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.     

Proliferative Activity of Liver Growth Factor is Associated with an Improvement of Cigarette Smoke-Induced Emphysema in Mice

Cigarette smoke (CS)-induced emphysema is a major component of chronic obstructive pulmonary disease (COPD). COPD treatment is based on the administration of bronchodilators and corticosteroids to control symptoms and exacerbations, however, to date, there are no effective therapies to reverse disease progression. Liver growth factor (LGF) is an albumin-bilirubin complex with mitogenic properties, whose therapeutic effects have previously been reported in a model of emphysema and several rodent models of human disease. To approach the therapeutic effect of LGF in a model of previously established emphysema, morphometric and lung function parameters, matrix metalloproteinase (MMP) activity and the expression of several markers, such as VEGF, PCNA, 3NT and Nrf2, were assessed in air-exposed and CS-exposed C57BL/6J male mice with and without intraperitoneal (i.p.) injection of LGF. CS-exposed mice presented a significant enlargement of alveolar spaces, higher alveolar internal area and loss of lung function that correlated with higher MMP activity, higher expression of 3NT and lower expression of VEGF. CS-exposed mice injected with LGF, showed an amelioration of emphysema and improved lung function, which correlated with lower MMP activity and 3NT expression and higher levels of VEGF, PCNA and Nrf2. Taken together, this study suggests that LGF administration ameliorates CS-induced emphysema, highlights the ability of LGF to promote alveolar cell proliferation and may be a promising strategy to revert COPD progression.     

Transforming growth factor‐beta 1 (TGF‐β1) induces angiogenesis through vascular endothelial growth factor (VEGF)‐mediated apoptosis

VEGF and TGF‐β1 induce angiogenesis but have opposing effects on endothelial cells. VEGF protects endothelial cells from apoptosis; TGF‐β1 induces apoptosis. We have previously shown that VEGF/VEGF receptor‐2 (VEGFR2) signaling mediates TGF‐β1 induction of apoptosis. This finding raised an important question: Does this mechanism stimulate or inhibit angiogenesis? Here we report that VEGF‐mediated apoptosis is required for TGF‐β1 induction of angiogenesis. In vitro the apoptotic effect of TGF‐β1 on endothelial cells is rapid and followed by a long period in which the cells are refractory to apoptosis induction by TGF‐β1. Inhibition of VEGF/VEGFR2 signaling abrogates formation of cord‐like structures by TGF‐β1 with an effect comparable to that of z‐VAD, an apoptosis inhibitor. Similarly, genetic deficiency of VEGF abolishes TGF‐β1 upregulation of endothelial cell differentiation and formation of vascular structures in embryoid bodies. In vivo TGF‐β1 induces endothelial cell apoptosis as rapidly as in vitro. Inhibition of VEGF blocks TGF‐β1 induction of both apoptosis and angiogenesis, an effect similar to that of z‐VAD. Thus, TGF‐β1 induction of angiogenesis requires a rapid and transient apoptotic effect mediated by VEGF/VEGFR2. This novel, unexpected role of VEGF and VEGFR2 indicates VEGF‐mediated apoptosis as a potential target to control angiogenesis. J. Cell. Physiol. 219: 449–458, 2009. © 2009 Wiley‐Liss, Inc.     

Mesenchymal stem cell–derived conditioned medium attenuate angiotensin II‐induced aortic aneurysm growth by modulating macrophage polarization

