ADAMTS5 deficiency in mice does not affect cardiac function.

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.

Antioxidant Treatment Improves Cardiac Dysfunction in a Murine Model of Premature Aging.

Bmal1-(brain and muscle ARNT-like protein-1) deficient (Bmal1) mice prematurely age because of an increased reactive oxygen species (ROS) production. These mice also show a decline in cardiac function with age. We investigated whether an antioxidant treatment can ameliorate the declining cardiac function in prematurely aged Bmal1 mice. Male Bmal1 and wild-type (Bmal1) mice were exposed for 15 weeks to a high fat and high cholesterol diet with or without the antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL; 5 mmol/L; in drinking water during the last 10 weeks). Echocardiographic analysis revealed that TEMPOL treatment of Bmal1 mice normalized cardiac function, as evidenced by a decrease in left ventricular diastolic and systolic internal diameters, and by an increase in fractional shortening and ejection fraction. The antioxidant did not affect cardiac function in Bmal1 mice. Although TEMPOL did not influence cardiac ROS levels in Bmal1 mice, it significantly protected Bmal1 cardiac telomeres from oxidation, as evidenced by a reduction in the telomere damage score (0.11 ± 0.012% vs. 0.16 ± 0.015%; P = 0.028). Thus, antioxidant treatment normalized cardiac function of Bmal1 mice, probably in part by scavenging ROS.

Pulmonary and hemostatic toxicity of multi-walled carbon nanotubes and zinc oxide nanoparticles after pulmonary exposure in Bmal1 knockout mice.

BACKGROUND: Pulmonary exposure to nanoparticles (NPs) may affect, in addition to pulmonary toxicity, the cardiovascular system such as procoagulant effects, vascular dysfunction and progression of atherosclerosis. However, only few studies have investigated hemostatic effects after pulmonary exposure. METHODS: We used Bmal1 (brain and muscle ARNT-like protein-1) knockout (Bmal1(-/-)) mice which have a disturbed circadian rhythm and procoagulant phenotype, to study the pulmonary and hemostatic toxicity of multi-walled carbon nanotubes (MWCNTs) and zinc oxide (ZnO) NPs after subacute pulmonary exposure. Bmal1(-/-) and wild-type (Bmal1(+/+)) mice were exposed via oropharyngeal aspiration, once a week, during 5 consecutive weeks, to a cumulative dose of 32 or 128 µg MWCNTs or 32 or 64 µg ZnO NPs. RESULTS: MWCNTs caused a pronounced inflammatory response in the lung with increased cell counts in the broncho-alveolar lavage and increased secretion of interleukin-1ß and cytokine-induced neutrophil chemo-attractant (KC), oxidative stress (increased ratio of oxidized versus reduced glutathione and decreased total glutathione) as well as anemic and procoagulant effects as evidenced by a decreased prothrombin time with increased fibrinogen concentrations and coagulation factor (F)VII. In contrast, the ZnO NPs seemed to suppress the inflammatory (decreased neutrophils in Bmal1(-/-) mice) and oxidative response (increased total glutathione in Bmal1(-/-) mice), but were also procoagulant with a significant increase of FVIII. The procoagulant effects, as well as the significant correlations between the pulmonary endpoints (inflammation and oxidative stress) and hemostasis parameters were more pronounced in Bmal1(-/-) mice than in Bmal1(+/+) mice. CONCLUSIONS: The Bmal1(-/-) mouse is a sensitive animal model to study the procoagulant effects of engineered NPs. The MWCNTs and ZnO NPs showed different pulmonary toxicity but both NPs induced procoagulant effects, suggesting different mechanisms of affecting hemostasis. However, the correlation analysis suggests a causal association between the observed pulmonary and procoagulant effects.

Effect of ageing on the murine venous circulation.

