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Objective A potential strategy to treat obesity – and the associated metabolic consequences – is to increase energy expenditure. This could be achieved by stimulating thermogenesis through activation of brown adipose tissue (BAT) and/or the induction of browning of white adipose tissue (WAT). Over the last years, it has become clear that several metalloproteinases play an important role in adipocyte biology. Here, we investigated the potential role of ADAMTS5. Methods Mice deficient in ADAMTS5 (Adamts5−/−) and wild-type (Adamts5+/+) littermates were kept on a standard of Western-type diet for 15 weeks. Energy expenditure and heat production was followed by indirect calorimetry. To activate thermogenesis, mice were treated with the β3-adrenergic receptor (β3-AR) agonist CL-316,243 or alternatively, exposed to cold for 2 weeks. Results Compared to Adamts5+/+ mice, Adamts5−/− mice have significantly more interscapular BAT and marked browning of their subcutaneous (SC) WAT. Thermogenic pathway analysis indicated, in the absence of ADAMTS5, enhanced β3-AR signaling via activation of the cAMP response element-binding protein (CREB). Additional β3-AR stimulation with CL-316,243 promoted browning of WAT in Adamts5+/+ mice but had no additive effect in Adamts5−/− mice. However, cold exposure induced more pronounced browning of WAT in Adamts5−/− mice. Conclusions These data indicate that ADAMTS5 plays a functional role in development of BAT and browning of WAT. Hence, selective targeting of ADAMTS5 could provide a novel therapeutic strategy for treatment/prevention of obesity and metabolic diseases., Highlights • Mice deficient in ADAMTS5 have elevated interscapular brown adipose tissue mass. • ADAMTS5 deficient mice show increased browning of their white adipose tissue. • The thermogenic profile is enhanced via adrenergic signaling and CREB activation. • ADAMTS5 seems an attractive therapeutic target for metabolic diseases.

Previous studies, predominantly based on increased or decreased plasma levels, have reported conflicting data on a potential functional role of ADAMTS13 in the pathogenesis of liver diseases, including non‑alcoholic steatohepatitis (NASH). The aim of the current study was to evaluate whether ADAMTS13 deficiency affects development of NASH. Therefore, male wild‑type (WT) and Adamts13 deficient (Adamts13‑/‑) mice were kept on a steatosis‑inducing diet devoid of methionine and choline (MCD) or a control diet (MCC) for 4 weeks. Induction of NASH did not affect plasma ADAMTS13 antigen levels of WT mice. MCD as compared with MCC feeding resulted in reduced body and liver weight with no differences between the genotypes. Plasma levels of the liver enzymes AST and ALT were significantly higher for MCD vs. MCC fed Adamts13‑/‑ and WT mice, however were not different between the genotypes. Liver triglyceride levels were also higher after MCD feeding, but were not different between WT and Adamts13‑/‑ mice. Adamts13‑/‑ mice on the two diets exhibited higher insulin sensitivity when compared with WT mice. On the MCC diet, the genotype did not show clear histological abnormalities in the liver, whereas severe steatosis and fibrosis were observed on MCD diet, however were comparable for both genotypes. This was supported by comparably enhanced hepatic expression in the two genotypes on MCD diet of the steatosis marker CD36 and of the fibrosis marker tissue inhibitor of metalloproteinase 1. Thus, the results of the current study do not support a functional role of ADAMTS13 in this murine model of NASH.

The aggrecanase ADAMTS5 (A Disintegrin and Metalloproteinase with ThromboSpondin type 1 motifs, member 5) and the cleavage of its substrate versican have been implicated in the development of heart valves. Furthermore, ADAMTS5 deficiency was shown to protect against diet-induced obesity, a known risk factor for cardiovascular disease. Therefore, in this study, we investigated the potential role of ADAMTS5 in cardiac function using ADAMTS5-deficient (Adamts5-/- ) mice and their wild-type (Adamts5+/+ ) counterparts exposed to a standard-fat or a high-fat diet (HFD). Eight-weeks-old Adamts5-/- and Adamts5+/+ mice were exposed to each diet for 15 weeks. Cardiac function and electrophysiology were analyzed by transthoracic echocardiogram and electrocardiogram at the end of the study. Cleavage of versican, as detected by the appearance of the DPEEAE neo-epitope on western blotting with protein extracts, was defective in the heart of HFD-treated Adamts5-/- as compared with Adamts5+/+ mice. ADAMTS5 deficiency led to statistically significant increases in diastolic posterior wall thickness (0.94 ± 0.023 vs. 0.82 ± 0.036 mm; P = 0.0056) and left ventricle volume (47 ± 4.5 vs. 31 ± 2.5 μL; P = 0.0043) in comparison to Adamts5+/+ mice, but only in animals on a HFD. Cardiac function parameters such as ejection fraction, fractional shortening, and stroke volume were unaffected by ADAMTS5 deficiency or diet. Electrocardiogram analysis revealed no ADAMTS5-specific changes in either diet group. Thus, in the absence of ADAMTS5, cleavage of versican in the cardiac extracellular matrix is impaired, but cardiac function, even upon exposure to a HFD, is not markedly affected. ispartof: CELL BIOLOGY INTERNATIONAL vol:43 issue:6 pages:593-604 ispartof: location:England status: published

