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.     

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.     

Reversal of elastase-induced pulmonary emphysema and promotion of alveolar epithelial cell proliferation by simvastatin in mice

Besides lowering cholesterol, statins exert multiple effects, such as anti-inflammatory activity and improvement of endothelial cell function. We examined whether simvastatin (SS) protects against the development of elastase-induced pulmonary emphysema in mice by using mean linear intercepts of alveoli (Lm) as a morphometric parameter of emphysema. After injection of intratracheal elastase on day 0, C57BL/6 mice were treated daily with SS (SS+ group) or PBS (SS- group) for 2 wk. A 21% decrease in Lm on day 7 was observed in the SS+ group vs. the SS- group. Anti-inflammatory effects of SS were observed as a decrease in percentage of neutrophils up to day 3, and in hydroxyproline concentration on day 3, in bronchoalveolar lavage fluid (BALF). SS also increased the number of proliferating cell nuclear antigen (PCNA)-positive alveolar epithelial cells between days 3 and 14. To confirm the role of statins in promoting proliferation of alveolar cells, mice were treated with SS (SS+) vs. PBS (SS-) for 12 days, starting 3 wk after elastase administration. After SS treatment, Lm decreased by 52% and PCNA-positive alveolar epithelial cells increased compared with the SS- group. Concentrations of vascular endothelial growth factor in BALF and endothelial nitric oxide synthase protein expression in pulmonary vessels tended to be higher in the SS+ group vs. the SS- group in this protocol. In conclusion, SS inhibited the development of elastase-induced pulmonary emphysema in mice. This therapeutic effect was due not only to anti-inflammation but also to the promotion of alveolar epithelial cell regeneration, partly mediated by restoring endothelial cell functions.     

Over-expression and refolding of isotopically labeled recombinant catalytic domain of human macrophage elastase (MMP-12) for NMR studies

Human macrophage elastase (MMP-12) plays an important role in inflammatory processes and is involved in a number of physiological or pathological situations, such as conversion of plasminogen into angiostatin, allergic airway inflammation, vascular remodeling or alteration, as well as emphysema, and has been justified as a novel drug target. Here, we report the over-expression in Escherichia coil, purification and refolding of MMP-12 catalytic domain for NMR studies. The primary sequence of expressed protein was identified by means of MALDI-TOF MS, and was confirmed by the MALDI-TOF MS data of trypsin-digested peptides. A significantly optimized protocol has been worked out to prepare 15N and/or 13C-labeled MMP-12 catalytic domain, and the yield of the purified protein is estimated to 10-12 mg from 0.5L of M9 minimal media. Finally, the 15N-1H HSQC spectrum of uniformly 15N-labeled MMP-12 catalytic domain indicates the presence of well-ordered and properly folded protein in a monomeric form.     

Pathogenic role of matrix metalloproteases and their inhibitors in asthma and chronic obstructive pulmonary disease and therapeutic relevance of matrix metalloproteases inhibitors.

Matrix metalloproteinases (MMPs) are an at least 23 member family of calcium and zinc dependent enzymes implicated in many physiological and pathological processes. Asthma, chronic obstructive pulmonary disease (COPD) and emphysema are diseases associated with an inflammation of the airways and lung parenchyma. In this review, we focus on the role played by MMPs in the pathogenesis of inflammation, airway remodelling and alveolar destruction, depicting the observational studies in humans and the experimental studies in animal models. During the course of asthma, MMP-2,-8,-9 and TIMP-1 are expressed at baseline and the allergen exposure or exacerbations of the disease lead to an increase of MMP-9 secretion being at this time much higher than that of TIMP-1, allowing temporarily a matrix damage, possibly followed by abnormal repair. Animal models suggest a predominant role for MMP-9 and MMP-12 in the pathogenesis of pulmonary inflammation and link an absence of MMP-2 to an increased parenchymal inflammation. In COPD and emphysema, human studies indicate an over-secretion of MMP-2,-8,-9 and animal models pointout MMP-1 and MMP-12 as being key players in the pathogenesis of emphysema. Taken together, these data identify specific MMP inhibition as appropriate target for therapeutic intervention in asthma or COPD/emphysema They also strongly argue against the widespread use of large spectrum non specific inhibitors that could be detrimental.     