Mesenchymal stem cells (MSCs) exhibit therapeutic benefits on aortic aneurysm (AA); however, the molecular mechanisms are not fully understood. The current study aimed to investigate the therapeutic effects and potential mechanisms of murine bone marrow MSC (BM‐MSCs)–derived conditioned medium (MSCs‐CM) on angiotensin II (AngII)‐induced AA in apolipoprotein E‐deficient (apoE^?/?) mice. Murine BM‐MSCs, MSCs‐CM or control medium were intravenously administrated into AngII‐induced AA in apoE^?/? mice. Mice were sacrificed at 2 weeks after injection. BM‐MSCs and MSCs‐CM significantly attenuated matrix metalloproteinase (MMP)‐2 and MMP‐9 expression, aortic elastin degradation and AA growth at the site of AA. These treatments with BM‐MSCs and MSCs‐CM also decreased Ly6c^high monocytes in peripheral blood on day 7 and M1 macrophage infiltration in AA tissues on day 14, whereas they increased M2 macrophages. In addition, BM‐MSCs and MSCs‐CM reduced MCP‐1, IL‐1Ra and IL‐6 expression and increased IL‐10 expression in AA tissues. In vitro, peritoneal macrophages were co‐cultured with BM‐MSCs or fibroblasts as control in a transwell system. The mRNA and protein expression of M2 macrophage markers were evaluated. IL‐6 and IL‐1β were reduced, while IL‐10 was increased in the BM‐MSC systems. The mRNA and protein expression of M2 markers were up‐regulated in the BM‐MSC systems. Furthermore, high concentration of IGF1, VEGF and TGF‐β1 was detected in MSCs‐CM. Our results suggest that MSCs‐CM could prevent AA growth potentially through regulating macrophage polarization. These results may provide a new insight into the mechanisms of BM‐MSCs in the therapy of AA.     

Recuperating lung decoction attenuates inflammation and oxidation in cigarette smoke‐induced COPD in rats via activation of ERK and Nrf2 pathways

Oxidative/antioxidative imbalance and chronic inflammation are the main contributors to the pathogenesis of chronic obstructive pulmonary disease (COPD). This study evaluated the effect of recuperating lung decoction (RLD) on inflammation and oxidative stress in rats with COPD induced by cigarette smoke and lipopolysaccharides (LPS). We used intravenous infusion of LPS combined with cigarette smoke exposure as a COPD rat model. We observed that RLD treatment increased the protein level of GSH and the ratio of GSH/GSSG but decreased 8‐OHdG and 4‐HNE in the serum. Furthermore, RLD significantly inhibited the expressions of IL‐1β, IL‐6, TNF‐α, and TGF‐β induced by cigarette smoke exposure, reduced the number of inflammatory cells in the bronchoalveolar lavage fluid, and alleviated the severity of cigarette smoke‐induced emphysema. Mechanistically, RLD treatment prevented disease through downregulation of phosphorylated‐ERK and Nrf2 expression, which regulates the production of proinflammatory cytokines. RLD treatment exerted a dramatic therapeutic effect on COPD. This study revealed a mechanism that RLD functions on the regulation of ERK signalling to inhibit inflammation.     

Macrophages modulate the viability and growth of human mesenchymal stem cells

Following myocardial infarction, tissue repair is mediated by the recruitment of monocytes and their subsequent differentiation into macrophages. Recent findings have revealed the dynamic changes in the presence of polarized macrophages with pro‐inflammatory (M1) and anti‐inflammatory (M2) properties during the early (acute) and late (chronic) stages of cardiac ischemia. Mesenchymal stem cells (MSCs) delivered into the injured myocardium as reparative cells are subjected to the effects of polarized macrophages and the inflammatory milieu. The present study investigated how cytokines and polarized macrophages associated with pro‐inflammatory (M1) and anti‐inflammatory (M2) responses affect the survival of MSCs. Human MSCs were studied using an in vitro platform with individual and combined M1 and M2 cytokines: IL‐1β, IL‐6, TNF‐α, and IFN‐γ (for M1), and IL‐10, TGF‐β1, TGF‐β3, and VEGF (for M2). In addition, polarization molecules (M1: LPS and IFN‐γ; M2: IL‐4 and IL‐13) and common chemokines (SDF‐1 and MCP‐1) found during inflammation were also studied. Indirect and direct co‐cultures were conducted using M1 and M2 polarized human THP‐1 monocytes. M2 macrophages and their associated cytokines supported the growth of hMSCs, while M1 macrophages and their associated cytokines inhibited the growth of hMSCs in vitro under certain conditions. These data imply that an anti‐inflammatory (M2) environment is more accommodating to the therapeutic hMSCs than a pro‐inflammatory (M1) environment at specific concentrations. J. Cell. Biochem. 114: 220–229, 2012. © 2012 Wiley Periodicals, Inc.     

