Keratinocyte growth factor protects against elastase-induced pulmonary emphysema in mice

Pulmonary emphysema is characterized by persistent inflammation and progressive alveolar destruction. The keratinocyte growth factor (KGF) favorably influences alveolar maintenance and repair and possesses anti-inflammatory properties. We aimed to determine whether exogenous KGF prevented or corrected elastase-induced pulmonary emphysema in vivo. Treatment with 5 mg x kg(-1) x day(-1) KGF before elastase instillation prevented pulmonary emphysema. This effect was associated with 1) a sharp reduction in bronchoalveolar lavage fluid total protein and inflammatory cell recruitment, 2) a reduction in the pulmonary expression of the chemokines CCL2 (or monocyte chemoattractant protein-1) and CXCL2 (or macrophage inflammatory protein-2alpha) and of the adhesion molecules ICAM-1 and VCAM-1, 3) a reduction in matrix metalloproteinase (MMP)-2 and MMP-9 activity at day 3, and 4) a major reduction in DNA damage detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) in alveolar cells at day 7. Treatment with KGF after elastase instillation had no effect on elastase-induced emphysema despite the conserved expression of the KGF receptor in the lungs of elastase-instilled animals as determined by immunohistochemistry. In vitro, KGF abolished the elastase-induced increase in CCL2, CXCL2, and ICAM-1 mRNA in the MLE-12 murine alveolar epithelial cell line. We conclude that KGF pretreatment protected against elastase-induced pulmonary inflammation, activation of MMPs, alveolar cell DNA damage, and subsequent emphysema in mice.     

Elimination of p19ARF‐expressing cells protects against pulmonary emphysema in mice

Senescent cells accumulate in tissues during aging and are considered to underlie several aging‐associated phenotypes and diseases. We recently reported that the elimination of p19^ARF‐expressing senescent cells from lung tissue restored tissue function and gene expression in middle‐aged (12‐month‐old) mice. The aging of lung tissue increases the risk of pulmonary diseases such as emphysema, and cellular senescence is accelerated in emphysema patients. However, there is currently no direct evidence to show that cellular senescence promotes the pathology of emphysema, and the involvement of senescence in the development of this disease has yet to be clarified. We herein demonstrated that p19^ARF facilitated the development of pulmonary emphysema in mice. The elimination of p19^ARF‐expressing cells prevented lung tissue from elastase‐induced lung dysfunction. These effects appeared to depend on reduced pulmonary inflammation, which is enhanced after elastase stimulation. Furthermore, the administration of a senolytic drug that selectively kills senescent cells attenuated emphysema‐associated pathologies. These results strongly suggest the potential of senescent cells as therapeutic/preventive targets for pulmonary emphysema.     

Successful establishment of primary small airway cell cultures in human lung transplantation

Background

Although both animal and human studies suggested the association between placenta growth factor (PlGF) and chronic obstructive pulmonary disease (COPD), especially lung emphysema, the role of PlGF in the pathogenesis of emphysema remains to be clarified. This study hypothesizes that blocking PlGF prevents the development of emphysema.

Methods

Pulmonary emphysema was induced in PlGF knock-out (KO) and wild type (WT) mice by intra-tracheal instillation of porcine pancreatic elastase (PPE). A group of KO mice was then treated with exogenous PlGF and WT mice with neutralizing anti-VEGFR1 antibody. Tumor necrosis factor alpha (TNF-α), matrix metalloproteinase-9 (MMP-9), and VEGF were quantified. Apoptosis measurement and immuno-histochemical staining for VEGF R1 and R2 were performed in emphysematous lung tissues.

Results

After 4 weeks of PPE instillation, lung airspaces enlarged more significantly in WT than in KO mice. The levels of TNF-α and MMP-9, but not VEGF, increased in the lungs of WT compared with those of KO mice. There was also increased in apoptosis of alveolar septal cells in WT mice. Instillation of exogenous PlGF in KO mice restored the emphysematous changes. The expression of both VEGF R1 and R2 decreased in the emphysematous lungs.

Conclusion

In this animal model, pulmonary emphysema is prevented by depleting PlGF. When exogenous PlGF is administered to PlGF KO mice, emphysema re-develops, implying that PlGF contributes to the pathogenesis of emphysema.     