BACKGROUND: Mice deficient in the circadian clock gene BMAL1 (Brain and Muscle ARNT-like protein-1) exhibit a hypercoagulable state and an enhanced arterial and venous thrombogenicity, which aggravates with age. We investigated the effect of BMAL1 deficiency in mice at a different age on the diurnal rhythm of factors involved in coagulation and fibrinolysis. MATERIALS AND METHODS: Hepatic, cardiac and brain tissues were isolated from 10- and 25-weeks-old Bmal1-deficient (BMAL1-/-) and wild-type (BMAL1+/+) mice at ZT2 and at ZT14 to analyze the mRNA expression level of genes involved in coagulation and fibrinolysis. RESULTS: Body weight and brain weight were significantly lower in all BMAL1-/- versus BMAL1+/+ mice. Bmal1 deficiency disturbed the diurnal rhythm of plasminogen activator inhibitor-1 (PAI-1) in liver and plasma, but not in cardiac or brain tissues. BMAL1+/+ livers showed diurnal fluctuations in factor (F)VII, FVII, protein S and anti-thrombin gene expression, which were not observed in BMAL1-/- tissues. Interestingly, tissue plasminogen activator (t-PA) expression was significantly upregulated in all BMAL1-/- versus BMAL1+/+ brains at both time points. Plasma t-PA-PAI-1 complex levels were however similar for all groups. CONCLUSION: Bmal1 deficiency affected the biphasic rhythm of coagulation and fibrinolysis factors predominantly in the liver. In the brain, Bmal1-dependent control of t-PA gene expression was documented for the first time. ispartof: EXPERIMENTAL GERONTOLOGY vol:118 pages:1-8 ispartof: location:England status: published

Obesity has become a global health-threat for every age group. It is well known that young mice (10-12 weeks of age) fed a western-type diet (WD) become obese and develop higher cholesterol levels and liver steatosis whereas insulin sensitivity is reduced. Less is known, however, about the effect of a WD on advanced-age mice. Therefore, 10 week-old (young) and 22 month-old (advanced-age), male C57BL/6JRj mice were kept on either a WD or a control diet (SFD) for 15 weeks. In contrast to young mice, advanced-age mice on WD did not show a higher body weight or adipose tissue (AT)-masses, suggesting a protection against diet-induced obesity. Furthermore, plasma adiponectin and leptin levels were not affected upon WD-feeding. A WD, however, did induce more hepatic lipid accumulation as well as increased hepatic expression of the macrophage marker F4/80, in advanced-age mice. There were no significant differences in mRNA levels of uncoupling protein-1 or F4/80 in brown AT (BAT) or of several intestinal integrity markers in colon suggesting that the protection against obesity is not due to excessive BAT or to impaired intestinal absorption of fat. Thus, advanced-age mice, in contrast to their younger counterparts, appeared to be protected against diet-induced obesity. ispartof: ADIPOCYTE vol:8 issue:1 pages:105-113 ispartof: location:United States status: published

Butein is a plant flavonoid chalcone, with presumed anti-adipogenic properties. It was reported to impair preadipocyte differentiation, limit adipose tissue (AT) development and enhance white AT browning in rodents. In this study, we investigated the hypothesis that these effects of butein may occur via reduction of ADAMTS5 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 5) expression. Murine 3T3-L1 or 3T3-F442A preadipocytes were differentiated into mature adipocytes in the presence of butein or vehicle. At regular time intervals RNA was collected for gene expression studies. Male hemizygous mice for Tg(Ucp1-luc2,-tdTomato)1Kajim (ThermoMouse) were exposed to butein or vehicle, after which ATs were analyzed for Adamts5 and uncoupling protein-1 (Ucp-1) mRNA level changes. During preadipocyte differentiation, butein (25 - 50 mM) did not affect Adamts5 or Ucp-1 expression. Oil Red O analysis and monitoring of differentiation markers failed to demonstrate effects of butein on the differentiation extent. Furthermore, butein administration to the ThermoMouse (10 or 20 mg/kg, 4 days) or to the C57BL6/Rj mice (20 mg/kg, 4 weeks) did not enhance Adamts5 or Ucp-1 expression. Thus, we could not demonstrate marked effects of butein on the preadipocyte differentiation extent or AT development and browning, nor on Adamts5 or Ucp-1 gene expression during these processes. ispartof: ADIPOCYTE vol:8 issue:1 pages:209-222 ispartof: location:United States status: published

BACKGROUND: An experimental imaging platform for longitudinal monitoring and evaluation of cardiac morphology-function changes has been long desired. We sought to establish such a platform by using a rabbit model of reperfused myocardial infarction (MI) that develops chronic left ventricle systolic dysfunction (LVSD) within 7 weeks. METHODS: Fifty-five New Zeeland white (NZW) rabbits received sham-operated or 60-min left circumflex coronary artery (LCx) ligation followed by reperfusion. Cardiac magnetic resonance imaging (cMRI), transthoracic echocardiography (echo), and blood samples were collected at baseline, in acute (48 hours or 1 week) and chronic (7 weeks) stage subsequent to MI for in vivo assessment of infarct size, cardiac morphology, LV function, and myocardial enzymes. Seven weeks post MI, animals were sacrificed and heart tissues were processed for histopathological staining. RESULTS: The success rate of surgical operation was 87.27%. The animal mortality rates were 12.7% and 3.6% both in acute and chronic stage separately. Serum levels of the myocardial enzyme cardiac Troponin T (cTnT) were significantly increased in MI rabbits as compared with sham animals after 4 hours of operation (P

Patients with heart disease have a higher risk to develop cardiac arrhythmias, either spontaneously or drug-induced. In this study, we have used a rabbit model of myocardial infarction (MI) with severe left ventricular systolic dysfunction (LVSD) to study potential drug-induced cardiac risks with N-(piperidin-2-ylmethyl)-2,5-bis(2,2,2-trifluoroethoxy)benzamide (flecainide). Upon ligation of the left circumflex arteries, male New Zealand White rabbits developed a large MI and moderate or severe LVSD 7 weeks after surgery, in comparison to SHAM-operated animals. Subsequently, animals were exposed to escalating doses of flecainide (0.25–4 mg/kg) or solvent. Electrocardiograms (ECG) were recorded before surgery, 1 and 7 weeks after surgery and continuously during the drug protocol. The ECG biomarker iCEB (index of Cardio-Electrophysiological Balance = QT/QRS ratio) was calculated. During the ECG recording at week 1 and week 7 post MI, rabbits had no spontaneous cardiac arrhythmias. When rabbits were exposed to escalating doses of flecainide, 2 out of 5 rabbits with MI and moderate LVSD versus 0 out of 5 solvent-treated rabbits developed arrhythmias, such as ventricular tachycardia/ventricular fibrillation. These were preceded by a marked decrease of iCEB just before the onset (from 4.09 to 2.42 and from 5.56 to 2.25, respectively). Furthermore, 1 out of 5 MI rabbits with moderate LVSD and 1 out of 7 MI rabbits with severe LVSD developed total atrioventricular block after flecainide infusion and died. This rabbit model of MI and severe LVSD may be useful for preclinical evaluation of drug (similar mechanism as flecainide)-induced arrhythmic risks, which might be predicted by iCEB.

