The purpose of this study was to investigate the gene expression of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) in circulating mononuclear cells harvested from septic shock patients on drotrecogin-α activated (DAA) in order to determine whether this treatment has any effect on the inflammation phase.
We conducted a prospective cohort study in two intensive care departments. Blood samples were collected at inclusion (T1) and 36 hours later (T2) to measure plasma cytokines and the changes in intracellular TNF-α, IL-10 and IFN-γ mRNA expressions using the real-time quantitative polymerase chain reaction (RT-qPCR). Thirty-two septic shock patients were included: 16 with DAA at 24 μg/kg/h for 96 hours (DAA+) and 16 control (DAA-) eligible but contraindicated for DAA because of low platelet count.
The basal characteristics were similar in both groups: mortality (50%), plasma cytokine concentrations, and baseline IFN-γ, TNF-α and IL-10 mRNA expressions (DAA+ vs. DAA-). At T2, there was a significant IFN-γ gene down-regulation in DAA+ but not in DAA- patients (-0.34 (-0.62; +1.54) vs. +1.41 (+0.35; +5.87), P = 0.008). In survivors, DAA administration was associated with a down-expression of both IFN-γ (-0.65 (-0.93; 0.48) vs. +0.7 (-0.04; +1.26), P = 0.01) and IL-10 (-0.78 (-0.92; -0.6) vs. -0.18 (-0.68; +0.46), P = 0.038). In the non-survivors, DAA infusion was associated with IL-10 over-expression when compared with survivors (+0.54 (-0.35; +11.52) vs. -0.78 (-0.92; -0.6), P < 0.001).
In this study, lack of IL-10 gene down-expression despite a 36-hour infusion of DAA is an ominous sign in septic shock patients suggesting that DAA is not able to reverse the outcome. Our results suggest that DAA can decrease the expression of anti-inflammatory cytokines in septic shock patients. IL-10 or IFN-γ gene down-expression could represent markers of DAA response.
Sepsis is defined as an invasion of microorganisms and/or their toxins into the blood associated the reaction of the organism to this invasion. Severe sepsis is a major cost driver in intensive care medicine. In Germany, prevalence data was assessed in the context of the German Prevalence Study. Severe sepsis has a prevalence of 35% in German intensive care units.
The following questions were analysed: is Drotrecogin alfa (activated) (DAA) effective in the treatment of patients with severe sepsis and a mixed risk of death, both in all patients and in different subgroups? Is DAA effective in the treatment of patients with severe sepsis and low risk of death? Is DAA cost effective in the treatment of patients with severe sepsis compared to placebo?
Only studies with adult patients are included. There are no other exclusion criteria. A systematic literature search is performed by the German Institute of Medical Documentation and Information (DIMDI). The literature search yielded as a total of 847 hits. After screening of the abstracts, 165 medical and 101 economic publications were chosen for full text appraisal.
Therapy with DAA appears to be cost effective in reducing 28-day-mortality in patients with severe sepsis and a high risk of death. A high risk of death is indicated by the presence of multiorgan failure (≥2) and/or an APACHE-II-Score ≥25. Therapy with DAA is not associated with a long-term reduction of mortality at later follow-up assessments. Therapy with DAA is not associated with a long-term reduction of mortality at later follow-up assessments. Therapy with DAA is cost-effective in patients with multiorgan failure and/or an APACHE II Score (≥25). In patients with a lower risk of death, DAA is not cost-effective. Costs associated with bleeding events have been rarely included in cost calculations.
DAA appears to reduce mortality in patients with severe sepsis and a high risk of death, but not in patients with a low risk of death. Bleeding events and mortality are considerable higher in studies in the usual care setting compared to clinical trials. In a number of subgroup analyses, both retrospectively and prospectively performed, DAA was not significantly associated with improved survival. The role of concurrent therapy with heparin is unclear, as DAA was only effective in reducing mortality in patients without heparin. There was no significant long-term survival benefit associated with DAA beyond the initial 28 days. Also, there is a lack of studies assessing prospectively functional ability, health-related quality of life, and morbidity in the long-term. In the subgroup of patients with a high risk of death, therapy with DAA ranges at the top level of generally accepted costs per LYG or QALY, in the subgroup of patients with low risk of death, cost effectiveness ratios were higher than those accepted for resource allocation.
Due to the lack of effectiveness of DAA in patients with severe sepsis and a low risk of death as well as with regard to the high bleeding rates in the usual care setting, indication for DAA therapy. In those subgroups with no significant survival benefit, prospective studies with adequate sample size are needed. With regard to the heterogeneity of severe sepsis, comorbidity and concurrent medication have to be taken into account in further studies. Studies with alternative study designs, for example studies comparing heparin alone or in combination with DAA to placebo, as well as studies conducted by different researchers are needed. Costs induced by bleeding events should also be taken into account in future studies, as bleeding events are the major complica-tion associated the DAA therapy.