The effect of hyaluronan on elastic fiber injury in vitro and elastase-induced airspace enlargement in vivo

This laboratory has previously described a method of preventing air-space enlargement in experimental pulmonary emphysema using aerosolized hyaluronan (HA). Although it was found that HA preferentially binds to elastic fibers (which undergo breakdown by elastases in emphysema), it remains to be shown that such attachment actually prevents damage to the fibers. In the current study, cell-free radiolabeled extracellular matrices, derived from rat pleural mesothelial cells, were used to test the ability of low molecular weight ( approximately 100 kDa) streptococcal HA to prevent elastolysis. Coating the matrices with HA significantly decreased elastolysis (P<0.05) induced by porcine pancreatic elastase (43%), human neutrophil elastase (53%), and human macrophage metalloelastase (80%). Concomitant in vivo studies examined the ability of an aerosol preparation of the streptococcal HA to prevent experimental emphysema induced by intratracheal administration of porcine pancreatic elastase. As seen with earlier studies involving bovine tracheal HA, a single aerosol exposure significantly decreased elastase-induced airspace enlargement, as measured by the mean linear intercept (107.5 vs 89.6 microm; P < 0. 05). Furthermore, repeated exposure to the HA aerosol for 1 month did not reveal any morphological changes in the lung. The results provide further evidence that aerosolized HA may be an effective means of preventing pulmonary emphysema and perhaps other lung diseases that involve elastic fiber injury.     

Elastase induces lung epithelial cell autophagy through placental growth factor: A new insight of emphysema pathogenesis

Chronic obstructive pulmonary disease (COPD) is a devastating disease, which is associated with increasing mortality and morbidity. Therefore, there is a need to clearly define the COPD pathogenic mechanism and to explore effective therapies. Previous studies indicated that cigarette smoke (CS) induces autophagy and apoptosis in lung epithelial (LE) cells. Excessive ELANE/HNE (elastase, neutrophil elastase), a factor involved in protease-antiprotease imbalance and the pathogenesis of COPD, causes LE cell apoptosis and upregulates the expression of several stimulus-responsive genes. However, whether or not elastase induces autophagy in LE cell remains unknown. The level of PGF (placental growth factor) is higher in COPD patients than non-COPD controls. We hypothesize that elastase induces PGF expression and causes autophagy in LE cells. In this study, we demonstrated that porcine pancreatic elastase (PPE) induced PGF expression and secretion in LE cells in vitro and in vivo. The activation of MAPK8/JNK1 (mitogen-activated protein kinase 8) and MAPK14/p38alpha MAPK signaling pathways was involved in the PGF mediated regulation of the TSC (tuberous sclerosis complex) pathway and autophagy in LE cells. Notably, PGF-induced MAPK8 and MAPK14 signaling pathways mediated the inactivation of MTOR (mechanistic target of rapamycin), the upregulation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β) and the increase of autophagosome formation in mice. Furthermore, the PPE-induced autophagy promotes further apoptosis in vitro and in vivo. In summary, elastase-induced autophagy promotes LE cell apoptosis and pulmonary emphysema through the upregulation of PGF. PGF and its downstream MAPK8 and MAPK14 signaling pathways are potential therapeutic targets for the treatment of emphysema and COPD.     

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.     