iPSC‐derived mesenchymal stem cells exert SCF‐dependent recovery of cigarette smoke‐induced apoptosis/proliferation imbalance in airway cells

Mesenchymal stem cells (MSCs) have emerged as a potential cell‐based therapy for pulmonary emphysema in animal models. Our previous study demonstrated that human induced pluripotent stem cell–derived MSCs (iPSC‐MSCs) were superior over bone marrow–derived MSCs (BM‐MSCs) in attenuating cigarette smoke (CS)‐induced airspace enlargement possibly through mitochondrial transfer. This study further investigated the effects of iPSC‐MSCs on inflammation, apoptosis, and proliferation in a CS‐exposed rat model and examined the effects of the secreted paracrine factor from MSCs as another possible mechanism in an in vitro model of bronchial epithelial cells. Rats were exposed to 4% CS for 1 hr daily for 56 days. At days 29 and 43, human iPSC‐MSCs or BM‐MSCs were administered intravenously. We observed significant attenuation of CS‐induced elevation of circulating 8‐isoprostane and cytokine‐induced neutrophil chemoattractant‐1 after iPSC‐MSC treatment. In line, a superior capacity of iPSC‐MSCs was also observed in ameliorating CS‐induced infiltration of macrophages and neutrophils and apoptosis/proliferation imbalance in lung sections over BM‐MSCs. In support, the conditioned medium (CdM) from iPSC‐MSCs ameliorated CS medium‐induced apoptosis/proliferation imbalance of bronchial epithelial cells in vitro. Conditioned medium from iPSC‐MSCs contained higher level of stem cell factor (SCF) than that from BM‐MSCs. Deprivation of SCF from iPSC‐MSC‐derived CdM led to a reduction in anti‐apoptotic and pro‐proliferative capacity. Taken together, our data suggest that iPSC‐MSCs may possess anti‐apoptotic/pro‐proliferative capacity in the in vivo and in vitro models of CS‐induced airway cell injury partly through paracrine secretion of SCF.     

Intratracheal mesenchymal stem cell administration attenuates monocrotaline-induced pulmonary hypertension and endothelial dysfunction

The administration of mesenchymal stem cells (MSCs) has been proposed for the treatment of pulmonary hypertension. However, the effect of intratracheally administered MSCs on the pulmonary vascular bed in monocrotaline-treated rats has not been determined. In the present study, the effect of intratracheal administration of rat MSCs (rMSCs) on monocrotaline-induced pulmonary hypertension and impaired endothelium-dependent responses were investigated in the rat. Intravenous injection of monocrotaline increased pulmonary arterial pressure and vascular resistance and decreased pulmonary vascular responses to acetylcholine without altering responses to sodium nitroprusside and without altering systemic responses to the vasodilator agents when responses were evaluated at 5 wk. The intratracheal injection of 3 x 10(6) rMSCs 2 wk after administration of monocrotaline attenuated the rise in pulmonary arterial pressure and pulmonary vascular resistance and restored pulmonary responses to acetylcholine toward values measured in control rats. Treatment with rMSCs decreased the right ventricular hypertrophy induced by monocrotaline. Immunohistochemical studies showed widespread distribution of lacZ-labeled rMSCs in lung parenchyma surrounding airways in monocrotaline-treated rats. Immunofluorescence studies revealed that transplanted rMSCs retained expression of von Willebrand factor and smooth muscle actin markers specific for endothelial and smooth muscle phenotypes. However, immunolabeled cells were not detected in the wall of pulmonary vessels. These data suggest that the decrease in pulmonary vascular resistance and improvement in response to acetylcholine an endothelium-dependent vasodilator in monocrotaline-treated rats may result from a paracrine effect of the transplanted rMSCs in lung parenchyma, which improves vascular endothelial function in the monocrotaline-injured lung.     