Wound healing impairment is a well-documented phenomenon in clinical and experimental diabetes, and in diabetic wound healing impaired fibroblast has been linked to increased levels of tumor necrosis factor-α (TNF-α). A number of signaling pathways including TNF-α/forkhead box O1 (FOXO1) and transforming growth factor-β1 (TGF-β1)/Smads in fibroblasts appear to play a cardinal role in diabetic wound healing. Dehydroabietic acid (DAA) is obtained from Commiphora oppbalsamum and inhibits the production of TNF-α in macrophages and adipocytes, decreases the level of TNF-α in obese diabetic KK-Ay mice, but its effect on diabetic wound healing is unknown. This study was to investigate the effect of DAA on TNF-α-stimulated human adult dermal fibroblasts. On the one hand, TNF-α significantly decreased the fibroblast proliferation and the expression of PCNA, Ki67 and cyclin D1, increased the fibroblast apoptosis, caspase-8/3 activity, expressions of cleaved caspase-8 and caspase-3, decreased the Bcl-2/Bax ratio and increased activation of the pro-apoptotic transcription factor FOXO1. All the above-mentioned cell responses were remarkably reversed by DAA. On the other hand, TNF-α also inhibited TGF-β1-induced the Smad3 signaling pathway what is closely related to the fibroblast migration and the differentiation of myofibroblasts. However, DAA significantly promoted the migration and increased the expression of α-smooth muscle actin and fibronectin under the stimulus of a combination of TNF-α and TGF-β1. In conclusion, DAA could reverse several cell responses stimulated by TNF-α, including the activation of FOXO1 and the TGF-β1/Smad3 signaling pathway. These results suggested that DAA could be useful in improving the diabetic wound healing.
Dehydroabietic acid; tumor necrosis factor-α; transforming growth factor-β1; forkhead box o1; diabetes; wound healing
There are no published data on the status of endogenous activated protein C (APC) in pulmonary embolism (PE), and no data on the effect of drotrecogin alfa (activated) (DAA) given in addition to therapeutic dose enoxaparin.
In this double-blind clinical trial, 47 patients with computed tomography (CT)-confirmed acute submassive PE treated with 1 mg/kg body weight of enoxaparin twice daily were randomized to groups receiving a 12-hour intravenous infusion of 6, 12, 18, or 24 μg/kg/hour of DAA or a placebo. Blood samples were drawn before starting DAA infusion, after 4, 8 and 12 hours (at the end of the infusion period), and on treatment days 2, 3, 4, 5 and 6.
Initial endogenous plasma activated protein C (APC) levels were 0.36 ± 0.48 ng/ml (<0.10 to 1.72 ng/ml) and remained in the same range in the placebo group. APC levels in patients treated with DAA were 13.67 ± 3.57 ng/ml, 32.71 ± 8.76 ng/ml, 36.13 ± 7.60 ng/ml, and 51.79 ± 15.84 ng/ml in patients treated with 6, 12, 18, and 24 μg/kg/hour DAA, respectively. In patients with a D-dimer level >4 mg/L indicating a high level of acute fibrin formation and dissolution, DAA infusion resulted in a more rapid drop in soluble fibrin, D-dimer, and fibrinogen/fibrin degradation products (FDP) levels, compared to enoxaparin alone. There was a parallel decline of soluble fibrin, D-dimer, FDP, and plasmin-plasmin inhibitor complex (PPIC) in response to treatment with enoxaparin ± DAA, with no evidence of a systemic profibrinolytic effect of the treatment.
In patients with acute submassive PE endogenous APC levels are low. DAA infusion enhances the inhibition of fibrin formation.
A genomic biomarker identifying patients likely to benefit from drotrecogin alfa (activated) (DAA) may be clinically useful as a companion diagnostic. This trial was designed to validate biomarkers (improved response polymorphisms (IRPs)). Each IRP (A and B) contains two single nucleotide polymorphisms that were associated with a differential DAA treatment effect.
DAA is typically given to younger patients with greater disease severity; therefore, a well-matched control group is critical to this multicenter, retrospective, controlled, outcome-blinded, genotype-blinded trial. Within each center, DAA-treated patients will be matched to controls treated within 24 months of each other taking into account age, APACHE II, cardiovascular, respiratory, renal, and hematologic dysfunction, mechanical ventilation status, medical/surgical status, and infection site. A propensity score will estimate the probability that a patient would have received DAA given their baseline characteristics. Two-phase data transfer will ensure unbiased selection of matched controls. The first transfer will be for eligibility and matching data and the second transfer for outcomes and genotypic data. The primary analysis will compare the effect of DAA in IRP + and IRP − groups on in-hospital mortality through day 28.
A design-based approach matching DAA-free to DAA-treated patients in a multicenter study of patients who have severe sepsis and high risk of death will directly compare control to DAA-treated groups for mortality by genotype. Results, which should be available in 2012, may help to identify the group of patients who would benefit from DAA and may provide a model for future investigation of sepsis therapies.
Drotrecogin alfa (activated); Pharmacogenomics biomarker; Predictive marker; Propensity score; Severe sepsis; Treatment selection; Sepsis; Drotrecogin alfa activated (DAA); Activated protein C; Genome wide association study; Survival
Drotrecogin alpha activated (DAA), trade name Xigris, is a recombinant human protein C that has been the subject of controversy since 2001 when it became the first biologic agent approved for the treatment of severe sepsis and septic shock. The PROWESS trial showed a 6.1% absolute reduction in 28-day mortality although these findings were not replicated in later trials, ultimately leading to the withdrawal of DAA in 2011. Observational trials, however, have consistently shown a mortality benefit with the use of DAA, leading to the questions, did DAA truly fail and if so, why? While these questions may never be definitively answered based on available evidence, several factors may explain the conflicting results.. In clinical practice, DAA may have been preferentially given to subjects more likely to survive. Contemporary treatments including early antibiotic administration and volume resuscitation may have mitigated the inflammatory processes leading to disordered coagulation and microvascular thrombosis and thus reduced or abolished the therapeutic opportunity for DAA. Later randomized clinical trials of DAA focused on the clinical phenotype of refractory shock largely due to a strong efficacy signal in this subset from PROWESS; however, this clinical phenotype may not be tightly linked, at least after contemporary early resuscitation strategies, to the mechanistic phenotype of dysregulated coagulation that may have been a better target for DAA. Future trials of biologic therapies in severe sepsis and septic shock should use a combination of clinical phenotype and biomarkers to identify responsive populations that may benefit from such therapies.