Prevention of elastase-induced emphysema in placenta growth factor knock-out mice

Background

Although both animal and human studies suggested the association between placenta growth factor (PlGF) and chronic obstructive pulmonary disease (COPD), especially lung emphysema, the role of PlGF in the pathogenesis of emphysema remains to be clarified. This study hypothesizes that blocking PlGF prevents the development of emphysema.

Methods

Pulmonary emphysema was induced in PlGF knock-out (KO) and wild type (WT) mice by intra-tracheal instillation of porcine pancreatic elastase (PPE). A group of KO mice was then treated with exogenous PlGF and WT mice with neutralizing anti-VEGFR1 antibody. Tumor necrosis factor alpha (TNF-α), matrix metalloproteinase-9 (MMP-9), and VEGF were quantified. Apoptosis measurement and immuno-histochemical staining for VEGF R1 and R2 were performed in emphysematous lung tissues.

Results

After 4 weeks of PPE instillation, lung airspaces enlarged more significantly in WT than in KO mice. The levels of TNF-α and MMP-9, but not VEGF, increased in the lungs of WT compared with those of KO mice. There was also increased in apoptosis of alveolar septal cells in WT mice. Instillation of exogenous PlGF in KO mice restored the emphysematous changes. The expression of both VEGF R1 and R2 decreased in the emphysematous lungs.

Conclusion

In this animal model, pulmonary emphysema is prevented by depleting PlGF. When exogenous PlGF is administered to PlGF KO mice, emphysema re-develops, implying that PlGF contributes to the pathogenesis of emphysema.     

The Effect of PPE-Induced Emphysema and Chronic LPS-Induced Pulmonary Inflammation on Atherosclerosis Development in APOE*3-LEIDEN Mice

Background

Chronic obstructive pulmonary disease (COPD) is characterized by pulmonary inflammation, airways obstruction and emphysema, and is a risk factor for cardiovascular disease (CVD). However, the contribution of these individual COPD components to this increased risk is unknown. Therefore, the aim of this study was to determine the contribution of emphysema in the presence or absence of pulmonary inflammation to the increased risk of CVD, using a mouse model for atherosclerosis. Because smoke is a known risk factor for both COPD and CVD, emphysema was induced by intratracheal instillation of porcine pancreatic elastase (PPE).

Methods

Hyperlipidemic APOE*3-Leiden mice were intratracheally instilled with vehicle, 15 or 30 µg PPE and after 4 weeks, mice received a Western-type diet (WTD). To study the effect of emphysema combined with pulmonary inflammation on atherosclerosis, mice received 30 µg PPE and during WTD feeding, mice were intranasally instilled with vehicle or low-dose lipopolysaccharide (LPS; 1 µg/mouse, twice weekly). After 20 weeks WTD, mice were sacrificed and emphysema, pulmonary inflammation and atherosclerosis were analysed.

Results

Intratracheal PPE administration resulted in a dose-dependent increase in emphysema, whereas atherosclerotic lesion area was not affected by PPE treatment. Additional low-dose intranasal LPS administration induced a low-grade systemic IL-6 response, as compared to vehicle. Combining intratracheal PPE with intranasal LPS instillation significantly increased the number of pulmonary macrophages and neutrophils. Plasma lipids during the study were not different. LPS instillation caused a limited, but significant increase in the atherosclerotic lesion area. This increase was not further enhanced by PPE.

Conclusion

This study shows for the first time that PPE-induced emphysema both in the presence and absence of pulmonary inflammation does not affect atherosclerotic lesion development.     

Exercise intolerance and systemic manifestations of pulmonary emphysema in a mouse model

Background

Systemic effects of chronic obstructive pulmonary disease (COPD) significantly contribute to severity and mortality of the disease. We aimed to develop a COPD/emphysema model exhibiting systemic manifestations of the disease.

Methods

Female NMRI mice were treated 5 times intratracheally with porcine pancreatic elastase (emphysema) or phosphate-buffered saline (control). Emphysema severity was quantified histologically by mean linear intercept, exercise tolerance by treadmill running distance, diaphragm dysfunction using isolated muscle strips, pulmonary hypertension by measuring right ventricular pressure, and neurohumoral activation by determining urinary norepinephrine concentration.