The effect of ageing on the morphology of veins, venous valves and arteries was investigated in male wild-type mice using an adapted procedure with injection of a silicone polymer Microfil(®) that preserves morphology of the vasculature. Throughout the hind limb the arterial, but not the venous, lumen area and wall thickness were significantly greater in 24-month as compared to 10-week-old C57BL/6 mice. Venous valves were most frequently located at the sapheno-femoral vein junction in the lower extremities, and appeared thicker at the base supported by structurally intact collagen fibers, and thinner towards the proximal end of the valve leaflet, with less organized collagen. Overall, valves were less supported by structurally intact collagen at 24 months as compared to 10 weeks. Endothelial expression of CD31, endothelial protein C receptor or von Willebrand factor (VWF) was not affected by age, while thrombomodulin expression was lower in aged versus young arteries. At both ages, expression of VWF was lower at venous valves versus veins. Evaluation of the blood coagulation profile revealed that aged mice had shortened prothrombin time, elevated plasma levels of factor (F)VII, FVIII and VWF and increased neutrophil and platelet counts. Thus, our data indicate that in mice with ageing, venous valves become more fragile, in association with a procoagulant and inflammatory blood phenotype. Taken together, we found that the procoagulant state in ageing, is accompanied by mild vascular changes.

Progression of the prothrombotic state in aging Bmal1-deficient mice.

OBJECTIVE: The goal of this study was to examine the functional relationship between aging endothelium and thrombogenicity in a mouse model of premature aging. METHODS AND RESULTS: Coagulation tests and factors, blood cell counts, aorta endothelial function, aorta gene expression, and FeCl(3)-induced thrombosis in mesenteric blood vessels were analyzed in 10- to 30-week-old brain and muscle ARNT-like protein-1 (Bmal1)-deficient (knockout [KO]) mice and wild-type littermates. Ten-week-old KO mice manifested shortened prothrombin times (9.7 versus 11.3 seconds in wild-type) and elevated plasma fibrinogen (264 versus 172 mg/dL). At 30 weeks, factor VII (198% versus 149%), and platelet counts (2049 versus 1354 K/µL) were increased in KO mice. Gene deficiency reduced the vasoactive nitric oxide production at 10 and 30 weeks and tended to reduce and increase the protein expression of thrombomodulin and von Willebrand factor, respectively, with aging. Shortened venular and arteriolar occlusion times on FeCl(3)-induced injury in 10-week-old KO mice confirmed higher thrombogenicity, culminating in priapism, observed in 60% of 25- to 30-week-old KO males. CONCLUSION: Endothelial dysfunction and a hypercoagulable state cause early arterial and venous thrombogenicity in Bmal1 KO mice. With aging, progressive endothelial dysfunction, rising platelet counts, and high factor VII further enhance thrombogenicity, provoking priapism.

Age-associated adaptations in murine adipose tissues.

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.

Deficiency of Bmal1 disrupts the diurnal rhythm of haemostasis.

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.

Does butein affect adipogenesis?

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.

Advanced-age C57BL/6JRj mice do not develop obesity upon western-type diet exposure.

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.

Adverse adipose phenotype and hyperinsulinemia in gravid mice deficient in placental growth factor.

Pregnancy-induced metabolic changes are regulated by signals from an expanded adipose organ. Placental growth factor (PlGF), acting through vascular endothelial growth factor receptor-1, may be among those signals. There is a steep rise in circulating PlGF during normal pregnancy, which is repressed in gravidas who develop preeclampsia. PlGF-deficiency in mice impairs adipose vascularization and development. Here we studied young-adult PlGF-deficient (PlGF(-/-)) and wild-type mice on a high-fat diet in the nongravid state and at embryonic day (E) 13.5 or E18.5 of gestation. Litter size and weight were normal, but E18.5 placentas were smaller in PlGF(-/-) pregnancies. PlGF(-/-) mice showed altered intraadipose dynamics, with the following: 1) less blood vessels and fewer brown, uncoupling protein (UCP)-1-positive, adipocytes in white sc and perigonadal fat compartments and 2) white adipocyte hypertrophy. The mRNA expression of beta(3)-adrenergic receptors, peroxisome proliferator-activated receptor-gamma coactivator-1alpha, and UCP-1 was decreased accordingly. Moreover, PlGF(-/-) mice showed hyperinsulinemia. Pregnancy-associated changes were largely comparable in PlGF(-/-) and wild-type dams. They included expanded sc fat compartments and adipocyte hypertrophy, whereas adipose expression of key angiogenesis/adipogenesis (vascular endothelial growth factor receptor-1, peroxisome proliferator-activated receptor-gamma(2)) and thermogenesis (beta(3)-adrenergic receptors, peroxisome proliferator-activated receptor-gamma coactivator-1alpha, and UCP-1) genes was down-regulated; circulating insulin levels gradually increased during pregnancy. In conclusion, reduced adipose vascularization in PlGF(-/-) mice impairs adaptive thermogenesis in favor of energy storage, thereby promoting insulin resistance and hyperinsulinemia. Pregnancy adds to these changes by PlGF-independent mechanisms. Disturbed intraadipose dynamics is a novel mechanism to explain metabolic changes in late pregnancy in general and preeclamptic pregnancy in particular.