Sepsis; septic shock; Systemic inflammatory response syndrome; Drotrecogin alfa activated; Protein C
Leptin-deficient ob/ob mice exhibit adipocyte hypertrophy and hyperplasia as well as elevated adipose tissue and systemic inflammation. Multipotent stem cells isolated from adult adipose tissue can differentiate into adipocytes ex vivo and thereby contribute toward increased adipocyte cell numbers, obesity, and inflamm ation. Currently, information is lacking regarding regulation of adipose stem cell numbers as well as leptin-induced inflammation and its signaling pathway in ob/ob mice.
Using leptin deficient ob/ob mice, we investigated whether leptin injection into ob/ob mice increases adipose stem cell numbers and adipose tissue inflammatory marker MCP-1 mRNA and secretion levels. We also determined leptin mediated signaling pathways in the adipose stem cells.
We report here that adipose stem cell number is significantly increased following leptin injection in ob/ob mice and with treatment of isolated stem cells with leptin in vitro. Leptin also up-regulated MCP-1 secretion in a dose- and time-dependent manner. We further showed that increased MCP-1 mRNA levels were due to increased phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) Ser727 but not STAT3 Tyr705 phosphorylation, suggesting differential regulation of MCP-1 gene expression under basal and leptin-stimulated conditions in adipose stem cells.
Taken together, these studies demonstrate that leptin increases adipose stem cell number and differentially activates STAT3 protein resulting in up-regulation of MCP-1 gene expression. Further studies of mechanisms mediating adipose stem cell hyperplasia and leptin signaling in obesity are warranted and may help identify novel anti-obesity target strategies.
Obesity; Adipose stem cell; Leptin
Significant debate continues over the efficacy of drotrecogin alpha activated (DAA) in sepsis. This updated meta-analysis provides an updated summary effect estimate and explores the reasons for outcome heterogeneity in placebo-controlled randomized clinical trials of DAA on 28-day all-cause mortality in patients with severe sepsis or septic shock.
Computer searches of MEDLINE, EMBASE, the Cochrane Library, ClinicalTrials.gov, published abstracts from major intensive care meetings and examination of reference lists were used to identify five placebo-controlled randomized clinical trials with 7,260 patients. The primary endpoint was 28-day all-cause mortality. Secondary outcomes were 28-day incidence of severe bleeding and intracranial hemorrhage.
DAA was not associated with improved 28-day all-cause mortality in patients with severe sepsis or septic shock (pooled relative risk (RR) of 0.97 [95% CI 0.83-1.14]), and is associated with an increase in serious bleeding. The significant heterogeneity in the pooled RR for 28-day mortality (I2 value of 59.4%, χ2 p-value 0.043) is no longer present with exclusion of the post-study amendment portion of PROWESS (I2 value of 0%, χ2 p-value 0.44 without PROWESS post-amendment). Using meta-regression, the best ranked predictor of outcome heterogeneity was baseline mortality in the placebo arm, which was among the highest in PROWESS.
DAA is not associated with improved survival in patients with severe sepsis or septic shock. Further studies should be done to determine whether changes in supportive therapy for sepsis explain the variable efficacy of DAA in randomized controlled clinical trials observed over time.
Severe sepsis; septic shock; Drotrecogin alfa (activated); meta-analysis
Unstable carotid plaques cause cerebral emboli. Leptin promotes atherosclerosis and vessel wall remodeling. We hypothesized that carotid atherosclerotic lesion instability is associated with local leptin synthesis.
Methods and Results
Carotid endarterectomy plaques from symptomatic (n=40) and asymptomatic patients with progressive stenosis (n=38) were analyzed for local expression of leptin, tumor necrosis factor (TNF)-α, and plasminogen activator inhibitor type 1. All lesions exhibited advanced atherosclerosis inclusive of thick- and thin-cap fibroatheromas or lesion rupture. Symptomatic lesions exhibited more plaque ruptures and macrophage infiltration (P=0.001 and P=0.05, respectively). Symptomatic plaques showed preferential leptin, TNF-α, and plasminogen activator inhibitor type 1 transcript (P=0.03, P=0.04, and P=0.05, respectively). Leptin mRNA and antigen in macrophages and smooth muscle cells were confirmed by in situ hybridization and immunohistochemistry. Plasma leptin levels were not significantly different between groups (P=1.0), whereas TNF-α was significantly increased in symptomatic patients (P=0.006). Human aortic smooth muscle cell culture stimulated by TNF-α, lipopolysaccharide, or lipoteichoic acid revealed 6-, 6.7-, and 6-fold increased secreted leptin antigen, respectively, at 72 hours (P<0.05).
Neurologically symptomatic patients overexpress leptin mRNA and synthesize leptin protein in carotid plaque macrophages and smooth muscle cells. Local leptin induction, presumably by TNF-α, could exert paracrine or autocrine effects, thereby contributing to the pathogenesis of lesion instability.