Results

Mean linear intercept was higher in emphysema (260.7 ± 26.8 μm) than in control lungs (24.7 ± 1.7 μm). Emphysema mice lost body weight, controls gained weight. Running distance was shorter in emphysema than in controls. Diaphragm muscle length was shorter in controls compared to emphysema. Fatigue tests of muscle strips revealed impaired relaxation in emphysema diaphragms. Maximum right ventricular pressure and norepinephrine were elevated in emphysema compared to controls. Linear correlations were observed between running distance changes and intercept, right ventricular weight, norepinephrine, and diaphragm length.

Conclusion

The elastase mouse model exhibited severe emphysema with consecutive exercise limitation, and neurohumoral activation. The model may deepen our understanding of systemic aspects of COPD.     

Is neutrophil elastase the missing link between emphysema and fibrosis? Evidence from two mouse models

Background

The separation of emphysema from fibrosis is not as clear-cut as it was thought in early studies. These two pathologies may be present at the same time in human lungs and in mice either instilled with elastolytic enzymes or bleomycin or exposed to cigarette-smoke. According to a current view, emphysema originates from a protease/antiprotease imbalance, and a role for antiproteases has also been suggested in the modulation of the fibrotic process. In this study we investigate in experimental animal models of emphysema and fibrosis whether neutrophil elastase may constitute a pathogenic link between these two pathologies.

Methods

This study was done in two animal models in which emphysema and fibrosis were induced either by bleomycin (BLM) or by chronic exposure to cigarette-smoke. In order to assess the protease-dependence of the BLM-induced lesion, a group mice was treated with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, a serine proteinase inhibitor active toward neutrophil elastase. Lungs from each experimental group were used for the immunohistochemical assessment of transforming growth factor-β (TGF-β) and transforming growth factor-α (TGF-α) and for determination of the mean linear intercept as well as the percent volume densities of fibrosis and of emphysematous changes. Additionally, the lungs were also assessed for desmosine content and for the determination of elastase levels in the pulmonary interstitium by means of immunoelectron microscopy.

Results

We demonstrate that in BLM-treated mice (i) the development of elastolytic emphysema precedes that of fibrosis; (ii) significant amount of elastase in alveolar interstitium is associated with an increased expression of TGF-β and TGF-α; and finally, (iii) emphysematous and fibrotic lesions can be significantly attenuated by using a protease inhibitor active against neutrophil elastase.Also, in a strain of mice that develop both emphysema and fibrosis after chronic cigarette-smoke exposure, the presence of elastase in alveolar structures is associated with a positive immunohistochemical reaction for reaction for both TGF-β and TGF-α.

Conclusion

The results of the present study strongly suggest that neutrophil elastase may represent a common pathogenic link between emphysema and fibrosis. Proteases and in particular neutrophil elastase could act as regulatory factors in the generation of soluble cytokines with mitogenic activity for mesenchymal cells resulting either in emphysema or in fibrosis or both.     

Intrapulmonary distal airway stem cell transplantation repairs lung injury in chronic obstructive pulmonary disease

ObjectivesChronic obstructive pulmonary disease (COPD) is characterized by irreversible lung tissue damage including chronic bronchitis and emphysema, which could further develop into respiratory failure. Many studies have revealed a potential regenerative function of the distal airway stem/progenitor cells (DASCs) after lung injury.Materials and MethodsMouse and human DASCs were expanded, analysed, and engrafted into injured mouse lungs. Single‐cell analyses were performed to reveal the differentiation path of the engrafted cells. Finally, human DASCs were transplanted into COPD mice induced by porcine pancreatic elastase (PPE) and lipopolysaccharide (LPS) administration.ResultsWe showed that isolated mouse and human DASCs could be indefinitely expanded and were able to further differentiate into mature alveolar structures in vitro. Single‐cell analysis indicated that the engrafted cells expressed typical cellular markers of type I alveolar cells as well as the specific secreted proteins. Interestingly, transplantation of human DASCs derived from COPD patients into the lungs of NOD‐SCID mice with COPD injury repaired the tissue damage and improved the pulmonary function.ConclusionsThe findings demonstrated that functional lung structure could be reconstituted by intrapulmonary transplantation of DASCs, suggesting a potential therapeutic role of DASCs transplantation in treatment for chronic obstructive pulmonary disease.     