Substrate specificity determinants of human macrophage elastase (MMP-12) based on the 1.1 A crystal structure

The macrophage elastase enzyme (MMP-12) expressed mainly in alveolar macrophages has been identified in the mouse lung as the main destructive agent associated with cigarette smoking, which gives rise to emphysema, both directly via elastin degradation and indirectly by disturbing the proteinase/antiproteinase balance via inactivation of the alpha1-proteinase inhibitor (alpha1-PI), the antagonist of the leukocyte elastase. The catalytic domain of human recombinant MMP-12 has been crystallized in complex with the broad-specificity inhibitor batimastat (BB-94). The crystal structure analysis of this complex, determined using X-ray data to 1.1 A and refined to an R-value of 0.165, reveals an overall fold similar to that of other MMPs. However, the S-shaped double loop connecting strands III and IV is fixed closer to the beta-sheet and projects its His172 side-chain further into the rather hydrophobic active-site cleft, defining the S3 and the S1-pockets and separating them from each other to a larger extent than is observed in other MMPs. The S2-site is planar, while the characteristic S1'-subsite is a continuous tube rather than a pocket, in which the MMP-12-specific Thr215 replaces a Val residue otherwise highly conserved in almost all other MMPs. This alteration might allow MMP-12 to accept P1' Arg residues, making it unique among MMPs. The active-site cleft of MMP-12 is well equipped to bind and efficiently cleave the AlaMetPhe-LeuGluAla sequence in the reactive-site loop of alpha1-PI, as occurs experimentally. Similarities in contouring and particularly a common surface hydrophobicity both inside and distant from the active-site cleft explain why MMP-12 shares many substrates with matrilysin (MMP-7). The MMP-12 structure is an excellent template for the structure-based design of specific inhibitors for emphysema therapy and for the construction of mutants to clarify the role of this MMP.     

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.     

A flavanone from Baccharis retusa (Asteraceae) prevents elastase-induced emphysema in mice by regulating NF-κB,oxidative stress and metalloproteinases

BackgroundPulmonary emphysema is characterized by irreversible airflow obstruction, inflammation, oxidative stress imbalance and lung remodeling, resulting in reduced lung function and a lower quality of life. Flavonoids are plant compounds with potential anti-inflammatory and antioxidant effects that have been used in folk medicine. Our aim was to determine whether treatment with sakuranetin, a flavonoid extracted from the aerial parts of Baccharis retusa, interferes with the development of lung emphysema.MethodsIntranasal saline or elastase was administered to mice; the animals were then treated with sakuranetin or vehicle 2 h later and again on days 7, 14 and 28. We evaluated lung function and the inflammatory profile in bronchoalveolar lavage fluid (BALF). The lungs were removed to evaluate alveolar enlargement, extracellular matrix fibers and the expression of MMP-9, MMP-12, TIMP-1, 8-iso-PGF-2α and p65-NF-κB in the fixed tissues as well as to evaluate cytokine levels and p65-NF-κB protein expression.ResultsIn the elastase-treated animals, sakuranetin treatment reduced the alveolar enlargement, collagen and elastic fiber deposition and the number of MMP-9- and MMP-12-positive cells but increased TIMP-1 expression. In addition, sakuranetin treatment decreased the inflammation and the levels of TNF-α, IL-1β and M-CSF in the BALF as well as the levels of NF-κB and 8-iso-PGF-2α in the lungs of the elastase-treated animals. However, this treatment did not affect the changes in lung function.ConclusionThese data emphasize the importance of oxidative stress and metalloproteinase imbalance in the development of emphysema and suggest that sakuranetin is a potent candidate that should be further investigated as an emphysema treatment. This compound may be useful for counteracting lung remodeling and oxidative stress and thus attenuating the development of emphysema.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0233-3) contains supplementary material, which is available to authorized users.     

