Administration Of Porcine Pancreatic Elastase Research Articles

A Japanese herbal medicine (kampo), hochuekkito (TJ-41), has anti-inflammatory effects on the chronic obstructive pulmonary disease mouse model

Chronic obstructive pulmonary disease (COPD) is a progressive disease that is characterized by chronic airway inflammation. A Japanese herbal medicine, hochuekkito (TJ-41), is prominently used for chronic inflammatory diseases in Japan. This study aimed to analyze the anti-inflammatory effect of TJ-41 in vivo and its underlying mechanisms. We created a COPD mouse model using intratracheal administration of porcine pancreatic elastase and lipopolysaccharide (LPS) and analyzed them with and without TJ-41 administration. A TJ-41-containing diet reduced inflammatory cell infiltration of the lungs in the acute and chronic phases and body weight loss in the acute phase. In vitro experiments revealed that TJ-41 treatment suppressed the LPS-induced inflammatory cytokines in BEAS-2B cells. Furthermore, TJ-41 administration activated the AMP-activated protein kinase (AMPK) pathway and inhibited the mechanistic target of the rapamycin (mTOR) pathway, both in cellular and mouse experiments. We concluded that TJ-41 administration reduced airway inflammation in the COPD mouse model, which might be regulated by the activated AMPK pathway, and inhibited the mTOR pathway.

Effects of elastase-induced emphysema on muscle and bone in mice.

Chronic obstructive pulmonary disease (COPD) causes sarcopenia and osteoporosis. However, the mechanisms underlying muscle and bone loss as well as the interactions between muscle and bone in the COPD state remain unclear. Therefore, we herein investigated the effects of the COPD state on muscle and bone in mice intratracheally administered porcine pancreatic elastase (PPE). The intratracheal administration of PPE to mice significantly reduced trabecular bone mineral density (BMD), trabecular bone volume, trabecular number, cortical BMD and cortical area. It also significantly decreased grip strength, but did not affect muscle mass or the expression of myogenic differentiation-, protein degradation- or autophagy-related genes in the soleus and gastrocnemius muscles. Among the myokines examined, myostatin mRNA levels in the soleus muscles were significantly elevated in mice treated with PPE, and negatively related to grip strength, but not bone parameters, in mice treated with or without 2 U PPE in simple regression analyses. Grip strength positively related to bone parameters in mice treated with or without PPE. In conclusion, we showed that a PPE model of COPD in mice exerts dominant effects on bone rather than skeletal muscles. Increased myostatin expression in the soleus muscles of mice in the COPD state may negatively relate to a reduction in grip strength, but not bone loss.

Dental Pulp Stem Cells Ameliorate Elastase-Induced Pulmonary Emphysema by Regulating Inflammation and Oxidative Stress.

Dental pulp stem cells (DPSCs) are considered excellent candidates for stem cell-based tissue regeneration. In this study, we aimed to evaluate the therapeutic effect of DPSCs in a mouse chronic obstructive pulmonary disease (COPD) model and to explore whether DPSCs reduce lung inflammation and oxidative stress by regulating the nuclear factor erythroid-2 related factor-2 (Nrf2) signaling pathway. DPSCs were isolated from dental pulp tissue by the tissue block method. Emphysema of C57BL/6 mice was induced by endotracheal administration of porcine pancreatic elastase (PPE). Then, the DPSCs were injected into the lungs through the trachea, and after 3 weeks of stem cell treatment, various efficacy tests were performed. The AniRes2005 animal lung function analytic system was used to detect lung function. Hematoxylin-eosin staining (H&E) and Victoria blue staining was used to assess emphysema severity. The animal tissues were detected by Western blot, RT‒qPCR, ELISA and oxidative stress related detection. In experimental COPD models, DPSCs transplantation improved lung function, body weight, and emphysema-like changes better than bone marrow mesenchyml stem cells (BM-MSCs). Compared with the COPD group, the levels of IL-1β, TNF-α and IL-6 in lung tissue and bronchoalveolar lavage fluid (BALF) were decreased after transplantation of DPSCs. DPSCs may be associated with lower malondialdehyde (MDA) levels, and higher catalase (CAT) and glutathione (GSH) levels. Western blot results showed that the expression of Nrf2 and its downstream factors increased after transplantation of DPSCs. The current study showed that DPSCs had good performance in the treatment of a mouse COPD model and could be a promising option for stem cell therapy. DPSCs may play antioxidant and anti-inflammatory roles in COPD by activating the Nrf2 signaling pathway.