Increased local levels of granulocyte colony-stimulating factor are associated with the beneficial effect of pre-elafin (SKALP/trappin-2/WAP3) in experimental emphysema

Few therapeutic options are offered to treat inflammation and alveolar wall destruction in emphysema. The effect of recombinant human pre-elafin, an elastase inhibitor, was evaluated in porcine pancreatic elastase (PPE)-induced emphysema in C57BL/6 mice. In a first protocol, mice received a single instillation of pre-elafin (17.5 pmol/mouse) at 1 h post-PPE and were sacrificed up to 72 h post-PPE. A single instillation of pre-elafin significantly reduced PPE-induced neutrophil accumulation in lungs, as assessed by bronchoalveolar lavage (BAL), by 51%, 71% and 67% at 24, 48 and 72 h, respectively. In a second protocol, mice also received a single dose of PPE, but pre-elafin three times a week for 2 weeks. After 2 weeks, pre-elafin significantly reduced the PPE-induced increase in BAL macrophage numbers, airspace dimensions and lung hysteresivity by 74%, 62% and 52%, respectively. Since G-CSF was previously shown to reduce emphysematous changes in mice, the BAL levels of this mediator were measured 6 h post-PPE in animals treated as described in the first protocol. Pre-elafin significantly increased G-CSF levels in PPE-exposed mice compared to sham- and PPE only-exposed animals. This suggests that the beneficial effects of pre-elafin could be mediated, at least in part, by its ability to increase G-CSF levels in the lung.     

Effects of different mesenchymal stromal cell sources and delivery routes in experimental emphysema

We sought to assess whether the effects of mesenchymal stromal cells (MSC) on lung inflammation and remodeling in experimental emphysema would differ according to MSC source and administration route. Emphysema was induced in C57BL/6 mice by intratracheal (IT) administration of porcine pancreatic elastase (0.1 UI) weekly for 1 month. After the last elastase instillation, saline or MSCs (1×10⁵), isolated from either mouse bone marrow (BM), adipose tissue (AD) or lung tissue (L), were administered intravenously (IV) or IT. After 1 week, mice were euthanized. Regardless of administration route, MSCs from each source yielded: 1) decreased mean linear intercept, neutrophil infiltration, and cell apoptosis; 2) increased elastic fiber content; 3) reduced alveolar epithelial and endothelial cell damage; and 4) decreased keratinocyte-derived chemokine (KC, a mouse analog of interleukin-8) and transforming growth factor-β levels in lung tissue. In contrast with IV, IT MSC administration further reduced alveolar hyperinflation (BM-MSC) and collagen fiber content (BM-MSC and L-MSC). Intravenous administration of BM- and AD-MSCs reduced the number of M1 macrophages and pulmonary hypertension on echocardiography, while increasing vascular endothelial growth factor. Only BM-MSCs (IV > IT) increased the number of M2 macrophages. In conclusion, different MSC sources and administration routes variably reduced elastase-induced lung damage, but IV administration of BM-MSCs resulted in better cardiovascular function and change of the macrophage phenotype from M1 to M2.     

Elastase induced lung epithelial cell apoptosis and emphysema through placenta growth factor