Functional role of ADAMTS5 in adiposity and metabolic health.

Previous studies with gene-deficient mice (ADAMTS5-P) revealed that ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin type 1 motifs, member 5) plays a functional role in adiposity and metabolic health. To confirm these observations, we have performed similar studies with an independently generated strain of ADAMTS5 deficient mice (ADAMTS5-J). Upon cold exposure as well as after high-fat diet feeding (diet-induced obesity or DIO model), these knockout (KO) mice developed less subcutaneous and gonadal white adipose tissue (WAT) as compared to their wild-type (WT) littermates (reduction was more pronounced in ADAMTS5-P mice). Enhanced browning of WAT, as monitored by expression of UCP-1 was seen in the ADAMTS5-J KO mice upon cold exposure but not in the DIO model (seen in both conditions with the ADAMTS5-P mice). Brown adipose tissue (BAT) mass was not different between KO and WT ADAMTS5-J mice, either upon cold exposure or in the DIO model (in contrast to the enhanced BAT mass with the ADAMTS5-P mice). Energy expenditure and thermogenesis were not significantly different between KO and WT ADAMTS5-J mice (in contrast to somewhat enhanced levels in ADAMTS5-P mice). Insulin sensitivity was improved in the ADAMTS5-J KO mice, and they were protected against non-alcoholic steatohepatitis in the DIO model (as the ADAMTS5-P mice). These data are thus similar for both strains of KO mice, confirming specificity of the phenotype, but some quantitative and qualitative differences are also observed.

Adiposity and metabolic health in mice deficient in intestinal alkaline phosphatase.

Intestinal alkaline phosphatase 3 (AKP3) is an enzyme that was reported to play a role in lipid metabolism and to prevent high fat diet-induced metabolic syndrome in mice. To investigate a potential functional role of AKP3 in diet-induced adiposity and metabolic health, we have kept male and female wild-type or AKP3 deficient mice on a high fat diet for 15 weeks to induce obesity and compared those with mice kept on standard fat diet. Body weight as well as adipose tissue mass were statistically significantly higher upon high fat diet feeding for mice of both genders and genotypes. Female mice of either genotype kept on high fat diet gained less weight, resulting in smaller adipose tissue depots with smaller adipocytes. However, AKP3 deficiency had no significant effect on body weight gain or adipose tissue mass and did not affect adipocyte size or density. Gene expression analysis revealed no effect of the genotype on inflammatory parameters in adipose tissue, except for tumor necrosis factor alpha, which was higher in mesenteric adipose tissue of female obese mice. Plasma glucose and insulin levels were also not affected in obese AKP3 deficient mice. Overall, our data do not support a functional role of AKP3 in adipose tissue development, or insulin sensitivity.

Monitoring reperfused myocardial infarction with delayed left ventricular systolic dysfunction in rabbits by longitudinal imaging.

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<0.05). According to cardiac morphology and function changes, 4 groups could be distinguished: sham rabbits (n=12), and MI rabbits with no (MI_NO_LVSD; n=10), moderate (MI_M_LVSD; n=9) and severe (MI_S_LVSD; n=15) LVSD. No significant differences in cardiac function or wall thickening between sham and MI_NO_LVSD rabbits were observed at both stages using both cMRI and echo methods. cMRI data showed that MI_M_LVSD rabbits exhibited a reduction of ejection fraction (EF) and an increase in end-systolic volume (ESV) at the acute phase, while at the chronic stage these parameters did not change further. Moreover, in MI_S_LVSD animals, these observations were more striking at the acute stage followed by a further decline in EF and increase in ESV at the chronic stage. Lateral wall thickening determined by cMRI was significantly decreased in MI_M_LVSD versus MI_NO_LVSD animals at both stages (P<0.05). As for MI_S_LVSD versus MI_M_LVSD rabbits, the thickening of anterior, inferior and lateral walls was significantly more decreased at both stages (P<0.05). Echo confirmed the findings of cMRI. Furthermore, these in vivo outcomes including those from vivid cine cMRI could be supported by exactly matched ex vivo histomorphological evidences. CONCLUSIONS: Our findings indicate that chronic LVSD developed over time after surgery-induced MI in rabbits can be longitudinally evaluated using non-invasive imaging techniques and confirmed by the entire-heart-slice histomorphology. This experimental LVSD platform in rabbits may interest researchers in the field of experimental cardiology and help strengthen drug development and translational research for the management of cardiovascular diseases.

