were also studied. Indirect and direct co‐cultures were conducted using M1 and M2 polarized human THP‐1 monocytes. M2 macrophages and their associated cytokines supported the growth of hMSCs, while M1 macrophages and their associated cytokines inhibited the growth of hMSCs in vitro under certain conditions. These data imply that an anti‐inflammatory (M2) environment is more accommodating to the therapeutic hMSCs than a pro‐inflammatory (M1) environment at specific concentrations. J. Cell. Biochem. 114: 220–229, 2012. © 2012 Wiley Periodicals, Inc.