Chronic pulmonary obstructive disease (COPD) is the fourth leading cause of death worldwide, however, the pathogenic factors and mechanisms are not fully understood. Pulmonary emphysema is one of the major components of COPD and is thought to result from oxidative stress, chronic inflammation, protease–antiprotease imbalance and lung epithelial (LE) cell apoptosis. In our previous studies, COPD patients were noted to have higher levels of placenta growth factor (PlGF) in serum and bronchoalveolar lavage fluid than controls. In addition, transgenic mice overexpressing PlGF developed pulmonary emphysema and exposure to PlGF in LE cells induced apoptosis. Furthermore, intratracheal instillation of porcine pancreatic elastase (PPE) on to PlGF wild type mice induced emphysema, but not in PlGF knockout mice. Therefore, we hypothesized that PPE generates pulmonary emphysema through the upregulation of PlGF expression in LE cells. The elevation of PlGF then leads to LE cell apoptosis. In the present study, we investigated whether PPE induces PlGF expression, whether PlGF induces apoptosis and whether the downstream mechanisms of PlGF are related to LE cell apoptosis. We found that PPE increased PlGF secretion and expression both in vivo and in vitro. Moreover, PlGF-induced LE cell apoptosis and PPE-induced emphysema in the mice were mediated by c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) pathways. Given these findings, we suggest that the increase in PlGF and PlGF-induced JNK and p38 MAPK pathways contribute to PPE-induced LE cell apoptosis and emphysema. Regulatory control of PlGF and agents against its downstream signals may be potential therapeutic targets for COPD.     

Evaluation of emphysema severity and progression in a rabbit model: comparison of hyperpolarized 3He and 129Xe diffusion MRI with lung morphometry.

The apparent diffusion coefficients (ADCs) of hyperpolarized (3)He and (129)Xe gases were measured in the lungs of rabbits with elastase-induced emphysema and correlated against the mean chord length from lung histology. In vivo measurements were performed at baseline and 2, 4, 6, and 8 wk after instillation of elastase (mild and moderate emphysema groups) or saline (control group). ADCs were determined from acquisitions that used two b values. To investigate the effect of b value on the results, b-value pairs of 0 and 1.6 s/cm(2) and 0 and 4.0 s/cm(2) were used for (3)He, and b-value pairs of 0 and 5.0 s/cm(2) and 0 and 10.0 s/cm(2) were used for (129)Xe. At 8 wk after instillation, the rabbits were euthanized, and the lungs were analyzed histologically and morphometrically. ADCs for the rabbits in the control group did not change significantly from baseline to week 8, whereas ADCs for the rabbits in the emphysema groups increased significantly (P < 0.05) for all gas and b-value combinations except (129)Xe with the b-value pair of 0 and 5.0 s/cm(2). The largest percent change in mean ADC from baseline to week 8 (15.3%) occurred with (3)He and the b-value pair of 0 and 1.6 s/cm(2) for rabbits in the moderate emphysema group. ADCs (all b values) were strongly correlated (r = 0.62-0.80, P < 0.001) with mean chord lengths from histology. These results further support the ability of diffusion-weighted MRI with hyperpolarized gases to detect regional and global structural changes of emphysema within the lung.     

Inflammation, endothelial injury, and persistent pulmonary hypertension in heterozygous BMPR2-mutant mice

Heterozygous bone morphogenetic protein receptor-II-knockout (BMPR2(+/-)) mice have a similar genetic trait like that in some idiopathic pulmonary arterial hypertension patients. To examine the effect of pulmonary endothelial injury in BMPR2(+/-) mice, we challenged the mice with two injections of monocrotaline combined with intratracheal instillation of replication-deficient adenovirus expressing 5-lipoxygenase (MCT+Ad5LO). After the challenge (1 wk), BMPR2(+/-) mice exhibited a doubling of right ventricular systolic pressure that was greater than that of wild-type mice and remained elevated for 3 wk before heart failure developed. Muscularization and thickening of small pulmonary arterioles was evident in the BMPR2(+/-) lungs at 2 wk after the challenge and became severe at 3 wk. Marked perivascular infiltration of T cells, B cells, and macrophages was associated with the remodeled vessels. Real-time PCR analysis showed that the expression of six endothelial cell markers in lung tissue was decreased to 20-40% of original levels at 1 wk after the challenge in both BMPR2(+/-) and wild-type mice and largely recovered in wild-type (50-80%) but not BMPR2(+/-) lungs (30-50%) at 3 wk after the challenge. Macrophage inflammatory protein-1alpha and fractalkine receptor expression doubled in BMPR2(+/-) compared with wild-type lungs. Expression of type I and type II BMP receptors, but not transforming growth factor-beta receptors, in the challenged BMPR2(+/-) and wild-type lungs showed a similar pattern of expression as that of endothelial markers. Apoptotic responses at 1 wk after MCT and Ad5LO challenge were also significantly greater in the BMPR2(+/-) lungs than the wild-type lungs. These data show that BMPR2(+/-) mice are more sensitive to MCT+Ad5LO-induced pulmonary hypertension than wild-type mice. Greater endothelial injury and an enhanced inflammatory response could be the underlying causes of the sensitivity and may work in concert with BMPR2 heterozygosity to promote the development of persistent pulmonary hypertension.     