SPARC preserves endothelial glycocalyx integrity, and protects against adverse cardiac inflammation and injury during viral myocarditis.

Myocardial damage as a consequence of cardiotropic viruses leads to a broad variety of clinical presentations and is still a complicated condition to diagnose and treat. Whereas the extracellular matrix protein Secreted Protein Acidic and Rich in Cysteine or SPARC has been implicated in hypertensive and ischemic heart disease by modulating collagen production and cross-linking, its role in cardiac inflammation and endothelial function is yet unknown. Absence of SPARC in mice resulted in increased cardiac inflammation and mortality, and reduced cardiac systolic function upon coxsackievirus-B3 induced myocarditis. Intra-vital microscopic imaging of the microvasculature of the cremaster muscle combined with electron microscopic imaging of the microvasculature of the cardiac muscle uncovered the significance of SPARC in maintaining endothelial glycocalyx integrity and subsequent barrier properties to stop inflammation. Moreover, systemic administration of recombinant SPARC restored the endothelial glycocalyx and consequently reversed the increase in inflammation and mortality observed in SPARC KO mice in response to viral exposure. Reducing the glycocalyx in vivo by systemic administration of hyaluronidase, an enzyme that degrades the endothelial glycocalyx, mimicked the barrier defects found in SPARC KO mice, which could be restored by subsequent administration of recombinant SPARC. In conclusion, the secreted glycoprotein SPARC protects against adverse cardiac inflammation and mortality by improving the glycocalyx function and resulting endothelial barrier function during viral myocarditis.

Evaluation of cardiac arrhythmic risks using a rabbit model of left ventricular systolic dysfunction.

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.

Nanoparticles in the lungs of old mice: Pulmonary inflammation and oxidative stress without procoagulant effects.

Pulmonary exposure to nanoparticles (NPs) has been shown to induce pulmonary as well as cardiovascular toxicity. These effects might be enhanced in elderly subjects as a result of a compromised immunity and/or declined organ functions. To study the adverse in vivo effects of NPs in a model for the elderly, we exposed 18-month-old C75Bl/6 mice to multi-walled carbon nanotubes (MWCNTs) or ZnO NPs by intratracheal instillation once a week during 5 consecutive weeks. Pulmonary and hemostatic toxicity was determined 24 h (T1) and 8 weeks (T2) after the last administration. Both NP types significantly increased the pulmonary macrophages at both time points. The MWCNTs and ZnO NPs also induced a pulmonary influx of neutrophils, which was even larger at T2 compared to T1. All NPs induced only a modest increase of pulmonary IL-1ß, IL-6 and KC levels. Both types of NPs also increased blood neutrophils. Red blood cells were not significantly affected. Both NPs significantly increased coagulation factor VIII levels at both time points. Histological analysis revealed the presence of MWCNTs in the alveolar macrophages up to 8 weeks after the last administration and the ZnO NPs induced a pronounced alveolar inflammation. In these 18-month-old mice, NPs caused pulmonary inflammation (without evidence of oxidative stress) accompanied by large increases in coagulation factor VIII up to 8 weeks after the last NP exposure. The persistence of the MWCNTs in the lungs resulted in translocation from the lungs to the left heart and the ZnO NPs induced a fibrosis-like pathology.

Loss of ADAMTS5 enhances brown adipose tissue mass and promotes browning of white adipose tissue via CREB signaling.

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 ß3-adrenergic receptor (ß3-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 ß3-AR signaling via activation of the cAMP response element-binding protein (CREB). Additional ß3-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.

Novel Role for Matrix Metalloproteinase 9 in Modulation of Cholesterol Metabolism.