Background Expansion of adipose tissue is dependent on adipogenesis, angiogenesis and extracellular matrix remodeling. A functional role in these processes was suggested for the gelatinase subfamily of the matrix metalloproteinases. Here, we have evaluated a potential role of gelatinase A (MMP-2) in adipogenesis. Methods Murine embryonic fibroblasts (MEF) were derived from wild-type or MMP-2 deficient mice. Genetic manipulation of Mmp2 (shRNA-knockdown or overexpression) was performed in 3T3-F442A preadipocytes. Cell cultures were subjected to an adipogenic medium. As an in vivo model for de novo adipogenesis, 3T3-F442A preadipocytes with or without knockdown were injected subcutaneously in Nude BALB/c mice kept on high fat diet. Results Mmp2 deficient MEF, as compared to controls, showed significantly impaired differentiation into mature adipocytes, as demonstrated by 90% reduced intracellular lipid content and reduced expression of pro-adipogenic markers. Moreover, selective Mmp2 knockdown in 3T3-F442A preadipocytes resulted in significantly reduced differentiation. In contrast, overexpression of Mmp2 resulted in markedly enhanced differentiation. In de novo formed fat pads resulting from preadipocytes with Mmp2 knockdown expression of aP2, Ppar-γ and adiponectin was significantly lower, and collagen was more preserved. The fat pad weights as well as size and density of adipocytes or blood vessels were, however, not significantly different from controls. Conclusion Our data directly support a functional role of MMP-2 in adipogenesis in vitro, and suggest a potential role in in vivo adipogenesis. General significance Selective modulation of MMP-2 levels affects adipogenesis.

Increasing energy expenditure by stimulating thermogenesis through activation of brown adipose tissue (BAT) and/or induction of browning of white adipose tissue (WAT) is considered a promising strategy to treat/prevent obesity and related metabolic diseases. Whereas WAT is adapted to store energy as triglycerides, BAT produces heat (non-shivering thermogenesis). In brown adipocytes, the uncoupling protein-1 (Ucp-1) regulates conversion of energy into heat by uncoupling ATP production from mitochondrial respiration. Also in WAT adaptive Ucp-1 positive adipocytes (brown in white: brite or beige) can arise, predominantly in subcutaneous (s) WAT. Therefore, this browning of sWAT can be visualized by makingsWAT protein extracts and byanalyzing protein levels of brown adipocyte-specific markers such as Ucp-1 and peroxisome proliferator-activated receptor gamma coactivator 1 α (Pgc1α) in theseextracts.

Obesity can be induced by exposing mice to a western-type diet high in sugar and fat for 15 weeks. Control animals receive a standard fat diet for 15 weeks.

Increasing energy expenditure by stimulating thermogenesis through activation of brown adipose tissue (BAT) and/or induction of browning of white adipose tissue (WAT) is considered a promising strategy to treat/prevent obesity and related metabolic diseases. Whereas WAT is adapted to store energy as triglycerides, BAT produces heat (non-shivering thermogenesis). In brown adipocytes, the uncoupling protein-1 (UCP-1) regulates conversion of energy into heat by uncoupling ATP production from mitochondrial respiration. Also in WAT adaptive UCP-1 positive adipocytes (brown in white: brite or beige) can arise, predominantly in subcutaneous (s) WAT. This browning of WAT is enhanced by exposure to cold.

ADAMTS5, the main aggrecanase, is a protein that seems to play a functional role in the development of brown and white adipose tissue (WAT) and browning of WAT. These published observations were made using the ADAMTS5-P mice originally generated by Pfizer, in collaboration with Lexicon Genetics (The Woodlands, TX, USA).

Processing murine tissues into paraffin sections.

Increasing energy expenditure by stimulating thermogenesis through activation of brown adipose tissue (BAT) and/or induction of browning of white adipose tissue (WAT) is considered a promising strategy to treat/prevent obesity and related metabolic diseases. Whereas WAT is adapted to store energy as triglycerides, BAT produces heat (non-shivering thermogenesis). In brown adipocytes, the uncoupling protein-1 (UCP-1) regulates conversion of energy into heat by uncoupling ATP production from mitochondrial respiration. Also in WAT adaptive UCP-1 positive adipocytes (brown in white: brite or beige) can arise, predominantly in subcutaneous (s) WAT. This browning of WAT is enhanced by exposure to cold temperatures.

Diet-induced obesity by administration of a western-type diet high in fat and sugar to mice may result in an elevation of plasma total and LDL-cholesterol levels, triglycerides and the levels of the liver enzymes alkaline phosphatases and aspartate (AST)andalanine aminotransferases (ALT).

ADAMTS5, the main aggrecanase, is a protein that seems to play a functional role in the development of brown and white adipose tissue (WAT) and browning of WAT. These observations were made using the ADAMTS5-P mice originally generated by Pfizer, in collaboration with Lexicon Genetics (The Woodlands, TX, USA). ADAMTS5-J mice were generated by Deltagen Inc. and are now commercially available through The Jackson Laboratory (Bar Harbor, USA). The targeting construct, containing an IRES-lacZ-neomycin cassette, substituted 134 nucleotides of exon 2.

Sirius Red staining is a usefull stainingto studycollagendistribution. In adipose tissue development during obesity the extracellular matrix is being remodeled and this can be analyzed using Sirius Red staining. In addition, fibrosis in livers from mice suffering from non-alcoholic steatohepatitis can be identified using this staining. Moreover, scar formation after the induction of a myocardial infarct can be illustrated using the Sirius Red staining.

Diet-induced obesity in mice through the administration of a western-type diet high in fat and sugar will be accompanied by an increase in hepatic triglyceride content.