Clinical Trial Registration
URL: www.Clinicaltrials.gov. Unique identifier: NCT00449306.
atherosclerosis; leptin; macrophages; smooth muscle cells; tumor necrosis factor-α
Severely septic patients continue to experience excessive morbidity and mortality despite recent advances in critical care. Although significant resources have been invested in new treatments, almost all have failed to improve outcomes. An improved understanding of sepsis pathophysiology, including the complex interactions between inflammatory, coagulation, and fibrinolytic systems, has accelerated the development of novel treatments. Recombinant human activated protein C (rhAPC), or drotrecogin alfa (activated) (DAA), is currently the only US Food and Drug Administration (FDA)-approved medicine for the treatment of severe sepsis, and only in patients with a high risk of death. This review will discuss the treatment of severe sepsis, focusing on recent discoveries and unresolved questions about DAA's optimal use. Increasing pharmacological experience has generated enthusiasm for investigating medicines already approved for other indications as treatments for severe sepsis. Replacement doses of hydrocortisone and vasopressin may reduce mortality and improve hypotension, respectively, in a subgroup of patients with catecholamine-refractory septic shock. In addition to discussing these new indications, this review will detail the provocative preliminary data from four promising treatments, including two novel modalities: antagonizing high mobility group box protein and inhibiting tissue factor (TF). Observational data from the uncontrolled administration of heparin or statins in septic patients will also be reviewed.
septic shock; drotrecogin alfa; vasopressin; new therapies; statins; high mobility group box protein
AIM: To investigate the expression level and effects of leptin in human hepatocellular carcinoma cells in vitro and to explore the correlation between them.
METHODS: Human hepatocellular carcinoma cell line HepG2 was cultured in vitro, and (the expression level) mRNA of leptin and leptin receptors in HepG2 were assessed using reverse transcription polymerase chain reaction (RT-PCR). Effects of different concentrations of leptin (50 ng/mL, 100 ng/mL, 200 ng/mL) on HepG2 were detected with colorimetric assay by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) after incubation periods of 24 h, 48 h, and 72 h. Flow cytometry was performed to assess cell cycle progression of different concentrations of leptin as stated above after each 24 h incubation period.
RESULTS: mRNA of leptin and leptin receptors (including short and long isoforms) were expressed in HepG2. The 72 h incubation of leptin at different concentrations (50 ng/mL, 100 ng/mL, 200 ng/mL) promoted proliferation of HepG2 in a concentration- and time-dependent manner. The experimental group shows significant statistical differences when compared to the controlled group which contained 0 ng/mL of leptin. As the concentration of leptin increases, significant fewer cells were detected in G0-G1 phase and more cells in S and G2-M phases.
CONCLUSION: Leptin and leptin receptor are simultaneously expressed in human hepatocellular carcinoma cell line HepG2. Addition of leptin (0 ng/mL-200 ng/mL) in 72 h periods indicated there is a concentration- and time-dependent correlation in the stimulation of HepG2 cell proliferation. The effect of proliferation by leptin is due to promotion of DNA synthesis and enhancement of mitotic activity. The relationship between leptin and human hepatocellular carcinoma cells might indicate that adipokine could be associated with the progression of human hepatocellular carcinoma.
Leptin; Hepatocellular carcinoma; Proliferation
Adipokines, e.g. TNFα, IL-6 and leptin increase insulin resistance, and consequent hyperinsulinaemia influences breast cancer progression. Beside its mitogenic effects, insulin may influence adipokine production from adipocyte stromal cells and paracrine enhancement of breast cancer cell growth. In contrast, adiponectin, another adipokine is protective against breast cancer cell proliferation and insulin resistance.
AMP-activated protein kinase (AMPK) activity has been found decreased in visceral adipose tissue of insulin-resistant patients. Lipopolysaccharides (LPS) link systemic inflammation to high fat diet-induced insulin resistance. Modulation of LPS-induced adipokine production by metformin and AMPK activation might represent an alternative way to treat both, insulin resistance and breast cancer.
Human preadipocytes obtained from surgical biopsies were expanded and differentiated in vitro into adipocytes, and incubated with siRNA targeting AMPKalpha1 (72 h), LPS (24 h, 100 μg/ml) and/or metformin (24 h, 1 mM) followed by mRNA extraction and analyses. Additionally, the supernatant of preadipocytes or derived-adipocytes in culture for 24 h was used as conditioned media to evaluate MCF-7 breast cancer cell proliferation.
Conditioned media from preadipocyte-derived adipocytes, but not from undifferentiated preadipocytes, increased MCF-7 cell proliferation (p < 0.01). Induction of IL-6 mRNA by LPS was reduced by metformin (p < 0.01), while the LPS-induced mRNA expression of the naturally occurring anti-inflammatory cytokine interleukin 1 receptor antagonist was increased (p < 0.01). Silencing of AMPKalpha1 enhanced LPS-induced IL-6 and IL-8 mRNA expression (p < 0.05).
Adipocyte-secreted factors enhance breast cancer cell proliferation, while AMPK and metformin improve the LPS-induced adipokine imbalance. Possibly, AMPK activation may provide a new way not only to improve the obesity-related adipokine profile and insulin resistance, but also to prevent obesity-related breast cancer development and progression.
Leptin and adiponectin (APN) are adipokines produced by adipocytes that participate in the modulation of immune and inflammatory responses. In Crohn's disease (CD), fat wrapping surrounding the inflamed intestine produces high levels of leptin and APN. In inflammatory bowel disease (IBD), apoptosis resistance of lamina propria T lymphocytes (LPL-T) is one of the mechanisms that maintains chronic inflammation. We addressed the mechanism by which leptin and APN regulate inflammation and apoptosis in IBD.
Immune cell infiltration, several factors expressed by adipose tissue (AT), and spontaneous release of cytokines by adipocytes were measured. The presence of APN and leptin in intestinal mucosa was detected and their effect on LPL-T apoptosis, signal transducer and activator of transcription 3 (STAT3), Suppressor of Cytokine Signaling 3 (SOCS3), Bcl-2 and Bcl-xL expression, and cytokine production was studied. In addition, the effects of globular and high-molecular-weight (HMW) APN on LPL-T cytokine production and apoptosis were studied.