The early and late intervention effects of collagen-binding FGF2 on elastase-induced lung injury

IntroductionChronic obstructive pulmonary disease (COPD) has high morbidity and mortality, with no effective treatment at present. Emphysema, a major component of COPD, is a leading cause of human death worldwide. Fibroblast growth factor 2 (FGF2) is implicated in the pathogenesis of pulmonary emphysema and may play an important role in the lung repair process after injury, but concerns remain with respect to its effectiveness. ObjectiveIn the present work, we sought to determine how the timing (early and late intervention) of sustained-release FGF2 system administration impacted its effectiveness on a porcine pancreatic elastase (PPE)-induced lung injury mouse model. MethodsTo examine the early intervention efficiency of collagen-binding FGF2 (CBD-FGF2), mice received intratracheally nebulized CBD-FGF2 with concurrent intratracheal injection of PPE. To explore the late intervention effect, CBD-FGF2 was intratracheally aerosolized after PPE administration, and lungs were collected after CBD-FGF2 treatment for subsequent analysis. ResultIn response to PPE, mice had significantly increased alveolar diameter, collagen deposition and expression of inflammatory factors and decreased lung function indices and expression of alveolar epithelium markers. Our results indicate that CBD-FGF2 administration was able to prevent and repair elastase-induced lung injury partly through the suppression of the inflammatory response and recovery of the alveolar epithelium. The early use of CBD-FGF2 for the prevention of PPE-induced emphysema showed better results than late therapeutic administration against established emphysema. ConclusionThese data provide insight regarding the prospective role of a drug-based option (CBD-FGF2) for preventing and curing emphysema.

Ninjin'yoeito ameliorated PPE-induced pulmonary emphysema and anxiety/depressive-like behavior in aged C57BL/6J mice.

The prevalence of chronic obstructive pulmonary disease (COPD) is increasing in the elderly. COPD is a chronic respiratory disease characterized by airway remodeling and alveolar emphysema. COPD patients are also at high risk for mental illnesses such as depression and anxiety. Ninjin’yoeito (NYT) is prescribed to patients with conditions such as post-illness and postoperative weakness, fatigue, poor appetite, skin rash, cold hands and feet, and anemia. In addition to traditional uses, NYT is also prescribed as a therapeutic drug for poor functioning of the digestive organs, respiratory organs, and urinary organs. NYT is also known to have an antioxidant effect. The objective of this study was to investigate whether NYT could ameliorate COPD-induced lung injury and anxiety/depression in aged C57BL/6J mice exposed to porcine pancreatic elastase (PPE). While intratracheal administration of PPE induced emphysema in elderly mice, long-term administration of NYT suppressed the pathology. NYT was also found to suppress the apoptosis and oxidative stress caused by PPE. In addition, long-term administration of NYT was found to ameliorate PPE-induced depressive-like behavior in three different behavioral studies. These results suggest that NYT has a therapeutic effect on emphysema and the behavioral abnormalities caused by PPE.

Risk Assessment for Use of a Porcine Circovirus-Contaminated Reagent in a Barrier Maintained Rodent Colony.

A proposal for the use of porcine pancreatic elastase (PPE) to develop a mouse model of pulmonary emphysema raised concerns about introducing contaminating porcine viruses into our barrier facility. Porcine Circovirus (PCV) is a known contaminant of vaccines and cell cultures that have been exposed to porcine-derived reagents. Endemic infection of PCV3 in laboratory mice has been reported, and some evidence supports natural PCV infection in wild mice. PPE samples from 2 different vendors tested positive for DNA from both PCV2 and 3. To allow model development with these reagents to proceed, we developed a protocol that would meet scientific objectives, minimize exposure of mice, and provide information on the potential for the virus to spread. Five d after BALB/c mice received intralaryngeal administration of PPE, lungs were harvested and analyzed for evidence of disease. Tissues from other major organs were submitted to test for disseminated PCV2 and 3 DNA. Similarly, tissues (including lungs) from direct contact nude sentinel mice were analyzed for the presence of the virus. To evaluate the possibility of endemic PCV2/3 infection, we also surveyed non-porcine reagent exposed mice on other studies. PCV2 and 3 was not detected in any of the tissues submitted. Although this study provided no evidence of infection and transmission of PCV2/3 from the contaminated PPE sample over the 5 d study, further work is needed to understand the risks and impact of introducing PCV contaminated cells or reagents into barrier maintained rodent colonies.