SummaryEnhanced expression of the aggrecanase ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin type 1 motifs; member 5) has been observed in adipose tissue (AT) of obese rodents. Here, we have investigated the role of ADAMTS5 in adipogenesis, AT expansion and associated angiogenesis. In vitro differentiation of precursor cells into mature adipocytes was studied using murine embryonic fibroblasts (MEF) derived from wild-type (Adamts5 +/+) and ADAMTS5 deficient (Adamts5 -/-) mice, or 3T3-F442A preadipocytes with stable gene silencing of Adamts5. De novo adipogenesis was monitored by injection of 3T3-F442A cells with or without Adamts5 knockdown in Nude mice. Furthermore, Adamts5+/+ and Adamts5/- mice were kept on a high-fat diet (HFD) to monitor AT development. Adamts5-/- MEF, as well as 3T3-F442A preadipocytes with Adamts5 knockdown, showed significantly reduced differentiation as compared to control cells. In mice, de novo formed fat pads arising from 3T3-F442A cells with Adamts5 knockdown were significantly smaller as compared to controls. After 15 or 25 weeks on HFD, total body weight and subcutaneous AT weight were similar for Adamts5++/+ and Adamts5-/- mice, but visceral/gonadal fat mass was significantly lower for Adamts5-/-mice. These data were confirmed by magnetic resonance imaging. In addition, the blood vessel density in adipose tissue was higher for Adamts5-/- mice kept on HFD. In conclusion, our data support the concept that ADAMTS5 promotes adipogenesis in vitro and in vivo, as well as development of visceral AT and associated angiogenesis in mice kept on HFD.

Expression of thrombospondin-2 (TSP-2), a matricellular protein with anti-angiogenic properties, is modulated in developing adipose tissue. To investigate a potential functional role of TSP-2 in adipose tissue angiogenesis and growth, TSP-2 deficient (TSP-2(-/-)) and wild-type littermate (TSP-2(+/+)) mice were kept on normal chow (standard fat diet (SFD)) or on high fat diet (HFD) for 15 weeks. TSP-2(-/-) mice kept on HFD had a significantly lower total body weight throughout the experimental period. Subcutaneous (SC) and gonadal (GON) fat mass were, however, not different, and their composition in terms of size and density of adipocytes and blood vessels was also comparable in both genotypes. Macrophage infiltration in SC or GON adipose tissues was not affected by TSP-2 deficiency. TSP-2 deficiency had no effect on adipose tissue mRNA expression of gelatinase A (MMP-2), whereas gelatinase B (MMP-9) was downregulated in SC and GON adipose tissues of TSP-2(-/-) mice on HFD. Glucose tolerance and insulin resistance tests were comparable for TSP-2(+/+) and TSP-2(-/-) mice. TSP-2 deficiency was not compensated by increased expression of TSP-1 in the TSP-2(-/-) mice. These data suggest that TSP-2, despite its reported anti-angiogenic properties, does not play an important functional role in adipose tissue related angiogenesis or associated fat development in mice.

Background CD36 is a membrane glycoprotein, contributing to the pathogenesis of metabolic disorders, like obesity, which has become a major health concern worldwide. Methods A potential functional role of the scavenger receptor CD36 was investigated in in vitro adipocyte differentiation and in vivo adipogenesis. Results During differentiation of 3T3-F442A preadipocytes into mature adipocytes, expression of CD36 was upregulated and CD36 gene silencing resulted in impaired differentiation, as monitored by Oil Red O staining and expression of adipogenic markers. De novo fat pad formation in NUDE mice following injection of preadipocytes was significantly reduced upon CD36 gene silencing as compared to control. This was associated with marked adipocyte hypotrophy and reduced adipose tissue adipocyte content. Macrophage infiltration in de novo fat tissues derived from preadipocytes with CD36 gene silencing was not significantly different from controls. Collagen content was significantly higher in de novo fat with CD36 gene silencing. In a nutritionally induced obesity model, total body weight as well as subcutaneous and gonadal adipose tissue mass were significantly lower in CD36 deficient mice as compared to wild-type littermates. General significance Thus, our data support a functional role of CD36 in promoting adipogenesis in vitro as well as in vivo.

To evaluate a potential role of Axl, the high-affinity receptor of growth arrest-specific protein 6 (GAS6) in adiposity, murine embryonic fibroblasts (MEF) derived from mice with genetic deficiency of Axl (Axl(-/-)) or wild-type littermates (Axl(+/+)) were differentiated into mature adipocytes. In addition, Axl(-/-) and Axl(+/+) mice were kept on standard fat diet (SFD) or on high-fat diet (HFD) for 15 weeks. Deficiency of Axl in MEF did not affect differentiation, as shown by a similar uptake of Oil Red O and expression of the adipogenic markers aP2 and peroxisome proliferator activator receptor γ (PPARγ) at the end of the differentiation. In the first 7 weeks of HFD feeding, Axl(-/-) mice gained less weight than their wild-type littermates. Weight gain for both genotypes on either SFD of HFD over 15 weeks was, however, not significantly different, resulting in comparable body weights, as well as subcutaneous (s.c.) and gonadal (GON) fat mass. Adipocyte size in the fat tissues was not affected by Axl deficiency. Gene expression analysis indicated that the absence of Axl in vivo may be compensated for by the other TAM family members Mer and Tyro3. Glucose and insulin tolerance tests (ITT) in Axl(-/-) and Axl(+/+) mice did not reveal significant differences in glucose homeostasis. Thus, Axl deficiency had no significant effect on adipogenesis in vitro or in vivo.

1. Because the development of adipose tissue involves remodelling of the extracellular matrix (ECM), which requires matrix metalloproteinase (MMP) activity, we examined whether MMP inhibitors may have the potential to affect adipose tissue mass in obese mice. 2. Administration of the relatively gelatinase-specific MMP inhibitor tolylsam ((R)-3-methyl-2-[4-(3-p-tolyl-[1,2,4]oxadiazol-5-yl)-benzenesulphonylamino]-butyric acid; 100 mg/kg per day) for 7 weeks to obese wild-type mice on a high-fat diet resulted in significantly lower bodyweight (P < 0.05), lower subcutaneous (SC) and gonadal (GON) adipose tissue mass (both P < 0.05) and smaller adipocytes in both SC (P < 0.005) and GON (P < 0.0005) adipose tissues. 3. Magnetic resonance imaging confirmed a lower total body fat content in tolylsam-treated mice (P < 0.0005). In addition, tolylsam treatment of wild-type mice was associated with a marked enhancement in metabolic rate. 4. Electron microscopy analysis of tissue sections at the end of the 7 week feeding period revealed significantly higher collagen accumulation in the ECM of SC adipose tissues of tolylsam-treated mice (P < 0.001). 5. Thus, the relatively gelatinase-specific MMP inhibitor tolylsam has the potential to affect fat tissue growth in obese mice.

Introduction PAI-1 is the main physiological inhibitor of t-PA and u-PA. Elevated PAI-1 levels have been implicated in the pathogenesis of several thrombotic and non-thrombotic diseases. The effect of PAI-1 inhibition can be studied in mouse models, when appropriate immunological tools are available. The majority of the available monoclonal antibodies against PAI-1 have been raised against human PAI-1. Even though some of these antibodies cross-react with non-glycosylated PAI-1 from different species, these antibodies often do not cross-react sufficiently with glycosylated mouse PAI-1. Moreover, the antibodies that cross-react with glycosylated mouse PAI-1 often have decreased inhibitory properties in the presence of vitronectin. Our objective was the generation of a panel of monoclonal antibodies reacting with vitronectin-bound glycosylated mouse PAI-1. Results Five monoclonal antibodies revealed binding to glycosylated mouse PAI-1 and exerted a strong (i.e. 58-80% inhibition of PAI-1 activity) inhibitory effect toward mouse PAI-1. Similar inhibitory effects were seen in the presence of a 33-fold molar excess of vitronectin. The PAI-1 inhibitory potential of the antibodies in vivo was demonstrated in a thromboembolism model, in which the evaluated antibodies significantly increased the percentage of mice with normal physical activity in comparison to mice treated with negative control antibody. Conclusions To the best of our knowledge this is the first panel of monoclonal antibodies that can inhibit mouse PAI-1 in the presence of vitronectin and that show a profibrinolytic effect in vivo . Therefore these antibodies provide excellent immunological tools to further investigate the role of PAI-1 in mouse models.

Type 2 diabetes is causally related to obesity and characterized by dysfunctional pancreatic beta cells. It is so far unclear whether direct interactions exist between adipocytes and beta cells and possibly raise any pathogenic relevance. In this study, we examined whether 9-day co-cultured 3T3-F442A (pre)adipocytes and primary rat pancreatic beta cells exert an influence on each other's function. In the presence of beta cells, 3T3-F442A cells became lipid-storing cells expressing markers of differentiated adipocytes and releasing adiponectin. This effect was attributed to the medium insulin levels (around 0.1 μM) and was associated with an elevated glucose consumption by the 3T3-F442A cells. The subsequent decrease in medium glucose concentration reduced the rate of insulin release by beta cells cultured at 10 mM glucose, and thus suppressed their degranulation during culture. These changes in beta cell function did not occur at 20 mM glucose and were reversible upon removal of the 3T3-F422A cells. They could not be reproduced by 3T3-F422A-conditioned medium containing varying adiponectin concentrations. These data indicate that insulin secreted by beta cells is sufficient to induce differentiation of preadipocytes without addition of exogenous adipogenic factors. Over 9 days culture, (pre)adipocytes did not directly and irreversibly affect beta cell functions.

Fibrinolysis and pericellular proteolysis depend on molecular coassembly of plasminogen and its activator on cell, fibrin, or matrix surfaces. We report here the existence of a fibrinolytic cross-talk mechanism bypassing the requirement for their molecular coassembly on the same surface. First, we demonstrate that, despite impaired binding of Glu-plasminogen to the cell membrane by ϵ-aminocaproic acid (ϵ-ACA) or by a lysine-binding site–specific mAb, plasmin is unexpectedly formed by cell-associated urokinase (uPA). Second, we show that Glu-plasminogen bound to carboxy-terminal lysine residues in platelets, fibrin, or extracellular matrix components (fibronectin, laminin) is transformed into plasmin by uPA expressed on monocytes or endothelial cell–derived microparticles but not by tissue-type plasminogen activator (tPA) expressed on neurons. A 2-fold increase in plasmin formation was observed over activation on the same surface. Altogether, these data indicate that cellular uPA but not tPA expressed by distinct cells is specifically involved in the recognition of conformational changes and activation of Glu-plasminogen bound to other biologic surfaces via a lysine-dependent mechanism. This uPA-driven cross-talk mechanism generates plasmin in situ with a high efficiency, thus highlighting its potential physiologic relevance in fibrinolysis and matrix proteolysis induced by inflammatory cells or cell-derived microparticles.

SummaryThis study was performed to follow up on the observation that gelatinase A (MMP-2) deficiency impairs adipose tissue development in mice. The aim was to evaluate the role of its functional homologue gelatinase B (MMP-9) in adipose tissue growth. MMP-9 antigen levels were determined in lean and in obese women before and after weight loss. MMP-9-deficient mice and wild-type littermates (genetic background 50% 129sv : 50% CDI or 99.975% C57Bl/6, ten generations backcrossed into C57Bl/6 background) were kept on a high-fat diet (HFD) for 15 weeks. Subcutaneous and gonadal fat pads were analysed in terms of weight and size/density of adipocytes and blood vessels. Obese women had higher MMP-9 serum levels than lean controls (383 ± 29 vs. 304 ± 27 ng/ml, p = 0.02); after weight reduction MMP-9 levels dropped to 334 ± 17 ng/ml (p = 0.1 vs. obese). However, MMP-9-deficient and littermate wild-type mice kept on HFD were indistinguishable in terms of body and fat weight. No effect of MMP-9 deficiency was observed on size or density of adipocytes or blood vessels in subcutaneous or gonadal fat depots. Similar observations were made when mice were kept on normal chow. In conclusion, in lean and obese women, body mass index correlates positively with MMP-9 serum levels (p < 0.0001). However, MMP-9 does not seem to play a major role in adipose tissue development in murine models of diet-induced obesity.

Ageing is associated with an increase in visceral obesity in men and women. Although wild-type mice with a C57Bl/6 genetic background are extensively used in studies on obesity and metabolism, little information is available on age-associated changes in their adipose tissues. We have evaluated development and composition of subcutaneous (SC) and gonadal (GON) adipose tissue in male C57Bl/6 mice at the ages of 10 weeks, 12 months or 24 months, while kept on normal chow. Total body weight as well as SC and GON fat mass significantly increased between 10 weeks and 12 months, but markedly decreased again up to 24 months of age. Adipocyte size in both fat depots and blood vessel size in GON fat followed this trend. Plasma leptin levels correlated positively with body weight and SC or GON fat mass. Both 12 and 24 months old mice displayed better insulin sensitivity as compared to 10 weeks old counterparts, reflected by significantly decreased plasma levels of insulin and/or glucose. Thus, ageing of C57Bl/6 male mice is associated with a biphasic pattern (increase up to 12 months followed by a decrease up to 24 months) of body weight, SC and GON fat mass, adipocyte and blood vessel size.

International audience; We characterized the interactions between plasminogen and neurons and investigated the associated effects on extracellular matrix proteolysis, cell morphology, adhesion, signaling and survival. Upon binding of plasminogen to neurons, the plasmin formed by constitutively expressed tissue plasminogen activator (tPA) degrades extracellular matrix proteins, leading to retraction of the neuron monolayer that detaches from the matrix. This sequence of events required both interaction of plasminogen with carboxy-terminal lysine residues and the proteolytic activity of plasmin. Surprisingly, 24 h after plasminogen addition, plasmin-detached neurons survived and remained associated in clusters maintaining focal adhesion kinase phosphorylation contrasting with other adherent cell types fully dissociated by plasmin. However, long-term incubation (72 h) with plasminogen was associated with an increased rate of apoptosis, suggesting that prolonged exposure to plasmin may cause neurotoxicity. Regulation of neuronal organization and survival by plasminogen may be of pathophysiological relevance, as plasminogen is expressed in the brain and/or extravasate during vascular accidents or inflammatory processes.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a role in development of obesity by contributing to adipogenesis, angiogenesis and extracellular matrix degradation. We have evaluated a potential functional role of TIMP-1, which inhibits most MMPs, in early stages of in vitro and in vivo adipogenesis. Overexpression of human TIMP-1 (hTIMP-1) in 3T3-F442A preadipocytes resulted in a somewhat slower differentiation into mature adipocytes, without affecting the total extent of differentiation. Local overexpression by injection of 3T3-F442A preadipocytes expressing hTIMP-1 in the back of NUDE mice kept on high fat diet (HFD) for 4 weeks, had no effect on de novo formed fat pad mass. The fat pads formed from the hTIMP-1 expressing cells did show a significantly larger blood vessel size as compared to control cells (57 + or - 4.8 microm(2) vs 38 + or - 1.4 microm(2), p=0.0017). No effect was observed on blood vessel density or on adipocyte size or density. Thus, local hTIMP-1 overexpression did not significantly affect early stages of adipogenesis as evaluated from the extent of in vitro adipocyte differentiation or of in vivo de novo adipose tissue formation.

We investigated the in vitro fibrinolytic properties of microplasmin, the isolated proteinase domain of plasmin, and its thrombolytic efficacy in a coronary artery thrombosis model in dogs. The amidolytic and fibrinolytic activity of recombinant microplasmin was compared with natural human plasmin. The thrombolytic efficacy of microplasmin was studied in a canine model of copper coil induced coronary artery thrombosis. Animals were randomly assigned to one of six treatment regimens, each with five animals per cohort. Four treatment groups received an intravenous bolus of microplasmin followed by an intravenous infusion of microplasmin for 1 h (1 mg/kg + 1.5 mg/kg/h with or without abciximab or 2 mg/kg + 3 mg/kg/h). In two treatment groups, microplasmin was administered intracoronary. Bolus administration was followed by a 1-h infusion if coronary flow was incompletely restored after the initial bolus administration (1 mg/kg + 1.5 mg/kg/h or 2 mg/kg + 3 mg/kg/h, respectively). The thrombolytic efficacy was documented by repeated angiographies and the coronary perfusion was assessed with the Thrombolysis in Myocardial Infarction (TIMI) grading. No significant differences between plasmin and microplasmin were observed with respect to the catalytic efficiencies towards the synthetic chromogenic substrates S-2403 or S-2444. The concentration required for 50% lysis of purified fibrin clots in 3 h, was approximately 100 nM for microplasmin compared to 20 nM for natural plasmin. Intravenous bolus administration of microplasmin restored TIMI 3 coronary flow in 0/5, 0/5, 1/5 and 2/5, respectively, whereas intracoronary bolus administration restored TIMI 3 coronary flow in 1/5 and 4/5 (1 mg/kg and 2 mg/kg, respectively) (ANOVA P0.05). TIMI 3 coronary flow was obtained in 0/5, 2/5, 2/5 and 3/5, respectively, during subsequent intravenous administration and in 5/5 and 4/5 in case of intracoronary administration (ANOVA P0.05). When compared to natural plasmin, the catalytic efficiency of microplasmin towards chromogenic substrates was similar, but the fibrinolytic potency of microplasmin towards fibrin clots was lower. Intracoronay administration of microplasmin effectively lysed coronary thrombosis.

Tissue-type plasminogen activator (t-PA) is approved for treatment of ischemic stroke patients, but it increases the risk of intracranial bleeding (ICB). Previously, we have shown in a mouse stroke model that stromelysin-1 (matrix metalloproteinase-3 [MMP-3]) induced in endothelial cells was critical for ICB induced by t-PA. In the present study, using bEnd.3 cells, a mouse brain–derived endothelial cell line, we showed that MMP-3 was induced by both ischemic stress and t-PA treatment. This induction by t-PA was prevented by inhibition either of low-density lipoprotein receptor–related protein (LRP) or of nuclear factor-κB activation. LRP was up-regulated by ischemic stress, both in bEnd.3 cells in vitro and in endothelial cells at the ischemic damage area in the mouse stroke model. Furthermore, inhibition of LRP suppressed both MMP-3 induction in endothelial cells and the increase in ICB by t-PA treatment after stroke. These findings indicate that t-PA deteriorates ICB via MMP-3 induction in endothelial cells, which is regulated through the LRP/nuclear factor-κB pathway.

SummaryObesity and oral estrogens are independent risk factors for venous thrombosis, and their combined effect is stronger than the sum of the isolated factors. It was the objective of this study to investigate the interaction between obesity and estrogens at the level of venous thrombotic tendency, coagulation and inflammation in a mouse model.Female C57Bl/6J mice were fed a standard fat diet (SFD) or a high fat diet (HFD) to induce nutritional obesity.After 14 weeks, while maintaining their diet, mice were orally treated eight days with 1 µg ethinylestradiol or vehicle (n=25 per group), and subsequently subjected to an inferior caval vein (ICV) thrombosis model.The ICV thrombosis model resulted in an increased thrombus weight in vehicle-treated HFD mice (3.0 ± 0.7 mg) compared to vehicle-treated SFD mice (1.4 ± 0.4 mg; p=0.064). Surprisingly, estrogens reduced thrombus weight, which was significant for the HFD group (0.8 ± 0.5 mg; p=0.013).As compared to SFD feeding, HFD feeding significantly increased plasma levels of coagulation factor VIII, combined factor II/VII/X (p

SummaryThe effect of rofecoxib (Vioxx), a cyclooxygenase (COX)-2 inhibitor, on adipose tissue development was studied in a murine model of diet-induced obesity. Oral administration of Vioxx for six weeks (34 mg/kg/day) to C57Bl/6 mice kept on high-fat diet (n = 19) resulted in a significant reduction in total body weight (p < 0.01) and of subcutaneous (p < 0.05) and gonadal (p< 0.01) adipose tissue mass, as compared to placebo-treated animals (n = 21).There was no significant difference in food intake between both groups (2.8 ± 0.09 vs.3.0 ± 0.10 g/day;p = 0.20).Administration ofVioxx resulted in reduced total cholesterol and high-density lipoprotein (HDL) cholesterol levels (p < 0.0001) and in enhanced levels of liver enzymes, as compared to placebo. In the gonadal but not in the subcutaneous adipose tissue, adipocytes were smaller after Vioxx treatment (p < 0.05).The macrophage content was significantly lower in gonadal adipose tissues of Vioxx-treated mice (p < 0.05), but not in the subcutaneous adipose tissues. This was, however, not associated with differences in adipose tissue levels of the pro-inflammatory tumor necrosis factor (TNF)-α. Blood vessel size or density in either fat pad were not affected by Vioxx treatment. Thus, in a nutritionally induced murine obesity model, oral administration of Vioxx, as compared to placebo, resulted in reduced adipose tissue development, associated with lower feeding efficiency and smaller adipocytes.

We sought to delineate mechanisms through which the lack of plasminogen activator inhibitor (PAI)-1 in the heart affects remodeling of the heart early after myocardial infarction (MI). MI was induced by coronary occlusion in 10-weeks old PAI-1 knockout (KO) and control mice. Three days after MI, systolic and diastolic function was assessed with high-resolution echocardiography, infarct size was determined biochemically and histologically and accumulation of acute inflammatory cells in zones of infarction was characterized by immunocytochemistry. PAI-1 KO mice exhibited markedly thickened diastolic left ventricular anterior walls (1.38 +/- 0.38 mm vs. 0.77 +/- 0.13 SD), more profound depression of global and regional cardiac function (19 vs. 22% fractional shortening), and greater evidence of diastolic dysfunction (average E wave amplitude = 568 vs. 675 mm/s) all of which were significant. Markedly greater extent of infarction was demonstrated biochemically and histologically in knockout mice compared with controls (76 vs. 29% of the left ventricle, P0.05) associated with striking hemorrhage and intense inflammation. Fibrosis normalized for infarct size was markedly reduced (0.006 vs. 0.022 microg hydroxyproline/mg dry weight). Thus, lack of PAI-1 in the heart exerted deleterious effects mediated, at least in part by increased inflammation and hemorrhage and attenuating of fibrosis.

The potential role of placental growth factor (PlGF) in early stages of adipogenesis was investigated in vivo using a murine model of obesity, as well as in vitro using cultured preadipocytes. PlGF-deficient (PlGF-/-) and wild-type (WT) mice, kept on high fat diet (HFD) for 3 weeks, had comparable body weight and weight of subcutaneous (SC) and gonadal (GON) adipose tissues. Blood vessel size and blood vessel density, normalized to adipocyte number, were not significantly different in SC and GON adipose tissues of both genotypes. Differentiation of embryonic fibroblasts derived from WT or PlGF-/- mice into mature adipocytes was comparable. Furthermore, addition of recombinant PlGF, of the PlGF neutralizing MAb PL5D11D4 or of the anti-Flk-1 MAb DC101 to cultured 3T3-F442A preadipocytes did not significantly affect their differentiation into mature adipocytes. Ex vivo blood vessel outgrowth following seeding of adipose tissue-derived microvessel fragments in 3D-collagen gels was not affected by PlGF deficiency. Thus, in murine model systems, PlGF does not seem to play an important role in early adipogenesis.