Long non‐coding RNA TUG1 promotes airway remodelling by suppressing the miR‐145‐5p/DUSP6 axis in cigarette smoke‐induced COPD

Chronic obstructive pulmonary disease (COPD) is a progressive lung disease that is primarily caused by cigarette smoke (CS)‐induced chronic inflammation. In this study, we investigated the function and mechanism of action of the long non‐coding RNA (lncRNA) taurine‐up‐regulated gene 1 (TUG1) in CS‐induced COPD. We found that the expression of TUG1 was significantly higher in the sputum cells and lung tissues of patients with COPD as compared to that in non‐smokers, and negatively correlated with the percentage of predicted forced expiratory volume in 1 second. In addition, up‐regulation of TUG1 was observed in CS‐exposed mice, and knockdown of TUG1 attenuated inflammation and airway remodelling in a mouse model. Moreover, TUG1 expression was higher in CS extract (CSE)‐treated human bronchial epithelial cells and lung fibroblasts, whereas inhibition of TUG1 reversed CSE‐induced inflammation and collagen deposition in vitro. Mechanistically, TUG1 promoted the expression of dual‐specificity phosphatase 6 (DUSP6) by sponging miR‐145‐5p. DUSP6 overexpression reversed TUG1 knockdown‐mediated inhibition of inflammation and airway remodelling. These findings suggested an important role of TUG1 in the pathological alterations associated with CS‐mediated airway remodelling in COPD. Thus, TUG1 may be a promising therapeutic target in CS‐induced airway inflammation and fibroblast activation.     

Cigarette smoke disrupts VEGF165-VEGFR-2 receptor signaling complex in rat lungs and patients with COPD: morphological impact of VEGFR-2 inhibition

VEGF is fundamental in the development and maintenance of the vasculature. VEGF(165) signaling through VEGF receptor (VEGFR)-2/kinase insert domain receptor (KDR) is a highly regulated process involving the formation of a tertiary complex with glypican (GYP)-1 and neuropilin (NRP)-1. Both VEGF and VEGFR-2 expression are reduced in emphysematous lungs; however, the mechanism of regulation of VEGF(165) signaling through the VEGFR-2 complex in response to cigarette smoke exposure in vivo, and in smokers with and without chronic obstructive pulmonary disease (COPD), is still unknown. We hypothesized that cigarette smoke exposure disrupts the VEGF(165)-VEGFR-2 complex, a potential mechanism in the pathogenesis of emphysema. We show that cigarette smoke exposure reduces NRP-1 and GYP-1 as well as VEGF and VEGFR-2 levels in rat lungs and that VEGF, VEGFR-2, GYP-1, and NRP-1 expression in the lungs of both smokers and patients with COPD are also reduced compared with nonsmokers. Moreover, our data suggest that specific inhibition of VEGFR-2 alone with NVP-AAD777 would appear not to result in emphysema in the adult rat lung. As both VEGF(165) and VEGFR-2 expression are reduced in emphysematous lungs, decreased GYP-1 and NRP-1 expression may yet further disrupt VEGF(165)-VEGFR-2 signaling. Whether or not this by itself is critical for inducing endothelial cell apoptosis and decreased vascularization of the lung seen in emphysema patients is still unclear at present. However, targeted therapies to restore VEGF(165)-VEGFR-2 complex may promote endothelial cell survival and help to ameliorate emphysema.     