Therapeutic effects of LASSBio-596 in an elastase-induced mouse model of emphysema.

Emphysema is an intractable pulmonary disease characterized by an inflammatory process of the airways and lung parenchyma and ongoing remodeling process in an attempt to restore lung structure. There is no effective drug therapy that regenerates lung tissue or prevents the progression of emphysema; current treatment is aimed at symptomatic relief. We hypothesized that LASSBio-596, a molecule with potent anti-inflammatory and immunomodulatory effects, might reduce pulmonary inflammation and remodeling and thus improve lung function in experimental emphysema. Emphysema was induced in BALB/c mice by intratracheal administration of porcine pancreatic elastase (0.1 IU) once weekly during 4 weeks. A control group received saline using the same protocol. After the last instillation of saline or elastase, dimethyl sulfoxide, or LASSBio-596 were administered intraperitoneally, once daily for 8 days. After 24 h, in elastase-induced emphysema animals, LASSBio-596 yielded: (1) decreased mean linear intercept, hyperinflation and collagen fiber content, (2) increased elastic fiber content, (3) reduced number of M1 macrophages, (4) decreased tumor necrosis factor-α, interleukin-1β, interleukin-6, and transforming growth factor-β protein levels in lung tissue, and increased vascular endothelial growth factor. These changes resulted in increased static lung elastance. In conclusion, LASSBio-596 therapy reduced lung inflammation, airspace enlargement, and small airway wall remodeling, thus improving lung function, in this animal model of elastase-induced emphysema.

Elevated permeability of the blood–brain barrier in mice intratracheally administered porcine pancreatic elastase

Chronic obstructive pulmonary disease (COPD) shows progressive, irreversible airflow limitation induced by emphysema and lung inflammation. The aim of the present study was to determine if COPD conditions induce blood–brain barrier (BBB) dysfunction. We found that the intratracheal administration of porcine pancreatic elastase (PPE; 3 U) induced alveolar enlargement, increased neutrophil number in bronchoalveolar lavage fluid, and decreased blood oxygen saturation in mice at 21 days after inhalation. In parallel with these lung damages, BBB permeability to sodium fluorescein and Evans blue albumin was markedly increased. Our findings demonstrate that COPD conditions are associated with risk for BBB impairment.

Ovary-dependent emphysema augmentation and osteopontin induction in adult female mice

Biological differences between the sexes greatly impact the development and severity of pulmonary disorders such as emphysema. Recent studies have demonstrated crucial roles for osteopontin (OPN, also known as SPP1) in lung inflammation and alveolar destruction in human and experimental emphysema, but the impact of gender on OPN action remains unknown. Here, we report ovary-dependent induction of Opn mRNA with augmentation of experimental emphysema in adult female mice. Both male and female mice developed emphysematous lungs following intra-tracheal administration of porcine pancreatic elastase; however, compared with male mice, female mice developed more severe injury-related inflammation and pathologic alterations of the lungs. Notably, we observed female-specific induction of the Opn gene upon lung injury. Ovariectomy blocked this induction, with attenuation of lung inflammation and alveolar destruction, demonstrating the essential role of ovaries in injury-related Opn induction and augmentation of emphysema in adult female mice. Lastly, pre-treatment of adult female mice with pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, which blocks ATP-mediated wound response, suppressed Opn mRNA induction upon lung injury, resulting in attenuation of enhanced lung inflammation. Together, our findings define a novel, ovary-dependent mechanism underlying gender-specific augmentation of emphysema through transcriptional control of the Opn gene.

Keratinocyte Growth Factor Gene Electroporation into Skeletal Muscle as a Novel Gene Therapeutic Approach for Elastase-Induced Pulmonary Emphysema in Mice.

Pulmonary emphysema is a progressive disease with airspace destruction and an effective therapy is needed. Keratinocyte growth factor (KGF) promotes pulmonary epithelial proliferation and has the potential to induce lung regeneration. The aim of this study was to determine the possibility of using KGF gene therapy for treatment of a mouse emphysema model induced by porcine pancreatic elastase (PPE). Eight-week-old BALB/c male mice treated with intra-tracheal PPE administration were transfected with 80 μg of a recombinant human KGF (rhKGF)-expressing FLAG-CMV14 plasmid (pKGF-FLAG gene), or with the pFLAG gene expressing plasmid as a control, into the quadriceps muscle by electroporation. In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells. Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection. Arterial blood gas analysis revealed that the PaO2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice. These results indicated that KGF gene therapy with electroporation stimulated lung epithelial proliferation and protected depression of pulmonary function in a mouse emphysema model, suggesting a possible method of treating pulmonary emphysema.