The effect of lysozyme on elastase-mediated injury

Previous studies by this laboratory demonstrated that lysozyme is increased in human pulmonary emphysema, and that it preferentially binds to elastic fibers, which undergo degradation in this disease. In the current investigation, the relationship between lysozyme and elastic fiber injury was further examined, both in vitro and in vivo. The effect of exogenously administered egg-white lysozyme on pancreatic elastase-induced injury was determined using a biosynthetically radiolabeled extracellular matrix preparation mainly composed of elastic fibers. Although matrix treated with lysozyme showed attachment of the protein to elastic fibers, there was no significant increase in elastolysis compared with untreated controls following exposure to either 1 microg/ml or 100 ng/ml of pancreatic elastase. However, lysozyme did impair the ability of hyaluronan (HA) to prevent elastase injury to elastic fibers. Matrix samples sequentially treated with lysozyme and HA, then incubated with 1 microg/ml or 100 ng/ml of pancreatic elastase, showed significantly increased elastolysis compared with those treated with HA alone. Since HA is closely associated with elastic fibers in vivo, the ability of lysozyme to enhance elastolysis was further tested in an animal model of emphysema induced by intratracheal administration of porcine pancreatic elastase. Animals exposed to aerosolized lysozyme prior to elastase administration showed significantly increased airspace enlargement. The mean linear intercept of the lysozyme-treated animals was 123 microm compared with 75 microm for controls receiving aerosolized water (P < 0.0001). These findings suggest that lysozyme may not be an innocuous component of the inflammatory response associated with pulmonary emphysema, but may actually play a role in the pathogenesis of the disease.     

Transgenic expression of matrix metalloproteinase-9 causes adult-onset emphysema in mice associated with the loss of alveolar elastin

Matrix metalloproteinase (MMP)-9 has been consistently identified in the lungs of patients with chronic obstructive pulmonary disease (COPD). However, its role in the development of the disease remains undefined. Mice that specifically express human MMP-9 in their macrophages were generated, and morphometric, biochemical, and histological analyses were conducted on the transgenic and littermate control mice over 1 yr to determine the effect of macrophage MMP-9 expression on emphysema formation and lung matrix content. Lung morphometry was normal in transgenic mice at 2 mo of age (mean linear intercept = 50+/-3 littermate mice vs. 51+/-2 transgenic mice). However, after 12 mo of age, the MMP-9 transgenic mice developed significant air space enlargement (mean linear intercept = 53+/-3 littermate mice vs. 61+/-2 MMP-9 transgenic mice; P<0.04). Lung hydroxyproline content was not significantly different between wild-type and transgenic mice, but MMP-9 did significantly decrease alveolar wall elastin at 1 yr of age (4.9+/-0.3% area of alveolar wall in the littermate mice vs. 3.3+/-0.3% area of alveolar wall in the MMP-9 mice; P<0.004). Thus these results establish a central role for MMP-9 in the pathogenesis of this disease by demonstrating that expression of this protease in macrophages can alter the extracellular matrix and induce progressive air space enlargement in mice.     

Development of selective inhibitors and substrate of matrix metalloproteinase-12

Four phosphinic peptide libraries with compounds having the general formula p-Br-Ph-(PO2-CH2)-Xaa'-Yaa'-Zaa'-NH2 have been prepared and screened against 10 matrix metalloproteinases (MMPs). We identified two phosphinic peptides with Ki values of 0.19 and 4.4 nM toward MMP-12 (macrophage elastase) that are more than 2-3 orders of magnitude less potent toward the other MMPs tested. These highly selective MMP-12 inhibitors contain a Glu-Glu motif in their Yaa'-Zaa' positions. Incorporation of this Glu-Glu motif into the sequence of a nonspecific fluorogenic peptide cleaved by MMPs provides a highly selective substrate for MMP-12. A model of one of these inhibitors interacting with MMP-12 suggests that the selectivity observed might be due, in part, to the presence of two unique polar residues in MMP-12, Thr239 and Lys177. These MMP-12-selective inhibitors may have important therapeutic applications to diseases in which MMP-12 has been suggested to play a key role, such as in emphysema, atherosclerosis, and aortic abdominal aneurysm.     

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.