Inhibition of histone deacetylase causes emphysema

In patients with chronic obstructive pulmonary disease (COPD), histone deacetylase (HDAC) expression and activity are reduced in the lung tissue. However, whether HDAC activity controls the maintenance of the lung alveolar septal structures has not been investigated. To explore the consequences of HDAC inhibition and address the question of whether HDAC inhibition causes lung cell apoptosis and emphysema, male Sprague-Dawley rats and human pulmonary microvascular endothelial cells (HPMVEC) were treated with trichostatin A (TSA), a specific inhibitor of HDACs. Chronic TSA treatment increased the alveolar air space area, mean linear intercept, and the number of caspase-3-positive cells in rat lungs. TSA suppressed hypoxia-inducible factor-1α (HIF-1α), VEGF, and lysyl oxidase (LOX) and increased microtubule-associated protein-1 light chain 3 (LC3), p53, and miR34a microRNA expression in both rat lungs and cultured HPMVEC. Gene silencing of HDAC2 using small interfering RNA (siRNA) in cultured HPMVEC resulted in the suppression of HIF-1α, VEGF, and LOX and an increase of p53 expression. These data indicate that HDAC inhibition causes emphysema and that HDAC-dependent mechanisms contribute to the maintenance of the adult lung structure. Our results also suggest that the increase in apoptosis, as a consequence of HDAC inhibition, is associated with decreased VEGF and HIF-1α expression.     

Thioredoxin-1 Protects against Neutrophilic Inflammation and Emphysema Progression in a Mouse Model of Chronic Obstructive Pulmonary Disease Exacerbation

Background

Exacerbations of chronic obstructive pulmonary disease (COPD) are characterized by acute enhancement of airway neutrophilic inflammation under oxidative stress and can be involved in emphysema progression. However, pharmacotherapy against the neutrophilic inflammation and emphysema progression associated with exacerbation has not been established. Thioredoxin-1 has anti-oxidative and anti-inflammatory properties and it can ameliorate neutrophilic inflammation through anti-chemotactic effects and prevent cigarette smoke (CS)-induced emphysema. We aimed to determine whether thioredoxin-1 can suppress neutrophilic inflammation and emphysema progression in a mouse model of COPD exacerbation and if so, to reveal the underlying mechanisms.

Results

Mice were exposed to CS and then challenged with polyinosine-polycytidylic acid [poly(I:C)], an agonist for virus-induced innate immunity. Airway neutrophilic inflammation, oxidative stress and lung apoptosis were enhanced in smoke-sensitive C57Bl/6, but not in smoke-resistant NZW mice. Exposure to CS and poly(I:C) challenge accelerated emphysema progression in C57Bl/6 mice. Thioredoxin-1 suppressed neutrophilic inflammation and emphysema progression. Poly(I:C) caused early neutrophilic inflammation through keratinocyte-derived chemokine and granulocyte-macrophage colony-stimulating factor (GM-CSF) release in the lung exposed to CS. Late neutrophilic inflammation was caused by persistent GM-CSF release, which thioredoxin-1 ameliorated. Thioredoxin-1 enhanced pulmonary mRNA expression of MAP kinase phosphatase 1 (MKP-1), and the suppressive effects of thioredoxin-1 on prolonged GM-CSF release and late neutrophilic inflammation disappeared by inhibiting MKP-1.

Conclusion

Using a mouse model of COPD exacerbation, we demonstrated that thioredoxin-1 ameliorated neutrophilic inflammation by suppressing GM-CSF release, which prevented emphysema progression. Our findings deepen understanding of the mechanisms underlying the regulation of neutrophilic inflammation by thioredoxin-1 and indicate that thioredoxin-1 could have potential as a drug to counteract COPD exacerbation.     

Cigarette smoke induces PTX3 expression in pulmonary veins of mice in an IL-1 dependent manner

Background

Chronic obstructive pulmonary disease (COPD) is associated with abnormal inflammatory responses and structural alterations of the airways, lung parenchyma and pulmonary vasculature. Since Pentraxin-3 (PTX3) is a tuner of inflammatory responses and is produced by endothelial and inflammatory cells upon stimuli such as interleukin-1β (IL-1β), we hypothesized that PTX3 is involved in COPD pathogenesis.