Intratracheally administered titanium dioxide or carbon black nanoparticles do not aggravate elastase-induced pulmonary emphysema in rats

ABSTRACT: BACKGROUND: Titanium dioxide (TiO2) and carbon black (CB) nanoparticles (NPs) have biological effects that could aggravate pulmonary emphysema. The aim of this study was to evaluate whether pulmonary administration of TiO2 or CB NPs in rats could induce and/or aggravate elastase-induced emphysema, and to investigate the underlying molecular mechanisms. METHODS: On day 1, Sprague-Dawley rats were intratracheally instilled with 25 U kg1 pancreatic porcine elastase or saline. On day 7, they received an intratracheal instillation of TiO2 or CB (at 100 and 500 mug) dispersed in bovine serum albumin or bovine serum albumin alone. Animals were sacrificed at days 8 or 21, and bronchoalveolar lavage (BAL) cellularity, histological analysis of inflammation and emphysema, and lung mRNA expression of heme oxygenase-1 (HO-1), interleukin-1beta (IL-1beta), macrophage inflammatory protein-2, monocyte chemotactic protein-1, and matrix metalloprotease (MMP)-1, and -12 were measured. In addition, pulmonary MMP-12 expression was also analyzed at the protein level by immunohistochemistry. RESULTS: TiO2 NPs per se did not modify the parameters investigated, but CB NPs increased perivascular/peribronchial infiltration, and macrophage MMP-12 expression, without inducing emphysema. Elastase administration increased BAL cellularity, histological inflammation, HO-1, IL-1beta and macrophage MMP-12 expression and induced emphysema. Exposure to TiO2 NPs did not modify pulmonary responses to elastase, but exposure to CB NPs aggravated elastase-induced histological inflammation without aggravating emphysema. CONCLUSIONS: TiO2 and CB NPs did not aggravate elastase-induced emphysema. However, CB NPs induced histological inflammation and MMP-12 mRNA and protein expression in macrophages.     

Thioredoxin prevents the development and progression of elastase-induced emphysema

Thioredoxin 1 (TRX1) is a redox (reduction/oxidation)-active protein that scavenges reactive oxygen species. Here we examined whether endogenous or exogenous administration of TRX1 prevented the development and progression of elastase-induced pulmonary emphysema. Mice were treated with intratracheal elastase via microspray on day 0, and were given recombinant human TRX1 (rhTRX1) every other day from days -1 to 21. To determine the effects of TRX1 on the progression of established emphysema, mice were treated intratracheally with elastase on day 0, and rhTRX1 was administered from days 14 to 21. Histopathologic examination was performed on day 21. TRX1-transgenic but not transgene-negative mice demonstrated a decrease in the physiological indicators of elastase-induced emphysema. TRX1 administration from days -1 to 19 significantly decreased the signs of elastase-induced emphysema. Moreover, TRX1 administration beginning 14 days after elastase treatment significantly slowed the progression of emphysema. TRX1 may be of clinical benefit for the treatment of COPD.     

TPPU enhanced exercise‐induced epoxyeicosatrienoic acid concentrations to exert cardioprotection in mice after myocardial infarction