Higher levels of several chemokines, cytokines, and growth factors were present in AT near active than near inactive disease. A significantly higher amount of inflammatory infiltrate was present in AT near active CD than near ulcerative colitis, controls, and near the inactive area of CD. There were no changes in the ratios of APN molecular weight in control and IBD adipocyte products. Leptin and APN inhibited anti-CD3-stimulated-LPL-T apoptosis and potentiated STAT3 phosphorylation, Bcl-2, and Bcl-xL expression in IBD and control mucosa. However, SOCS3 expression was suppressed only in IBD. Both globular and HMW APN have similar effects on LPL-T cytokine production and apoptosis. Leptin and APN enhanced interleukin (IL)-10 production by anti-CD3-stimulated LPL-T in IBD only. APN, but not leptin, increased anti-CD3-induced IL-6 levels in LPL-T only in IBD patients. IL-10 exerts its anti-inflammatory activity in the presence of SOCS3 suppression by leptin or APN.
Leptin and APN maintain the inhibition of anti-CD3-stimulated LPL-T apoptosis by enhancing Bcl-2 and Bcl-xL overexpression and promoting STAT3 phosphorylation while suppressing SOCS3.
Bone remodelling and increased subchondral densification are important in osteoarthritis (OA). Modifications of bone vascularization parameters, which lead to ischemic episodes associated with hypoxic conditions, have been suspected in OA. Among several factors potentially involved, leptin and dickkopf-related protein 2 (DKK2) are good candidates because they are upregulated in OA osteoblasts (Obs). Therefore, in the present study, we investigated the hypothesis that hypoxia may drive the expression of leptin and DKK2 in OA Obs.
Obs from the sclerotic portion of OA tibial plateaus were cultured under either 20% or 2% oxygen tension in the presence or not of 50 nM 1,25-dihydroxyvitamin D3 (VitD3). The expression of leptin, osteocalcin, DKK2, hypoxia-inducible factor 1α (Hif-1α) and Hif-2α was measured by real-time polymerase chain reaction and leptin production was measured by enzyme-linked immunosorbent assay (ELISA). The expression of Hif-1α, Hif-2α, leptin and DKK2 was reduced using silencing RNAs (siRNAs). The signalling pathway of hypoxia-induced leptin was investigated by Western blot analysis and with mitogen-activated protein kinase (MAPK) inhibitors.
The expression of leptin and DKK2 in Obs was stimulated 7-fold and 1.8-fold, respectively (P <0.05) under hypoxia. Interestingly, whereas VitD3 stimulated leptin and DKK2 expression 2- and 4.2-fold, respectively, under normoxia, it stimulated their expression by 28- and 6.2-fold, respectively, under hypoxia (P <0.05). The hypoxia-induced leptin production was confirmed by ELISA, particularly in the presence of VitD3 (P <0.02). Compared to Obs incubated in the presence of scramble siRNAs, siHif-2α inhibited VitD3-stimulated leptin mRNA and protein levels by 70% (P =0.004) and 60% (P <0.02), respectively, whereas it failed to significantly alter the expression of DKK2. siHif-1α has no effect on these genes. Immunoblot analysis showed that VitD3 greatly stabilized Hif-2α under hypoxic conditions. The increase in leptin expression under hypoxia was also regulated, by p38 MAPK (P <0.03) and phosphoinositide 3-kinase (P <0.05). We found that the expression of leptin and DKK2 were not related to each other under hypoxia.
Hypoxic conditions via Hif-2 regulation trigger Obs to produce leptin, particularly under VitD3 stimulation, whereas DKK2 is regulated mainly by VitD3 rather than hypoxia.
Intermittent hypoxia (IH), resulted from recurring episodes of upper airway obstruction, is the hallmark feature and the most important pathophysiologic pathway of obstructive sleep apnea (OSA). IH is believed to be the most important factor causing systemic inflammation. Studies suggest that insulin resistance (IR) is positively associated with OSA. In this study, we hypothesized that the recurrence of IH might result in cellular and systemic inflammation, which was manifested through the levels of proinflammatory cytokines and adipokines after IH exposure, and because IR is linked with inflammation tightly, this inflammatory situation may implicate an IR status.
We developed an IH 3T3-L1 adipocyte and rat model respectively, recapitulating the nocturnal oxygen profile in OSA. In IH cells, nuclear factor kappa B (NF-κB) DNA binding reactions, hypoxia-inducible factor-1α (HIF-1α), glucose transporter-1 (Glut-1), necrosis factor alpha (TNF-α), interleukin (IL) -6, leptin, adiponectin mRNA transcriptional activities and protein expressions were measured. In IH rats, blood glucose, insulin, TNF-α, IL-6, leptin and adiponectin levels were analyzed.
The insulin and blood glucose levels in rats and NF-κB DNA binding activities in cells had significantly statistical results described as severe IH>moderate IH>mild IH>sustained hypoxia>control. The mRNA and protein levels of HIF-1α and Glut-1 in severe IH group were the highest. In cellular and animal models, both the mRNA and protein levels of TNF-α, IL-6 and leptin were the highest in severe IH group, when the lowest in severe IH group for adiponectin.
Oxidative stress and the release of pro-inflammatory cytokines/adipokines, which are the systemic inflammatory markers, are associated with IH closely and are proportional to the severity of IH. Because IR and glucose intolerance are linked with inflammation tightly, our results may implicate the clinical relationships between OSA and IR.
Increasing evidence support the regulatory role of leptin in osteoarthritis (OA). As high circulating concentrations of leptin disrupt the physiological function of the adipokine in obese individuals, the current study has been undertaken to determine whether the elevated levels of leptin found in the joint from obese OA patients also induce changes in the chondrocyte response to leptin.
Chondrocytes isolated from OA patients with various body mass index (BMI) were treated with 20, 100 or 500 ng/ml of leptin. The expression of cartilage-specific components (aggrecan, type 2 collagen), as well as regulatory (IGF-1, TGFβ, MMP-13, TIMP 2) or inflammatory (COX-2, iNOS, IL-1) factors was investigated by real-time PCR to evaluate chondrocyte responsiveness to leptin. Furthermore, the effect of body mass index (BMI) on leptin signalling pathways was analyzed with an enzyme-linked immunosorbent assay for STATs activation.
Leptin at 20 ng/ml was unable to modulate gene expression in chondrocytes, except for MMP-13 in obese OA patients. Higher leptin levels induced the expression of IGF-1, type 2 collagen, TIMP-2 and MMP-13. However, the activity of the adipokine was shown to be critically dependent on both the concentration and the BMI of the patients with a negative association between the activation of regulated genes and BMI for 100 ng/ml of adipokine, but a positive association between chondrocyte responsiveness and BMI for the highest leptin dose. In addition, the gene encoding MMP-13 was identified as a target of leptin for chondrocytes originated from obese patients while mRNA level of TIMP-2 was increased in leptin-treated chondrocytes collected from normal or overweight patients. The adipokine at 500 ng/ml triggered signal transduction through a STAT-dependent pathway while 100 ng/ml of leptin failed to activate STAT 3 but induced STAT 1α phosphorylation in chondrocytes obtained from obese patients.
The current study clearly showed that characteristics of OA patients and more expecially obesity may affect the responsiveness of cultured chondrocytes to leptin. In addition, the BMI-dependent effect of leptin for the expression of TIMP-2 and MMP-13 may explain why obesity is associated with an increased risk for OA.
BACKGROUND—Leptin regulates feeding behaviour and therefore may be a mediator of anorexia associated with acute and chronic inflammation. Recently, leptin mRNA and leptin protein were found in the gastric epithelium.
AIM—The aim of the present study was to examine the effect of Helicobacter pylori infection on gastric leptin expression to investigate the pathophysiological role of gastric leptin.
METHODS—Surgically resected human stomach tissues were subjected to immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) to check for the presence of leptin in the human gastric epithelium. A total of 201 H pylori positive patients with chronic gastritis underwent eradication therapy for H pylori and were examined for the effect of infection cure in terms of body mass index (BMI) and serum leptin levels. Biopsy specimens from the gastric fundic mucosa were obtained from 40 of the 201 patients before and three months after eradication therapy. These samples were subjected to quantitative RT-PCR to examine the effect of eradication therapy on leptin expression in the gastric fundic mucosa.
RESULTS—Leptin immunoreactive cells were detected in the lower half of the gastric fundic glands and a leptin PCR product was also found in the gastric fundic mucosa. H pylori infection significantly increased gastric leptin expression. In addition, cure of H pylori infection significantly reduced gastric leptin expression, with a concomitant increase in BMI. In contrast, serum leptin levels did not change significantly after cure of H pylori infection.
CONCLUSION—Leptin is present in the human gastric mucosa. Gastric leptin may play a role in weight gain after eradication of H pylori infection. Gastric leptin may have a local rather than systemic action.
Keywords: Helicobacter pylori; leptin; gastric mucosa; body mass index; gastritis
The effect of hypoxia, induced by incubation under low (1%) oxygen tension or by exposure to CoCl2, on the expression and secretion of inflammation-related adipokines was examined in human adipocytes. Hypoxia led to a rapid and substantial increase (greater than sevenfold by 4 h of exposure to 1% O2) in the hypoxia-sensitive transcription factor, HIF-1α, in human adipocytes. This was accompanied by a major increase (up to 14-fold) in GLUT1 transporter mRNA level. Hypoxia (1% O2 or CoCl2) led to a reduction (up to threefold over 24 h) in adiponectin and haptoglobin mRNA levels; adiponectin secretion also decreased. No changes were observed in TNFα expression. In contrast, hypoxia resulted in substantial increases in FIAF/angiopoietin-like protein 4, IL-6, leptin, MIF, PAI-1 and vascular endothelial growth factor (VEGF) mRNA levels. The largest increases were with FIAF (maximum 210-fold), leptin (maximum 29-fold) and VEGF (maximum 23-fold); these were reversed on return to normoxia. The secretion of IL-6, leptin, MIF and VEGF from the adipocytes was also stimulated by exposure to 1% O2. These results demonstrate that hypoxia induces extensive changes in human adipocytes in the expression and release of inflammation-related adipokines. Hypoxia may underlie the development of the inflammatory response in adipocytes, leading to obesity-associated diseases.
Adipokines; Adipose tissue; Hypoxia; Inflammation; Obesity; Metabolic syndrome
Emerging evidence has suggested a critical role of leptin in hepatic inflammation and fibrogenesis, however, the precise mechanisms underlying the profibrogenic action of leptin in the liver has not been well elucidated. Therefore, the present study was designed to investigate the expression and functions of leptin receptors (Ob-R) in hepatic sinusoidal cells. Hepatic stellate cells (HSCs), Kupffer cells and sinusoidal endothelial cells (SECs) were isolated from rat livers by in situ collagenase perfusion followed by differential centrifugation technique, and expression of Ob-Ra and Ob-Rb, short and long Ob-R isoforms, respectively, were analyzed by RT-PCR. Ob-Ra mRNA was detected ubiquitously in HSCs and SECs. In contrast, Ob-Rb was detected clearly only in SECs and Kupffer cells, but not in 7-day cultured HSCs. Indeed, tyrosine-phosphorylation of STAT-3, a downstream event of Ob-Rb signaling, was observed in SECs, but not in HSCs, 1 hr after incubation with leptin. Further, leptin increased AP-1 DNA binding activity and TGF-beta 1 mRNA levels in Kupffer cells and SECs, whereas leptin failed to increase TGF-beta 1 mRNA in HSCs. These findings indicated that SECs and Kupffer cells, but not HSCs, express functional leptin receptors, through which leptin elicits production of TGF-beta 1. It is hypothesized therefore that leptin, produced systemically from adipocytes and locally from HSCs, up-regulates TGF-beta 1 thereby facilitate tissue repairing and fibrogenesis in the sinusoidal microenvironment.
Lipofibroblasts of the developing rat lung resemble adipocytes and produce soluble growth factors which stimulate neighboring alveolar type II epithelial cell differentiation. The mature adipocyte secretes leptin, a cytokine with an expanding array of functions. Leptin is expressed by the developing rat lung beginning on E17–E18, increasing by 7–10 fold by E20(term= E22). Leptin is expressed by fetal lung fibroblasts of both rat and human (WI38) origins; the leptin receptor is expressed by fetal rat lung type II cells and by human adult lung epithelial cells (H441). Expression of leptin by mesenchymal cells and its cognate receptor by epithelial cells of the developing alveolus is consistent with a paracrine signaling loop, as demonstrated by our experimental data: (1) leptin stimulates the de novo synthesis of surfactant phospholipid by both fetal rat type II cells (400%/100 ng/ml/24h) and by adult human airway epithelial H441 cells (85%/100ng/24h) in culture; (2) leptin is secreted by the mature (E21) lipofibroblast in cell culture in amounts which significantly stimulate type II cell surfactant phospholipid synthesis in vitro, and leptin secretion is stimulated by PTHrP; (3) epithelial cell secretions such as parathyroid hormone-related protein (PTHrP) and prostaglandin E2, and dexamethasone all significantly stimulate leptin mRNA expression by fetal lung fibroblasts; (4) treatment of E18 fetal rat lung explants with PTHrP or leptin stimulates the de novo synthesis of surfactant phospholipid (2–2.5-fold/24h) and up-regulates the expression of Surfactant Protein-B(>25-fold/24h), an effect which is blocked by leptin antibody; (5) PTHrP receptor antagonist blocks the expression of leptin mRNA by explants, but does not inhibit stimulation of surfactant phosphospholipid or SP-B expression, indicating that fibroblast-dependent PTHrP stimulation of type II cell maturation requires leptin expression by lipofibroblasts. This is the first demonstration of a paracrine loop mediated by endogenously produced growth factors of both epithelial and mesenchymal origins cooperatively interacting to induce alveolar acinar lung development.
lung development; surfactant; type II cell; lipofibroblast; leptin
Leptin, a hormone produced mainly by adipose tissue, regulates food intake and energy expenditure. It is involved in inflammatory diseases such as chronic obstructive pulmonary disease (COPD) and its deficiency is associated with increased susceptibility to the infection. The leptin receptor is expressed in the lung and in the neutrophils.
We measured the levels of leptin, tumor necrosis factor alpha (TNF-α) and soluble form of intercellular adhesion molecule-1 (sICAM-1) in sputum and plasma from 27 smoker and former smoker patients with stable COPD using ELISA methods. Further we analyzed leptin and its receptor expression in sputum cells from 16 COPD patients using immunocytochemistry.
In plasma of COPD patients, leptin was inversely correlated with TNF-α and positively correlated with the patient weight, whereas the levels of sICAM-1 were positively correlated with TNF-α. In sputum of COPD patients leptin levels were correlated with forced expiratory volume in 1 second/forced vitality capacity. Additionally, increased levels of sputum leptin and TNF-α were observed in COPD former smokers rather than smokers. Further the expression of leptin receptor in sputum neutrophils was significantly higher in COPD former smokers than in smokers, and the expression of leptin and its receptor was positively correlated in neutrophils of COPD former smokers.
Our findings suggest a role of leptin in the local and systemic inflammation of COPD and, taking into account the involvement of neutrophils in this inflammatory disease, describe a novel aspect of the leptin/leptin receptor pathway in the regulation of host defense after smoking cessation.
COPD; smokers; inflammation; leptin; neutrophils
In addition to regulating body weight, leptin is also recognized for its role in the regulation of immune function and inflammation. The purpose of this study was to investigate the effect of leptin on Prevotella (P.) intermedia lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in differentiated THP-1 cells, a human monocytic cell line.
LPS from P. intermedia ATCC 25611 was prepared by the standard hot phenol-water method. THP-1 cells were incubated in the medium supplemented with phorbol myristate acetate to induce differentiation into macrophage-like cells. The amount of TNF-α and interleukin-8 secreted into the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). TNF-α and Ob-R mRNA expression levels were determined by semi-quantitative reverse transcription-polymerase chain reaction analysis.
Leptin enhanced P. intermedia LPS-induced TNF-α production in a dose-dependent manner. Leptin modulated P. intermedia LPS-induced TNF-α expression predominantly at the transcriptional level. Effect of leptin on P. intermedia LPS-induced TNF-α production was not mediated by the leptin receptor.
The ability of leptin to enhance P. intermedia LPS-induced TNF-α production may be important in the establishment of chronic lesion accompanied by osseous tissue destruction observed in inflammatory periodontal disease.
Leptin; Lipopolysaccharide; Prevotella intermedia; Tumor necrosis factor-α
Emerging evidence suggests that angiogenic and pro-inflammatory cytokine leptin might be implicated in ocular neovascularization. However, the potential of inhibiting leptin function in ophthalmic cells has never been explored. Here we assessed mitogenic, angiogenic, and signaling leptin activities in retinal and corneal endothelial cells and examined the capability of a specific leptin receptor (ObR) antagonist, Allo-aca, to inhibit these functions.
Methods and Results
The experiments were carried out in monkey retinal (RF/6A) and bovine corneal (BCE) endothelial cells. Leptin at 50-250 ng/mL stimulated the growth of both cell lines in a dose-dependent manner. The maximal mitogenic response (35±7 and 27±3% in RF6A and BCE cells, respectively) was noted at 24 h of 250 ng/mL leptin treatments. Leptin-dependent proliferation was reduced to base levels with 10 and 100 nM Allo-aca in BCE and RF6A cells, respectively. In both cell lines, leptin promoted angiogenic responses, with the maximal increase in tube formation (163±10 and 133±8% in RF6A and BCE cultures, respectively) observed under a 250 ng/mL leptin treatment for 3 h. Furthermore, in both cell lines 250 ng/mL leptin modulated the activity or expression of several signaling molecules involved in proliferation, inflammatory activity and angiogenesis, such as STAT3, Akt, and ERK1/2, COX2, and NFκB. In both cell lines, leptin-induced angiogenic and signaling responses were significantly inhibited with 100 nM Allo-aca. We also found that leptin increased its own mRNA and protein expression in both cell lines, and this autocrine effect was abolished by 100-250 nM Allo-aca.
Our data provide new insights into the role of leptin in ocular endothelial cells and represent the first original report on targeting ObR in ophthalmic cell models.
The effect of smoking on leptin regulation is controversial. Smoking may induce low-grade inflammation. Recent series of studies indicated the critical role of macrophage migration in the establishment of adipose tissue inflammation. In this study, we aimed to see the effects of smoking and inflammation on leptin regulation both at cellular and epidemiological levels.
We compared the concentration of inflammatory markers and serum leptin levels among Japanese male subjects. Additionally, leptin and intercellular adhesion molecule (ICAM) -1 gene expression was assessed in adipocytes co-cultured with or without macrophages in the presence or absence of nicotine and/or lipopolysaccharide (LPS).
In subjects with BMI below 25 kg/m2, both WBC counts and soluble-ICAM-1 levels are significantly higher in smokers than in non-smokers. However, leptin concentration did not differ according to smoking status. However, in subjects with BMI over 25 kg/m2, smokers exhibited significantly lower serum leptin level as well as higher WBC counts and s-ICAM-1 concentration as compared with non-smokers. Leptin gene expression was markedly suppressed in adipocytes co-cultured with macrophages than in adipocyte culture alone. Furthermore, nicotine further suppressed leptin gene expression. ICAM-1 gene expression was markedly up-regulated in adipocytes co-cultured with macrophages when stimulated with LPS.
Adipose tissue inflammation appears to down-regulate leptin expression in adipose tissues. Nicotine further suppresses leptin expression. Thus, both smoking and inflammation may diminish leptin effect in obese subjects. Therefore, obese, but not normal weight, smokers might be more resistant to weight loss than non-smokers.
Leptin; Smoking; Low-grade inflammation; Nicotine; ICAM-1
Elevated circulating leptin is present in human heart failure, and leptin deficiency is linked to worse outcomes in chronic ischemic injury. In the present observational study, we tested the hypothesis that cardiac leptin production and signaling are increased in the failing human heart, and that mechanical unloading with a ventricular assist device (VAD) reverses these changes.
Methods and Results
All studies were performed using human cardiac tissue obtained from 1) hearts not matched for transplantation (non-failing), 2) at time of cardiac transplant (failing), or 3) paired samples at the time of VAD implant (pre-VAD) and removal (post-VAD). Expression of brain naturetic peptide, leptin, leptin receptor and tumor necrosis factor-alpha (TNFα) mRNA was measured, and the protein expression of leptin and its receptor was examined by Western blot and immunofluorescent staining of cardiac sections. Assessment of leptin signaling was performed by measuring the phosphorylation state of the leptin receptor. The phosphorylation state of signal transducer and activator of transcription (STAT)-3 and AMP-activated kinase (AMPK) proteins were also measured. All data are expressed as mean ± stand error of the mean with statistical significance in failing relative to non-failing groups determined by student's independent t-test, and significance between pre- and post-VAD groups determined by paired t-test. In failing human hearts, the mRNA expression of leptin and its receptor were increased 5.4±0.3 (p<0.05) and 4.5±0.3 (p<0.05) fold, respectively, with similar changes in protein. The phosphorylation state of the leptin receptor and STAT-3 proteins were both increased 1.4±0.1 fold (p<0.05), and the level of phosphorylated AMPK protein was increased 1.9±0.2 fold (p<0.05). Mechanical unloading of the failing human heart with a VAD resulted in no change in TNFα expression, but a marked decrease in leptin production to 1.7±0.1% (p<0.05), and leptin receptor expression to 3.0±0.2% (p<0.05), of pre-VAD levels. Phosphorylation of the leptin receptor, STAT-3 and AMPK were also decreased to 45±7%, 75±8%, and 58±8 % of pre-VAD values, respectively (all p<0.05).
These results indicate that the failing human heart increases expression of leptin and its receptor, and that mechanical unloading downregulates this increase. Further, a cardioprotective role for leptin in the failing human heart is suggested through the activation of STAT-3 and AMPK signaling.
leptin; leptin receptor; ventricular assist device; AMP kinase; heart failure