Methods and Results

We evaluated whether cigarette smoke (CS) triggers pulmonary and systemic PTX3 expression in vivo in a murine model of COPD. Using immunohistochemical (IHC) staining, we observed PTX3 expression in endothelial cells of lung venules and veins but not in lung arteries, airways and parenchyma. Moreover, ELISA on lung homogenates and semi-quantitative scoring of IHC-stained sections revealed a significant upregulation of PTX3 upon subacute and chronic CS exposure. Interestingly, PTX3 expression was not enhanced upon subacute CS exposure in IL-1RI KO mice, suggesting that the IL-1 pathway is implicated in CS-induced expression of vascular PTX3. Serum PTX3 levels increased rapidly but transiently after acute CS exposure.To elucidate the functional role of PTX3 in CS-induced responses, we examined pulmonary inflammation, protease/antiprotease balance, emphysema and body weight changes in WT and Ptx3 KO mice. CS-induced pulmonary inflammation, peribronchial lymphoid aggregates, increase in MMP-12/TIMP-1 mRNA ratio, emphysema and failure to gain weight were not significantly different in Ptx3 KO mice compared to WT mice. In addition, Ptx3 deficiency did not affect the CS-induced alterations in the pulmonary (mRNA and protein) expression of VEGF-A and FGF-2, which are crucial regulators of angiogenesis.

Conclusions

CS increases pulmonary PTX3 expression in an IL-1 dependent manner. However, our results suggest that either PTX3 is not critical in CS-induced pulmonary inflammation, emphysema and body weight changes, or that its role can be fulfilled by other mediators with overlapping activities.     

Mesenchymal stem cell transplantation increases expression of vascular endothelial growth factor in papain-induced emphysematous lungs and inhibits apoptosis of lung cells

BackgroundPulmonary emphysema is characterized by loss of alveolar structures. We have found that bone marrow (BM) mesenchymal stem cell (MSC) transplantation ameliorates papain-induced pulmonary emphysema. However, the underlying mechanism is not completely understood. It has been shown that blocking the vascular endothelial growth factor (VEGF) signaling pathway leads to apoptosis of lung cells and pulmonary emphysema, and MSC are capable of secreting VEGF. We hypothesized that MSC transplantation may have a protective effect on pulmonary emphysema by increasing VEGF-A expression and inhibiting apoptosis of lung cells.MethodsWe examined the morphology and expression of VEGF-A in rat lung after papain treatment and MSC transplantation. We also used a co-culture system in which MSC and cells prepared from papain-treated lungs or control lungs were cultured together. The levels of VEGF-A in cells and culture medium were determined, and apoptosis of cultured lung cells was evaluated.ResultsVEGF-A expression in rat lungs was decreased after papain treatment, which was partly rescued by MSC transplantation. MSC production of VEGF-A was increased when MSC were co-cultured with cells prepared from papain-treated lungs. Furthermore, the apoptosis of papain-treated lung cells was inhibited when co-cultured with MSC. The induction of MSC production of VEGF-A by papain-treated lung cells was inhibited by adding anti-tumor necrosis factor (TNF)-α antibody to the medium.ConclusionsThe protective effect of MSC transplantation on pulmonary emphysema may be partly mediated by increasing VEGF-A expression and inhibiting the apoptosis of lung cells. TNF-α released from papain-treated lung cells induces MSC to secret VEGF-A.     

Interleukin‐6 downregulation with mesenchymal stem cell differentiation results in loss of immunoprivilege