Exercise training (ET) is a safe and efficacious therapeutic approach for myocardial infarction (MI). Given the numerous benefits of exercise, exercise‐induced mediators may be promising treatment targets for MI. C57BL/6 mice were fed 1‐trifluoromethoxyphenyl‐3‐(1‐propionylpiperidine‐4‐yl) urea (TPPU), a novel soluble epoxide hydrolase inhibitor (sEHI), to increase epoxyeicosatrienoic acid (EET) levels, for 1 week before undergoing MI surgery. After 1‐week recovery, the mice followed a prescribed exercise programme. Bone marrow‐derived endothelial progenitor cells (EPCs) were isolated from the mice after 4 weeks of exercise and cultured for 7 days. Angiogenesis around the ischaemic area, EPC functions, and the expression of microRNA‐126 (miR‐126) and its target gene Spred1 were measured. The results were confirmed in vitro by adding TPPU to EPC culture medium. ET significantly increased serum EET levels and promoted angiogenesis after MI. TPPU enhanced the effects of ET to reduce the infarct area and improve cardiac function after MI. ET increased EPC function and miR‐126 expression, which were further enhanced by TPPU, while Spred1 expression was significantly down‐regulated. Additionally, the protein kinase B/glycogen synthase kinase 3β (AKT/GSK3β) signalling pathway was activated after the administration of TPPU. EETs are a potential mediator of exercise‐induced cardioprotection in mice after MI. TPPU enhances exercise‐induced cardiac recovery in mice after MI by increasing EET levels and promoting angiogenesis around the ischaemic area.     

Role of CD69 in the pathogenesis of elastase-induced pulmonary inflammation and emphysema

Cluster of differentiation 69 (CD69), known as an early activation marker of lymphocytes, has been demonstrated to regulate inflammatory events in various disease models. Although the increased number of CD69-expressed T lymphocytes in the lungs of patients with chronic obstructive pulmonary disease (COPD) has been reported, a functional role of CD69 in the pathogenesis of COPD remains unknown. To address to this question, CD69-deficient (CD69KO) mice and wild-type (WT) mice were subjected to a mouse model of porcine pancreatic elastase (PPE)-induced pulmonary inflammation and emphysema. In the two genotypes, PPE increased counts of macrophages, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF) and induced emphysematous changes in the lung, whereas those two pathological signs were significantly enhanced in CD69KO mice compared to WT mice. Moreover, the PPE-induced levels of IL-17 and IL-6 in BALF were significantly higher in CD69KO mice than in WT mice at the acute inflammatory phase. Immunofluorescent studies showed that IL-17 and IL-6 were predominantly expressed in CD4⁺ and γδ T cells and macrophages, respectively. Concomitant administration of IL-17- and IL-6-neutralizing antibodies significantly attenuated the PPE-induced emphysematous changes in the two genotypes. These findings suggest that CD69 negatively regulates the development of PPE-induced emphysema in part at least through modulating function of IL-17-producing T cells.     

Nrf2 attenuates inflammatory response in COPD/emphysema: Crosstalk with Wnt3a/β‐catenin and AMPK pathways

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and abnormal inflammatory response. Wnt/β‐catenin and AMP‐activated protein kinase (AMPK) have been shown to modulate lung inflammatory responses and injury. However, it remains elusive whether Wnt/β‐catenin and AMPK modulate nuclear factor erythroid‐2 related factor‐2 (Nrf2)‐mediated protective responses during the development of emphysema. Here we showed that treatment with a Wnt pathway activator (LiCl) reduced elastase‐induced airspace enlargement and cigarette smoke extract (CSE)‐induced lung inflammatory responses in WT mice, which was associated with increased activation of Nrf2 pathway. Interestingly, these effects of LiCl were not observed in Nrf2^?/? mice exposed to elastase. In normal human bronchial epithelial (NHBE) cells, Wnt3a overexpression up‐regulated, whereas Wnt3a knockdown further down‐regulated the levels of Nrf2 and its target proteins heme oxygenase‐1 (HO‐1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) by CSE treatment. In contrast, Nrf2 deficiency did not have any effects on Wnt/β‐catenin pathway in mouse lungs and NHBE cells. Both elastase and CSE exposures reduced AMPK phosphorylation. A specific AMPK activator metformin increased Wnt3a, β‐catenin, Nrf2 phosphorylation and activation but reduced the levels of IL‐6 and IL‐8 in NHBE cells and mouse lungs exposed to CSE. Furthermore, Nrf2 deficiency abolished the protection of metformin against CSE‐induced increase in IL‐6 and IL‐8 in NHBE cells. In conclusion, Nrf2 mediates the protective effects of both Wnt3a/β‐catenin and AMPK on lung inflammatory responses during the development of COPD/emphysema. These findings provide potential therapeutic targets for the intervention of COPD/emphysema.