SummaryThe homozygous factorV Leiden mutation is associated with enhanced venous thrombotic risk. Obesity is a major risk factor for development of thrombotic cardiovascular disease. It was the objective of this study to investigate whether obesity affects the thrombotic risk associated with the mutation. Male mice with homozygous factor V Leiden mutation (Arg 504 to Gln) (FVQ/Q) and corresponding wild-type (WT) mice were kept on a standard fat diet (SFD) or high fat diet (HFD) for 14 weeks, and femoral artery thrombosis was induced by FeCl3 treatment. As compared to SFD, HFD feeding for 14 weeks resulted in significantly higher body weight and fat mass associated with adipocyte hypertrophy, which were, however, similar for both geno types. In the FeCl3-induced arterial thrombosis model, FVQ/Q mice kept on SFD had a 40% shorter occlusion time (p = 0.015) and 40% lower blood flow (p = 0.03), as compared to WT mice. However, on HFD the occlusion time and blood flow were not significantly different for both genotypes. This finding could not be explained by differential changes of coagulation factors in either genotype fed on SFD or HFD. In conclusion, on SFD, but not on HFD, the factorV Leiden mutation is associated with enhanced thrombotic tendency after FeCl3 injury of the femoral artery, suggesting that in this model obesity rescues the increased thrombotic risk associated with the factorV Leiden mutation.

Intestinal chemosensory signaling pathways involving the gustatory G-protein, gustducin, and bitter taste receptors (TAS2R) have been implicated in gut hormone release. Alterations in gut hormone profiles may contribute to the success of bariatric surgery. This study investigated the involvement of the gustatory signaling pathway in the development of diet-induced obesity and the therapeutic potential of targeting TAS2Rs to induce body weight loss. α-gustducin-deficient (α-gust-/-) mice became less obese than wild type (WT) mice when fed a high-fat diet (HFD). White adipose tissue (WAT) mass was lower in α-gust-/- mice due to increased heat production as a result of increases in brown adipose tissue (BAT) thermogenic activity, involving increased protein expression of uncoupling protein 1. Intra-gastric treatment of obese WT and α-gust-/- mice with the bitter agonists denatonium benzoate (DB) or quinine (Q) during 4 weeks resulted in an α-gustducin-dependent decrease in body weight gain associated with a decrease in food intake (DB), but not involving major changes in gut peptide release. Both WAT and 3T3-F442A pre-adipocytes express TAS2Rs. Treatment of pre-adipocytes with DB or Q decreased differentiation into mature adipocytes. In conclusion, interfering with the gustatory signaling pathway protects against the development of HFD-induced obesity presumably through promoting BAT activity. Intra-gastric bitter treatment inhibits weight gain, possibly by directly affecting adipocyte metabolism. ispartof: PLoS One vol:10 issue:12 ispartof: location:United States status: published

Sepsis is associated with enhanced production of tissue-type plasminogen activator (tPA). We investigated the function of endogenous tPA in the immune responses to Escherichia coli-induced abdominal sepsis using tPA gene-deficient (tPA−/−) and normal wild-type (WT) mice. tPA−/− mice demonstrated an impaired defense against E. coli peritonitis as indicated by higher bacterial loads at the primary site of the infection, enhanced dissemination, and reduced survival. The protective function of tPA was independent of plasmin since plasminogen gene-deficient (Plg−/−) mice were indistinguishable from WT mice. Relative to WT mice, tPA−/− mice demonstrated similar neutrophil counts in the peritoneal cavity despite much higher bacterial loads and higher local concentrations of neutrophil attracting chemokines, suggesting a reduced migratory response. In line, tPA−/− mice demonstrated a reduced thioglycolate-induced neutrophil influx into the peritoneal cavity and i.p. injection of WT mice with a replication-defective adenoviral vector expressing tPA caused an enhanced cell migration to the peritoneal cavity during E. coli peritonitis. These findings identify a novel protective function of tPA in abdominal sepsis caused by E. coli that seems independent of its role in the generation of plasmin.

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes responsible for extracellular protein degradation in the cardiovascular system. Originally known to play pivotal roles in tissue morphogenesis and wound healing, they have been shown to participate in the complex remodeling processes in blood vessels and the myocardium. The biological activity of MMPs is regulated at different levels: (1) gene expression, (2) activation of precursor proenzyme forms by other MMPs or non-MMP proteins, including thrombin and plasmin, (3) complex formation with surface and extracellular matrix (ECM) components and (4) inhibition by endogenous tissue inhibitors of MMPs, the TIMPs. Murine models with gain or loss of gene function of different MMPs and TIMPS have provided a wealth of experimental data on their critical role in pathological conditions ranging from atherosclerosis, vascular injury, and restenosis to left ventricular function and structural remodeling following chronic pressure and volume overload and ischemia-reperfusion injury.

SummaryIn order to evaluate a potential functional role of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) in development of obesity, we studied the effect of overexpression of human TIMP-1 (hTIMP-1) in C57Bl/6J mice in vivo and in 3T3-F442A preadipocytes in vitro. Stable long-term overexpression of hTIMP-1 in mice was achieved by adenoviral gene transfer, yielding plasma levels exceeding 250 ng/ml at eight weeks after injection. Mice overexpressing hTIMP-1 and kept on a high fat diet for 14 weeks had body weights, adipose tissue weights, and adipocyte diameters that were somewhat, but not significantly, lower than those of control mice. Similar observations were made after overexpression of hTIMP-1 in mice with lipectomy of the subcutaneous adipose tissue, kept on a high fat diet for 20 weeks. In both in vivo models, blood vessels in the adipose tissues were significantly smaller after hTIMP-1 gene transfer than in control mice. Overexpression of hTIMP-1 in 3T3-F442A preadipocytes had no effect on their subsequent differentiation into mature adipocytes. Thus, overexpression of hTIMP-1 in mice had no significant effect on ongoing adipogenesis or adipose tissue development, although the blood vessel size in adipose tissues was reduced.

Obesity is a common disorder and related diseases such as diabetes, atherosclerosis, hypertension, cardiovascular disease and cancer are a major cause of mortality and morbidity in Western-type societies. Development of obesity is associated with extensive modifications in adipose tissue involving adipogenesis, angiogenesis and extracellular matrix proteolysis. The fibrinolytic (plasminogen/plasmin) and matrix metalloproteinase (MMP) systems cooperate in these processes. A nutritionally induced obesity model in transgenic mice has been used extensively to study the role of the fibrinolytic and MMP systems in the development of obesity. These studies support a role of both systems in adipogenesis and obesity; the role of specific members of these families, however, remains to be determined.