Regional fractional ventilation mapping in spontaneously breathing mice using hyperpolarized ¹²⁹Xe MRI.

The feasibility of ventilation imaging with hyperpolarized (HP) (129) Xe MRI has been investigated for quantitative and regional assessment of ventilation in spontaneously breathing mice. The multiple breath ventilation imaging technique was modified to the protocol of spontaneous inhalation of HP (129) Xe delivered continuously from a (129) Xe polarizer. A series of (129) Xe ventilation images was obtained by varying the number of breaths before the (129) Xe lung imaging. The fractional ventilation, r, was successfully evaluated for spontaneously breathing mice. An attempt was made to detect ventilation dysfunction in the emphysematous mouse lung induced by intratracheal administration of porcine pancreatic elastase (PPE). As a result, the distribution of fractional ventilation could be visualized by the r map. Significant dysfunction of ventilation was quantitatively identified in the PPE-treated group. The whole-lung r value of 0.34 ± 0.01 for control mice (N = 4) was significantly reduced, to 0.25 ± 0.07, in PPE-treated mice (N = 4) (p = 0.038). This study is the first application of multiple breath ventilation imaging to spontaneously breathing mice, and shows that this methodology is sensitive to differences in the pulmonary ventilation. This methodology is expected to improve simplicity as well as noninvasiveness when assessing regional ventilation in small rodents.

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.

Impact of inflammation, emphysema, and smoking cessation on V/Q in mouse models of lung obstruction

BackgroundChronic obstructive pulmonary disease (COPD) is known to greatly affect ventilation (V) and perfusion (Q) of the lung through pathologies such as inflammation and emphysema. However, there is little direct evidence regarding how these pathologies contribute to the V/Q mismatch observed in COPD and models thereof. Also, little is known regarding how smoking cessation affects V/Q relationships after inflammation and airspace enlargement have become established. To this end, we have quantified V/Q on a per-voxel basis using single photon emission computed tomography (SPECT) in mouse models of COPD and lung obstruction.MethodsThree distinct murine models were used to investigate the impact of different pathologies on V/Q, as measured by SPECT. Lipopolysaccharide (LPS) was used to produce neutrophilic inflammation, porcine pancreatic elastase (PPE) was used to produce emphysema, and long-term cigarette smoke (CS) exposure and cessation were used to investigate the combination of these pathologies.ResultsCS exposure resulted in an increase in mononuclear cells and neutrophils, an increase in airspace enlargement, and an increase in V/Q mismatching. The inflammation produced by LPS was more robust and predominantly neutrophilic, compared to that of cigarette smoke; nevertheless, inflammation alone caused V/Q mismatching similar to that seen with long-term CS exposure. The emphysematous lesions caused by PPE administration were also capable of causing V/Q mismatch in the absence of inflammation. Following CS cessation, inflammatory cell levels returned to those of controls and, similarly, V/Q measures returned to normal despite evidence of persistent mild airspace enlargement.ConclusionsBoth robust inflammation and extensive airspace enlargement, on their own, were capable of producing V/Q mismatch. As CS cessation resulted in a return of V/Q mismatching and inflammatory cell counts to control levels, lung inflammation is likely a major contributor to V/Q mismatch observed in the cigarette smoke exposure model as well as in COPD patients. This return of V/Q mismatching to control values also took place in the presence of mild airspace enlargement, indicating that emphysematous lesions must be of a larger volume before affecting the lung significantly. Early smoking cessation is therefore critical before emphysema has an irreversible impact on gas exchange.

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

BackgroundChronic 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).MethodsHyperlipidemic 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.ResultsIntratracheal 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.ConclusionThis 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.

Comprehensive analysis of elastase-induced pulmonary emphysema in mice: Effects of ambient existing particulate matters