Allogeneic mesenchymal stem cell (MSC) transplantation improves cardiac function, but cellular differentiation results in loss of immunoprivilege and rejection. To explore the mechanism involved in this immune rejection, we investigated the influence of interleukin‐6 (IL‐6), a factor secreted by MSCs, on immune privilege after myogenic, endothelial and smooth muscle cell differentiation induced by 5‐azacytidine, VEGF, and transforming growth factor‐β (TGF‐β), respectively. Both RT‐PCR and ELISA showed that myogenic differentiation of MSCs was associated with significant downregulation of IL‐6 expression (P < 0.01), which was also observed following endothelial (P < 0.01) and smooth muscle cell differentiation (P < 0.05), indicating that IL‐6 downregulation was dependent on differentiation but not cell phenotype. Flow cytometry demonstrated that IL‐6 downregulation as a result of myogenic differentiation was associated with increased leucocyte‐mediated cell death in an allogeneic leucocyte co‐culture study (P < 0.01). The allogeneic reactivity associated with IL‐6 downregulation was also observed following MSC differentiation to endothelial and smooth muscle cells (P < 0.01), demonstrating that leucocyte‐mediated cytotoxicity was also dependent on differentiation but not cell phenotype. Restoration of IL‐6 partially rescued the differentiated cells from leucocyte‐mediated cell death. These findings suggest that rejection of allogeneic MSCs after implantation may be because of a reduction in cellular IL‐6 levels, and restoration of IL‐6 may be a new target to retain MSC immunoprivilege.     

Xanthine oxidoreductase is a critical mediator of cigarette smoke-induced endothelial cell DNA damage and apoptosis

Cigarette smoke (CS) exposure is unquestionably the most frequent cause of emphysema in the United States. Accelerated pulmonary endothelial cell (EC) apoptosis is an early determinant of lung destruction in emphysema. One of the pathogenic causes of emphysema is an alveolar oxidant and antioxidant imbalance. The enzyme xanthine oxidoreductase (XOR) has been shown to be a source of reactive oxygen species (ROS) in a multitude of diseases (S. Sakao et al., FASEB J. 21, 3640–3652; 2007). The contribution of XOR to CS-induced apoptosis is not well defined. Here we demonstrate that C57/bl6 mice exposed to CS have increased pulmonary XOR activity and protein levels compared to filtered-air-exposed controls. In addition, we demonstrate that primary pulmonary human lung microvascular endothelial cells exposed to cigarette smoke extract undergo increased rates of caspase-dependent apoptosis that are reliant on XOR activity, ROS production, and p53 function/expression. We also demonstrate that exogenous XOR is sufficient to increase p53 expression and induce apoptosis, suggesting that XOR is an upstream mediator of p53 in CS-induced EC apoptosis. Furthermore, we show that XOR activation results in DNA double-strand breaks that activate the enzyme ataxia telangiectasia mutated, which phosphorylates histone H2AX and upregulates p53. In conclusion, CS increases XOR expression, and the enzyme is both sufficient and necessary for p53 induction and CS-induced EC apoptosis.     

Cirsium japonicum var. maackii and apigenin block Hif‐2α‐induced osteoarthritic cartilage destruction

Although Hif‐2α is a master regulator of catabolic factor expression in osteoarthritis development, Hif‐2α inhibitors remain undeveloped. The aim of this study was to determine whether Cirsium japonicum var. maackii (CJM) extract and one of its constituents, apigenin, could attenuate the Hif‐2α‐induced cartilage destruction implicated in osteoarthritis progression. In vitro and in vivo studies demonstrated that CJM reduced the IL‐1β‐, IL‐6, IL‐17‐ and TNF‐α‐induced up‐regulation of MMP3, MMP13, ADAMTS4, ADAMTS5 and COX‐2 and blocked osteoarthritis development in a destabilization of the medial meniscus mouse model. Activation of Hif‐2α, which directly up‐regulates MMP3, MMP13, ADAMTS4, IL‐6 and COX‐2 expression, is inhibited by CJM extract. Although cirsimarin, cirsimaritin and apigenin are components of CJM and can reduce inflammation, only apigenin effectively reduced Hif‐2α expression and inhibited Hif‐2α‐induced MMP3, MMP13, ADAMTS4, IL‐6 and COX‐2 expression in articular chondrocytes. IL‐1β induction of JNK phosphorylation and IκB degradation, representing a critical pathway for Hif‐2α expression, was completely blocked by apigenin in a concentration‐dependent manner. Collectively, these effects indicate that CJM and one of its most potent constituents, apigenin, can lead to the development of therapeutic agents for blocking osteoarthritis development as novel Hif‐2α inhibitors.