BACKGROUND: The development of atherosclerosis is strongly linked to disorders of cholesterol metabolism. Matrix metalloproteinases (MMPs) are dysregulated in patients and animal models with atherosclerosis. Whether systemic MMP activity influences cholesterol metabolism is unknown. METHODS AND RESULTS: We examined MMP-9-deficient (Mmp9-/-) mice and found them to have abnormal lipid gene transcriptional responses to dietary cholesterol supplementation. As opposed to Mmp9+/+ (wild-type) mice, Mmp9-/- mice failed to decrease the hepatic expression of sterol regulatory element binding protein 2 pathway genes, which control hepatic cholesterol biosynthesis and uptake. Furthermore, Mmp9-/- mice failed to increase the expression of genes encoding the rate-limiting enzymes in biliary cholesterol excretion (eg, Cyp7a and Cyp27a). In contrast, MMP-9 deficiency did not impair intestinal cholesterol absorption, as shown by the 14C-cholesterol and 3H-sitostanol absorption assay. Similar to our earlier study on Mmp2-/- mice, we observed that Mmp9-/- mice had elevated plasma secreted phospholipase A2 activity. Pharmacological inhibition of systemic circulating secreted phospholipase A2 activity (with varespladib) partially normalized the hepatic transcriptional responses to dietary cholesterol in Mmp9-/- mice. Functional studies with mice deficient in other MMPs suggested an important role for the MMP system, as a whole, in modulation of cholesterol metabolism. CONCLUSIONS: Our results show that MMP-9 modulates cholesterol metabolism, at least in part, through a novel MMP-9-plasma secreted phospholipase A2 axis that affects the hepatic transcriptional responses to dietary cholesterol. Furthermore, the data suggest that dysregulation of the MMP system can result in metabolic disorder, which could lead to atherosclerosis and coronary heart disease.

Protein kinase CKIIalpha interacts with the Bcr moiety of Bcr/Abl and mediates proliferation of Bcr/Abl-expressing cells.

The Bcr protein was originally identified because of its fusion to Abl as a consequence of the Philadelphia chromosome translocation found in chronic myelogenous and acute lymphoblastic leukemias. The Bcr moiety is essential for the transforming activity of the Bcr/Abl oncogene. In search of physiologically relevant Bcr and Bcr/Abl-interacting proteins, we performed an interaction screen in yeast using the entire Bcr protein as bait. We here report that the alpha catalytic subunit of protein kinase CKII strongly and specifically forms a complex with Bcr in yeast in mouse lysates. The region in Bcr responsible for CKIIalpha binding was localized to residues 242-413. CKIIalpha was previously shown to be involved in leukemogenesis and tumorigenesis using different experimental approaches including mouse models. Inhibition of Bcr/Abl P190 in lymphoma cells from Bcr/Abl transgenic mice using imatinib reduced CKIIalpha activity. A highly selective inhibitor of CKIIalpha, 4,5,6,7-tetrabromo-2-benzotriazole, inhibited the growth of murine lymphoid cells with induced P210 Bcr/Abl expression and of P190 lymphoma cells. Our results demonstrate that CKIIalpha plays an important role in the proliferation of Bcr/Abl expressing cells, and suggests that inhibitors of CKIIalpha may have therapeutic potential in the treatment of Bcr/Abl-positive leukemia patients.

BCR/ABL P190 transgenic mice develop leukemia in the absence of Crkl.

The Bcr/Abl fusion protein directly causes chronic myelogenous leukemia and Philadelphia-chromosome positive acute lymphoblastic leukemia. Multiple independent studies have implicated Crkl, a small adapter protein, in transduction of oncogenic signals of Bcr/Abl and Crkl tyrosine-phosphorylation is used as a diagnostic tool for Philadelphia-positive leukemia. To evaluate the contribution of Crkl to this type of leukemia, we generated mutant mice that lack Crkl expression. We found that the overall survival of P190 BCR/ABL crkl-/- mice was comparable to that of genetically matched P190 BCR/ABL crkl +/+ mice. Both genotypes developed lymphoid lineage leukemia/lymphoma. Western blot analysis of -/- and +/+ lymphomas showed that the related Crk protein was tyrosine phosphorylated and could be found complexed with Bcr-Abl P190. These data indicate that possible therapeutic approaches that target Crkl may be complicated by the presence of pathways that compensate for lack of Crkl function.