Neutrophil peptidylarginine deiminase 4 plays a systemic role in obesity-induced chronic inflammation in mice.

BACKGROUND: Obesity is an increasing problem in our current society and is expected to keep rising in incidence. With its multiorigin, complex pathophysiology, it is difficult to treat and easy to acquire unnoticeably. During obesity, it has been established that the body is in a constant state of low-grade inflammation, thereby causing changes in immune cell physiology. OBJECTIVES: Here, we investigated the influence of neutrophils, more specifically as a result of peptidylarginine deiminase 4 (PAD4) activity and the release of neutrophil extracellular traps (NETs), during obesity-induced chronic inflammation. METHODS: Wild-type mice were placed on a high-fat diet (HFD) and investigated over a period of 10 weeks for NET formation and its impact on the heart. Neutrophil-selective PAD4 knockout (Ne-PAD4-/-) mice were studied in parallel. RESULTS: As a result of high fat intake, we observed clear alteration in the priming status of isolated neutrophils toward NET release, including early stages of speck formation and histone citrullination of apoptosis-associated speck-like protein containing a CARD. Ne-PAD4-/- mice deficient in NET formation did not increase bodyweight to the same extent as their littermate controls, with Ne-PAD4-/- mice being leaner after 10 weeks of HFD feeding. Interestingly, obesity progression led to cardiac remodeling and diastolic dysfunction in wild-type mice after 10 weeks, while this remodeling and subsequent decrease in function were absent in Ne-PAD4-/- mice. Surprisingly, HFD did not alter NET content or thrombus formation in the inferior vena cava stenosis model. CONCLUSION: Detrimental physiological effects, the result of obesity progression, can in part be attributed to neutrophil PAD4 and NETs in response to chronic inflammation.

Neutrophil peptidylarginine deiminase 4 is essential for detrimental age-related cardiac remodelling and dysfunction in mice.

Mice fully deficient in peptidylarginine deiminase 4 (PAD4) enzyme have preserved cardiac function and reduced collagen deposition during ageing. The cellular source of PAD4 is hypothesized to be neutrophils, likely due to PAD4's involvement in neutrophil extracellular trap release. We investigated haematopoietic PAD4 impact on myocardial remodelling and systemic inflammation in cardiac ageing by generating mice with Padi4 deletion in circulating neutrophils under the MRP8 promoter (Ne-PAD4-/-), and ageing them for 2 years together with littermate controls (PAD4fl/fl). Ne-PAD4-/- mice showed protection against age-induced fibrosis, seen by reduced cardiac collagen deposition. Echocardiography analysis of structural and functional parameters also demonstrated preservation of both systolic and diastolic function with MRP8-driven PAD4 deletion. Furthermore, cardiac gene expression and plasma cytokine levels were evaluated. Cardiac genes and plasma cytokines involved in neutrophil recruitment were downregulated in aged Ne-PAD4-/- animals compared to PAD4fl/fl controls, including decreased levels of C-X-C ligand 1 (CXCL1). Our data confirm PAD4 involvement from circulating neutrophils in detrimental cardiac remodelling, leading to cardiac dysfunction with old age. Deletion of PAD4 in MRP8-expressing cells impacts the CXCL1-CXCR2 axis, known to be involved in heart failure development. This supports the future use of PAD4 inhibitors in cardiovascular disease. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Neutrophils Protect Against Staphylococcus aureus Endocarditis Progression Independent of Extracellular Trap Release.