Pulmonary exposure of rodents to porcine pancreatic elastase (PPE) induces lesions that morphologically resemble human panacinar emphysema. However, there has been little work on the comprehensive analysis of this model. The present study was designed to extensively examine the biological effects of PPE on inflammation, cell damage, emphysematous change, and cholinergic reactivity in the lungs of mice. Furthermore, we evaluated the effects of pulmonary exposure to diesel exhaust particles (DEP) on the disease model. Intratracheal administration of PPE induced (1) proinflammatory response in the lungs that was characterized by significant infiltration of leukocytes such as macrophages, eosinophils, and lymphocytes and an increased level of interleukin-1β in lung homogenates, (2) lung cell damage, indicated by higher levels of total protein, lactate dehydrogenase, and alkaline phosphatase in lung homogenates, (3) emphysema-related morphological changes including airspace enlargement and progressive destruction of alveolar wall structures, and (4) airway responsiveness to methacholine in the context of the compliance value of the respiratory system in a dose-dependent manner showing an overall trend. A single intratracheal administration of DEP did not significantly facilitate the hallmark of the disease. This is the first study to extensively analyze PPE-induced lung emphysema in mice with evaluation of the effects of DEP. Furthermore, this bioassay may be applied to future investigations that evaluate new therapeutic agents or risk factors for pulmonary emphysema.

Curcumin attenuates elastase- and cigarette smoke-induced pulmonary emphysema in mice

Curcumin, a yellow pigment obtained from turmeric (Curcumina longa), is a dietary polyphenol that has been reported to possess anti-inflammatory and antioxidant properties. The effect of curcumin against the development of pulmonary emphysema in animal models is unknown. The aim of this study was to determine whether curcumin is able to attenuate the development of pulmonary emphysema in mice. Nine-week-old male C57BL/6J mice were treated with intratracheal porcine pancreatic elastase (PPE) or exposed to mainstream cigarette smoke (CS) (60 min/day for 10 consecutive days or 5 days/wk for 12 wk) to induce pulmonary inflammation and emphysema. Curcumin (100 mg/kg) or vehicle was administrated daily by oral gavage 1 h and 24 h before intratracheal PPE treatment and daily thereafter throughout a 21-day period in PPE-exposed mice and 1 h before each CS exposure in CS-exposed mice. As a result, curcumin treatment significantly inhibited PPE-induced increase of neutrophils in bronchoalveolar lavage fluid at 6 h and on day 1 after PPE administration, with an increase in antioxidant gene expression at 6 h and significantly attenuated PPE-induced air space enlargement on day 21. It was also found that curcumin treatment significantly inhibited CS-induced increase of neutrophils and macrophages in bronchoalveolar lavage fluid after 10 consecutive days of CS exposure and significantly attenuated CS-induced air space enlargement after 12 wk of CS exposure. In conclusion, oral curcumin administration attenuated PPE- and CS-induced pulmonary inflammation and emphysema in mice.

Induction of the myofibroblast phenotype following elastolytic injury to mouse lung

The repair of alveolar structures following endotracheal administration of porcine pancreatic elastase (PPE) to mice involves the coordinated deposition of new matrix elements. We determined the induction of the myofibroblast phenotype following elastolytic injury to mouse lung by examining the expression of alpha-smooth muscle actin (alpha-SMA) by immunohistochemistry. We also examined elastin and alpha1(I) collagen mRNA expression by in situ hybridization. Changes in airspace dimensions were assessed by determining mean linear intercept. In untreated mice, alpha-SMA was localized to vascular structures and large airways, with no detectable expression in alveolar units. PPE induced alpha-SMA expression in damaged areas surrounding large vessels, in septal remnants, and in the opening ring of alveolar ducts. Elastin and alpha1(I) collagen mRNA expression were up-regulated in residual alveolar structures and septal walls. PPE dose-response studies indicated that alpha1(I) collagen and elastin mRNA expression were not induced in areas of normal lung adjacent to damaged lung. The administration of low dose PPE resulted in increased alpha-SMA protein and elastin mRNA expression in the cells comprising the opening ring of alveolar ducts. Our data suggest that repair mechanisms following elastolytic injury are confined to overtly damaged alveolar structures and involve the induction of the myofibroblast phenotype.

Elastase-induced changes in lung function: relationship to morphometry and effect of drugs

Intratracheal administration of porcine pancreatic elastase (PPE) produced a dose related decline in lung function, as assessed by changes in dynamic lung compliance (C dyn) in New Zealand White rabbits. This occured within 24 h of administration and persisted for 56 days ( n=6). These lung function changes were accompanied by histological evidence of emphysema in the lungs and were not mimicked by intratracheal administration of the proteolytic enzyme trypsin. Neither the lung function nor the histological changes induced by elastase could be prevented or reversed by either the glucocorticosteroid, dexamethasone, or all trans retinoic acid (ATRA). Our data suggest that local administration of elastase to the lungs of rabbits may provide a convenient way to assess the effects of drugs on the changes induced by elastase in airways.

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.

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.