Oxidant-antioxidant imbalance as a potential contributor to the progression of human pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonia. IPF is a disease with poor prognosis and an aggressive nature, and poses major challenges to clinicians. Thus, a large part of research in the area has focused on the pathogenesis on IPF. Characteristic features in IPF include fibrotic lesions devoid of inflammatory cell infiltrates. There are experimental models of lung fibrosis (e.g., bleomycin-induced fibrosis), but they typically contain a prominent inflammatory pattern in the lung, which leads to relatively diffuse lung fibrosis. Nonetheless, experimental models have provided important information about the progression and pathways contributing to the lung fibrosis, including activation of transforming growth factor beta (TGF-beta). Both patient material and experimental models of lung fibrosis have displayed marked elevation of several markers of oxidant burden and signs for disturbed antioxidant/oxidant balance. Several studies also suggest that reactive oxygen species can cause activation of growth-regulatory cytokines, including TGF-beta. In addition, there are indications that endogenous and exogenous antioxidants/redox modulators can influence fibrogenesis, protect the lung against fibrosis, and prevent its progression. Factors that restore the antioxidant capacity and prevent sustained activation of growth-regulatory cytokines may have a therapeutic role in IPF.     

The circadian clock regulates rhythmic activation of the NRF2/glutathione-mediated antioxidant defense pathway to modulate pulmonary fibrosis

The disruption of the NRF2 (nuclear factor erythroid-derived 2-like 2)/glutathione-mediated antioxidant defense pathway is a critical step in the pathogenesis of several chronic pulmonary diseases and cancer. While the mechanism of NRF2 activation upon oxidative stress has been widely investigated, little is known about the endogenous signals that regulate the NRF2 pathway in lung physiology and pathology. Here we show that an E-box-mediated circadian rhythm of NRF2 protein is essential in regulating the rhythmic expression of antioxidant genes involved in glutathione redox homeostasis in the mouse lung. Using an in vivo bleomycin-induced lung fibrosis model, we reveal a clock “gated” pulmonary response to oxidative injury, with a more severe fibrotic effect when bleomycin was applied at a circadian nadir in NRF2 levels. Timed administration of sulforaphane, an NRF2 activator, significantly blocked this phenotype. Moreover, in the lungs of the arrhythmic Clock^Δ19 mice, the levels of NRF2 and the reduced glutathione are constitutively low, associated with increased protein oxidative damage and a spontaneous fibrotic-like pulmonary phenotype. Our findings reveal a pivotal role for the circadian control of the NRF2/glutathione pathway in combating oxidative/fibrotic lung damage, which might prompt new chronotherapeutic strategies for the treatment of human lung diseases, including idiopathic pulmonary fibrosis.     

Role for cystic fibrosis transmembrane conductance regulator protein in a glutathione response to bronchopulmonary pseudomonas infection

The lung maintains an elevated level of glutathione (GSH) in epithelial lining fluid (ELF) compared to serum. The mechanism(s) by which the lung maintains high levels of ELF GSH and factors that modulate them are largely unexplored. We hypothesized that lung cystic fibrosis transmembrane conductance regulator protein (CFTR) modulates GSH efflux in response to extracellular stress, which occurs with lung infections. Mice were challenged intratracheally with Pseudomonas aeruginosa, and on the third day of infection bronchoalveolar lavage fluid was obtained and analyzed for cytokines and antioxidants. Lung tissue antioxidants and enzyme activities were also assessed. P. aeruginosa lung infection increased levels of inflammatory cytokines and neutrophils in the ELF. This corresponded with a marked threefold increase in GSH and a twofold increase in urate levels in the ELF of P. aeruginosa-infected wild-type mice. A twofold increase in urate levels was also observed among lung tissue antioxidants of P. aeruginosa-infected wild-type mice. There were no changes in markers of lung oxidative stress associated with the P. aeruginosa lung infection. In contrast with wild-type mice, the CFTR knockout mice lacked a significant increase in ELF GSH when challenged with P. aeruginosa, and this correlated with a decrease in the ratio of reduced to oxidized GSH in the ELF, a marker of oxidative stress. These data would suggest that the lung adapts to infectious agents with elevated ELF GSH and urate. Individuals with lung diseases associated with altered antioxidant transport, such as cystic fibrosis, might lack the ability to adapt to the infection and present with a more severe inflammatory response.     

Pathogenetic mechanisms in usual interstitial pneumonia/idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive, usually fatal, form of interstitial lung disease characterized by failure of alveolar re-epithelialization, persistence of fibroblasts/myofibroblasts, deposition of extracellular matrix, and distortion of lung architecture which ultimately results in respiratory failure. Clinical IPF is associated with a histopathological pattern of usual interstitial pneumonia (UIP) on surgical lung biopsy. Therapy for this disease with glucocorticoids and other immunomodulatory agents is largely ineffective and recent trials of newer anti-fibrotic agents have been disappointing. While the inciting event(s) leading to the initiation of scar formation in UIP remain unknown, recent advances in our understanding of the mechanisms underlying both normal and aberrant wound healing have shed some light on pathogenetic mechanisms that may play significant roles in this disease. Unlike other fibrotic diseases of the lung, such as those associated with collagen vascular disease, occupational exposure, or chemotherapeutic agents, UIP is not associated with a significant inflammatory response; rather, dysregulated epithelial-mesenchymal interactions predominate. Identification of pathways crucial to fibrogenesis might offer potentially novel therapeutic targets to slow or halt the progression of IPF. This review focuses on evolving concepts of cellular and molecular mechanisms in the pathogenesis of UIP/IPF.     

Environmental, inhaled and ingested causes of pulmonary fibrosis

Pulmonary fibrosis is a general term that refers to a collection of connective tissue around alveolar structures. There are over 200 disorders where the lungs may be involved in a fibrotic response. To determine the cause of pulmonary fibrosis requires an in depth understanding of the pathogenesis of pulmonary fibrosis and breadth of knowledge of the causative agents and associated disorders that may lead to pulmonary fibrosis. A comprehensive evaluation of the patient is absolutely essential, starting with detailed history that includes an occupational and environmental history because fibrogenic exposures can occur in many settings. Equally important is a history of ingestion of pharmaceutical or nonpharmaceutical substances. A physical examination and judicious investigations are always a part of any comprehensive clinical assessment but they are not commonly helpful in elucidating the cause of most pulmonary fibrotic disorders. Although, a chest film is invariably done, a logical and strongly recommended next step is a high-resolution computed tomography (HRCT). HRCT provides a better assessment of the radiological pattern, may suggest a diagnosis as well as direct the site, and type of lung biopsy. If the history and investigations do not lead to a diagnosis then a lung biopsy is required. Prevention or removal of the inciting agent is critical to the treatment of these disorders and in some instances corticosteroids may be of help.     

Fatal lung fibrosis associated with immunodeficiency and gonadal dysgenesis in 46XX sisters--a new syndrome

Inherited immune deficiencies are a heterogeneous group of diseases that can be either isolated, with the immune defect being the exclusive manifestation, or associated with other abnormalities. We report on two sisters, born to consanguineous parents of Sri-Lankan descent, who presented in infancy with immunodeficiency, gonadal dysgenesis, and fatal lung fibrosis. Immune studies demonstrated combined humoral and cellular abnormalities including reduced immunoglobulin production, an absence of lymphoid tissue, markedly reduced T-lymphocyte numbers and function and reduced newly thymus-derived T-cells. Both infants succumbed to rapidly progressive lung fibrosis. Autopsy showed dysgenetic gonads bearing no discernible oocytes. In both, karyotypes were normal female (46,XX). Comparative genome hybridization and analysis of genes known to be associated with severe immune defects in infancy or gonadal dysgenesis showed no abnormality. The distinct findings in these two sisters have not been reported before and thus suggest a hitherto unknown autosomal recessive condition that includes immune dysfunction, gonadal dysgenesis, and pulmonary fibrosis.     

Diagnosis and classification of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis is a difficult disease to diagnose. Idiopathic pulmonary fibrosis is a member of a class of diseases known as idiopathic interstitial pneumonias. