Monique Verdier

The inflammatory receptor CD40 is expressed on human adipocytes: contribution to crosstalk between lymphocytes and adipocytes.

Aims/hypothesisObesity is associated with adipose tissue inflammation. The CD40 molecule, TNF receptor superfamily member 5 (CD40)/CD40 ligand (CD40L) pathway plays a role in the onset and maintenance of the inflammatory reaction, but has not been studied in human adipose tissue. Our aim was to examine CD40 expression by human adipocytes and its participation in adipose tissue inflammation.MethodsCD40 expression was investigated in human whole adipose tissue and during adipocyte differentiation by real-time PCR, Western blot and immunohistochemistry. The CD40/CD40L pathway was studied using recombinant CD40L (rCD40L) in adipocyte culture and neutralising antibodies in lymphocyte/adipocyte co-culture.ResultsCD40 mRNA levels in subcutaneous adipose tissue were higher in the adipocyte than in the stromal–vascular fraction. CD40 expression was upregulated during adipocyte differentiation. Addition of rCD40L to adipocytes induced mitogen activated protein kinase (MAPK) activation, stimulated inflammatory adipocytokine production, and decreased insulin-induced glucose transport in parallel with a downregulation of IRS1 and GLUT4 (also known as SCL2A4). rCD40L decreased the expression of lipogenic genes and increased lipolysis. CD40 mRNA levels were significantly higher in subcutaneous adipose tissue than in visceral adipose tissue of obese patients and were positively correlated with BMI, and with IL6 and leptin mRNA levels. Lymphocyte/adipocyte co-culture led to an upregulation of proinflammatory adipocytokines and a downregulation of leptin and adiponectin. Physical separation of the two cell types attenuated these effects, suggesting the involvement of a cell–cell contact. Blocking the CD40/CD40L interaction with neutralising antibodies reduced IL-6 secretion from adipocytes.Conclusions/interpretationAdipocyte CD40 may contribute to obesity-related inflammation and insulin resistance. T lymphocytes regulate adipocytokine production through both the release of soluble factor(s) and heterotypic contact with adipocytes involving CD40.

Chronic plasminogen activator inhibitor-1 (PAI-1) overexpression dampens CD25+ lymphocyte recruitment after lipopolysaccharide endotoxemia in mouse lung

Summary.  Background: Plasma plasminogen activator inhibitor‐1 (PAI‐1) level rises during sepsis and confers a worse prognosis. PAI‐1 participation to sepsis has been poorly documented and was mainly associated with fibrin deposits. Beside fibrin deposits, increased tissue PAI‐1 expression may contribute to the poor outcome of endotoxemia through other mechanisms. Objective and methods: During lipopolysaccharide (LPS) challenge, the role of PAI‐1 in the early phase of inflammation was examined in the lungs of transgenic mice that either overexpress or lack the PAI‐1 gene (PAI‐1Tg or PAI‐1−/−). Results: Analysis of leukocytes revealed that neutrophil and macrophage infiltrations did not differ for PAI‐1Tg and wild‐type (WT) mice. Remarkably, CD25+ lymphocyte infiltration was totally blunted in PAI‐1Tg lungs and inversely correlated with fibrin depositions. In parallel, mRNA levels of the regulatory T cell (Treg) markers FoxP3, CTLA‐4, and GITR were significantly lower in PAI‐1Tg than in WT lungs after LPS challenge. These data are supported by opposite results in PAI‐1−/− lungs. The systemic compartments (spleen and peripheral blood) showed no decrease in CD25+, CD4+ CD25+ lymphocytes, and Treg markers in PAI‐1Tg mice after LPS injection compared with WT mice. In addition, plasma and lung concentrations of interleukin‐6 (IL‐6) and macrophage inflammatory protein‐1α (MIP‐1α) were significantly higher in PAI‐1Tg mice than WT mice. Conclusion: Our results suggest that chronic tissue PAI‐1 overexpression influences the early phase of the inflammatory response during endotoxemia through the control of T lymphocyte traffic.

The Plasminogen Activation System Modulates Differently Adipogenesis and Myogenesis of Embryonic Stem Cells

Regulation of the extracellular matrix (ECM) plays an important functional role either in physiological or pathological conditions. The plasminogen activation (PA) system, comprising the uPA and tPA proteases and their inhibitor PAI-1, is one of the main suppliers of extracellular proteolytic activity contributing to tissue remodeling. Although its function in development is well documented, its precise role in mouse embryonic stem cell (ESC) differentiation in vitro is unknown. We found that the PA system components are expressed at very low levels in undifferentiated ESCs and that upon differentiation uPA activity is detected mainly transiently, whereas tPA activity and PAI-1 protein are maximum in well differentiated cells. Adipocyte formation by ESCs is inhibited by amiloride treatment, a specific uPA inhibitor. Likewise, ESCs expressing ectopic PAI-1 under the control of an inducible expression system display reduced adipogenic capacities after induction of the gene. Furthermore, the adipogenic differentiation capacities of PAI-1−/− induced pluripotent stem cells (iPSCs) are augmented as compared to wt iPSCs. Our results demonstrate that the control of ESC adipogenesis by the PA system correspond to different successive steps from undifferentiated to well differentiated ESCs. Similarly, skeletal myogenesis is decreased by uPA inhibition or PAI-1 overexpression during the terminal step of differentiation. However, interfering with uPA during days 0 to 3 of the differentiation process augments ESC myotube formation. Neither neurogenesis, cardiomyogenesis, endothelial cell nor smooth muscle formation are affected by amiloride or PAI-1 induction. Our results show that the PA system is capable to specifically modulate adipogenesis and skeletal myogenesis of ESCs by successive different molecular mechanisms.

Polymorphism A36G of the tumor necrosis factor receptor 1 gene is associated with PAI-1 levels in obese women

The tumor necrosis factor (TNF) pathway may be implicated in etiopathogenesis of PAI-1 overexpression during obesity. The aim of this study was to investigate the influence of polymorphism A36G of the TNF receptor 1 (TNFRSF1A +36A/G) on plasma concentrations of PAI-1 in 163 obese (31 with the metabolic syndrome, MetS) and 150 lean, healthy women. Genotypic and allele frequencies did not significantly differ between obese and lean subjects. TNFRSF1A genotypes were significantly associated with sTNFR1 plasma levels in obese women only (p < 0.01); TNFRSF1A +36G/G obese carriers exhibited higher sTNFR1 and PAI-1 levels than A carriers (p < 0.01 and p < 0.05, respectively). In obese women, the presence of the MetS significantly potentiated the elevation of sTNFR1 and PAI-1 levels observed in the TNFRSF1A + 36G/G carriers. Our results suggest that association between TNFRSF1A +36G/G genotype and the MetS renders obese women more prone to activation of the TNF pathway reflected by high circulating sTNFR1 and PAI-1 levels.

CD28 deletion improves obesity-induced liver steatosis but increases adiposity in mice

Background/objectives:Lymphocytes have a critical role in visceral adipose tissue (AT) inflammation. The CD28 costimulatory molecule is required for lymphocyte activation and for the development of a functional regulatory T cells (Tregs) compartment; however, its role during obesity is unknown.Methods:During diet-induced obesity, we investigated the effects of selective interference with CD28 signaling using knockout mice (Cd28KO) and a CTLA4-Ig fusion protein inhibiting CD28-B7 interactions.Results:Cd28 deficiency decreased pathogenic T cells and Treg content within AT without changing the macrophages number. Cd28KO epididymal but not subcutaneous fat was characterized by enlarged adipocytes, reduced levels of inflammatory cytokines and increased Glut4, adiponectin and lipogenic enzyme mRNA levels. This was associated with reduced inflammation, fat accumulation and enhanced glucose metabolism in liver. Weight gain and fasting glucose tolerance were not affected. CTLA4-Ig injections reduced the number of T cells in epididymal AT (epiAT) but not the inflammatory cytokines levels and failed to improve liver fat accumulation.Conclusions:Deletion of CD28 creates a new pro/anti-inflammatory balance in epiAT and liver and exerts a protective effect against hepatic steatosis.

Early matrix metalloproteinase-9 concentration in the first 48 h after aneurysmal subarachnoid haemorrhage predicts delayed cerebral ischaemia: An observational study.

BACKGROUND Delayed cerebral ischaemia from vasospasm is an important cause of complications and death after aneurysmal subarachnoid haemorrhage. There is currently no established biomarker for identifying patients at high risk of delayed cerebral ischaemia. OBJECTIVE Considering the important role of inflammation in the pathogenesis of delayed cerebral ischaemia, we investigated whether matrix metalloproteinase-9 (MMP-9) may be an efficient biomarker for predicting elayed cerebral ischaemia after subarachnoid haemorrhage. DESIGN Single-centre prospective observational study. SETTING Neuroscience Critical Care Unit of a teaching hospital. PARTICIPANTS Thirty consecutive patients with severe subarachnoid haemorrhage requiring external ventricular drainage were enrolled during 2013 and 2014. INTERVENTIONS Blood and cerebrospinal fluid (CSF) were sampled within the first 24 h and between 48 and 72 h after admission. We evaluated the activity and concentrations of MMP-9 and endothelin-1 with zymography and ELISA. Patients were allocated to groups with delayed cerebral ischaemia (n = 16) or without delayed cerebral ischaemia (n = 14). RESULTS Within 24 h, median [interquartile range] MMP-9 concentrations in CSF were significantly higher in patients with delayed cerebral ischaemia (47 [21 to 102] ng ml−1) than in those without delayed cerebral ischaemia (4 [2 to 13] ng ml−1, P = 0.001). CSF MMP-9 activity and endothelin-1 concentrations were correlated (r = 0.6, P = 0.02). The areas under the receiver operating characteristic curves were 0.73 (95% confidence interval [0.53 to 0.87]) and 0.91 (95% confidence interval [0.75 to 0.98]) for MMP-9 concentrations in plasma and CSF, respectively, at 24 h to predict delayed cerebral ischaemia CSF MMP-9 concentrations more than 14.3 ng ml−1 at 24 h predicted the occurrence of delayed cerebral ischaemia with a sensitivity and specificity of 88 and 86%, respectively. After multivariate logistic analysis, only CSF MMP-9 concentrations at 24 h predicted the occurrence of delayed cerebral ischaemia (P = 0.01). CONCLUSION MMP-9 concentrations in both plasma and CSF, measured within 48 h after subarachnoid haemorrhage, were highly predictive of the occurrence of delayed cerebral ischaemia within the first 2 weeks. TRIAL REGISTRATION Clinicaltrials.gov identifier: NCT02397759.

Immediate Postnatal Overfeeding in Rats Programs Aortic Wall Structure Alterations and Metalloproteinases Dysregulation in Adulthood.

BACKGROUND Alterations in the nutritional perinatal environment, such as intrauterine growth retardation with subsequent postnatal catch-up growth, program cardiovascular disease in adulthood, possibly through alterations in matrix metalloproteinase (MMP)-2 and -9. However, experimental evidences demonstrating that changes in the nutritional perinatal environment can program MMP-2 and -9 with subsequent alterations of vessel wall are lacking. AIM The current study evaluated whether immediate postnatal overfeeding is able to alter vascular morphological indexes and circulating and/or vascular MMP2-2 and -9 status. METHODS Aortic morphology (wall thickness and percentage of incomplete elastin lamellae) and circulating and aortic MMP-2 and -9 activity (measured by gelatin zymography) and aortic MMP-2 and -9 mRNA (measured by reverse transcription polymerase chain reaction (RT-PCR)) were studied in adult male rats overfed (OF) or normofed (NF) during the immediate postnatal period. RESULTS Postnatal overfeeding induced early onset obesity. Adult OF rats presented with increased blood pressure and circulating MMP-2 and -9 activity. In the thoracic aorta, postnatal overfeeding increased wall thickness and decreased elastin integrity (as demonstrated by an increased percentage of incomplete elastin lamellae). OF rats showed enhanced aortic MMP-2 activity and MMP-9 mRNA levels. Circulating and aortic MMP-2 activity correlated positively with the percentage of incomplete elastin lamellae and aortic wall thickness, respectively. CONCLUSION Our data demonstrate for the first time that immediate postnatal nutritional programming induces increases in circulating and aortic MMP-2 activity with parallel aortic wall alterations, such as decreased elastin integrity and enhanced thickening, showing that this experimental model is suitable for the study of perinatal nutritional programming of vascular functions.

OP6: Endothelium-independent vascular reactivity in high-fat diet-fed rats: role of vascular wall and perivascular adipose tissue oxidative stress

Background and aims visceral obesity is a risk factor for cardiovascular diseases. Perivascular adipose tissue (PVAT) is a visceral fat depot close to the vessel wall which therefore could directly influence vascular reactivity. Diet-induced alterations in endothelium-dependent vascular reactivity have been well investigated. Diet-induced changes in endothelium-independent vascular reactivity have received less attention. Material and Methods we studied in 5-month-old rats fed low-fat (LFD) or high-fat diet (HFD) from weaning vascular reactivity using phenylephrine (PE)-stimulated isolated endothelium-removed aortic rings without or with their PVAT (PVAT - or PVAT +, respectively), Results HFD induced an increase in mesenteric and PVAT fat mass, and enhanced systemic and mesenteric AT Tbars while it decreased aortic and PVAT Tbars. When PVAT - rings were incubated with increasing doses of PE, maximal contraction and EC50 were not different between groups. The presence of PVAT decreased the maximal contraction to the same extent in rings obtained from LFD- or HFD-fed rats. Incubation with catalase suppressed the anticontractile properties of PVAT, indicating that this effect was mediated through H2O2. Transfer experiments of PVAT conditioned medium demonstrated that PVAT was the site of H2O2 synthesis. In LFD PVAT- rings, oxidative stress-associated procontractile activity was generated by xanthine oxidase and cytochrome c oxidase. In HFD PVAT- rings oxidative stress-associated procontractile activity increased and was generated, in addition to the above-mentioned enzymes, by NADPH oxidase. Catalase-induced H2O2 dismutation had prorelaxing properties, comparable in rings obtained from LFD or HFD fed rats. Superoxide dismutase (SOD) was devoid of effect in LFD rings, whereas it had a prorelaxing activity in HFD rings. These observations suggest that, in addition to H2O2, O2.- has a procontractile activity in the aortic wall of HFD rats. In aorta HFD increased the mRNAs coding for NADPH oxidase (p47 phox, p67phox, and NOX4), xanthine oxidase, SOD and catalase, suggesting an increased dismutation activity. PVAT modified the effect of the various drugs tested. The increased anticontractile activity of PVAT found after reactive oxygen species scavenging was reduced in HFD fed rats compared with LFD fed animals. Inhibition of O2.- dismutation resulting from DETC-induced SOD blocking led to a procontractile effect of PVAT in both groups, this effect being reduced in HFD compared to LFD rats. NADPH blockade did not affect the anticontractile properties of PVAT in both groups. Xanthine oxidase blockade amplified the anticontractile effect of PVAT in LFD animals while it reversed this effect in HFD rats. In rings obtained from LFD animals cytochrome c oxidase blockade reversed the anticontractile effect of PVAT in LFD animals whereas it was devoid of effect in HFD rats. In PVAT, HFD increased the mRNAs coding for cytochrome c oxidase, glutathione peroxidase and UCP-1 and -3. Conclusion our data show that HFD-induced obesity was associated with NADPH-dependent increased oxidative stress-associated procontractile activity in the aortic wall. Such phenomenon was counteracted by enhanced dismutation activity in the aortic wall and decreased procontractile activity provided by the PVAT. As a consequence, these observations predict that any defect in the above-mentioned counterregulatory mechanisms can have deleterious functional consequences.