BACKGROUND: Infective endocarditis (IE) is characterized by an infected thrombus at the heart valves. How bacteria bypass the immune system and cause these thrombi remains unclear. Neutrophils releasing NETs (neutrophil extracellular traps) lie at this interface between host defense and coagulation. We aimed to determine the role of NETs in IE immunothrombosis. METHODS: We used a murine model of Staphylococcus aureus endocarditis in which IE is provoked on inflamed heart valves and characterized IE thrombus content by immunostaining identifying NETs. Antibody-mediated neutrophil depletion and neutrophil-selective PAD4 (peptidylarginine deiminase 4)-knockout mice were used to clarify the role of neutrophils and NETs, respectively. S. aureus mutants deficient in key virulence factors related to immunothrombosis (nucleases or staphylocoagulases) were investigated. RESULTS: Neutrophils releasing NETs were present in infected thrombi and within cellular infiltrates in the surrounding vasculature. Neutrophil depletion increased occurrence of IE, whereas neutrophil-selective impairment of NET formation did not alter IE occurrence. Absence of S. aureus nuclease, which degrades NETs, did not affect endocarditis outcome. In contrast, absence of staphylocoagulases (coagulase and von Willebrand factor binding protein) led to improved survival, decreased bacteremia, smaller infiltrates, and decreased tissue destruction. Significantly more NETs were present in these vegetations, which correlated with decreased bacteria and cell death in the adjacent vascular wall. CONCLUSIONS: Neutrophils protect against IE independent of NET release. Absence of S. aureus coagulases, but not nucleases, reduced IE severity and increased NET levels. Staphylocoagulase-induced fibrin likely hampers NETs from constraining infection and the resultant tissue damage, a hallmark of valve destruction in IE.

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.

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.

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.

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.

Role of thrombospondin-2 in murine adipose tissue angiogenesis and development.

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.

Axl deficiency does not affect adipogenesis or adipose tissue development.

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.

Fumagillin reduces adipose tissue formation in murine models of nutritionally induced obesity.

The effect of fumagillin (a methionine aminopeptidase-type 2 (Met-AP2) inhibitor, with antiangiogenic properties) was investigated in murine models of diet-induced obesity. Eleven-week-old male C57Bl/6 mice (group 1) were given fumagillin by oral gavage at a dose of 1 mg/kg/day during 4 weeks while fed a high-fat diet (HFD) (20.1 kJ/g), and control mice (group 2) received solvent and were pair-fed. At the end of the experiment, body weights in group 1 were significantly lower as compared to group 2 (P < 0.0005). The subcutaneous (SC) and gonadal (GON) fat mass was also significantly lower in group 1 (P < 0.005 and P < 0.05, respectively). Adipocytes were smaller in adipose tissues of mice in group 1, associated with higher adipocyte density. Blood vessel density normalized to adipocyte density was lower in group 1 adipose tissues. However, in mice with established obesity monitored to maintain the same body weight and fat mass as controls, short-term fumagillin administration was also associated with adipocyte hypotrophy (P = 0.01) without affecting blood vessel size or density. Thus, treatment with fumagillin impaired diet-induced obesity in mice, associated with adipocyte hypotrophy but without marked effect on adipose tissue angiogenesis.

Tiplaxtinin impairs nutritionally induced obesity in mice.

To investigate the effect of tiplaxtinin, designed as a synthetic inhibitor of plasminogen activator inhibitor-1 (PAI-1), on obesity, male C57Bl/6 mice (13-14 weeks old) were kept on a high-fat diet (20.1 kJ/g) for four weeks without or with addition of tiplaxtinin (PAI-039) at a dose of 2 mg/g food. At the time of sacrifice, body weights were significantly lower in the inhibitor-treated mice (p < 0.0005). The weights of the isolated subcutaneous and gonadal fat deposits were also significantly lower (both p < 0.0005), associated with adipocyte hypotrophy. Inhibitor-treated adipose tissues displayed similar blood vessel size, but a higher blood vessel density. Fasting glucose and insulin levels, as well as glucose-tolerance tests were not significantly affected by the inhibitor treatment, whereas plasma triglyceride levels were significantly reduced (p = 0.02) and LDL-cholesterol levels significantly enhanced (p = 0.0002). Insulin-tolerance tests revealed significantly lower glucose levels at the end of the test in the inhibitor treated mice (p = 0.03). Thus, in this model of diet-induced obesity in mice administration of tiplaxtinin resulted in impaired adipose tissue development.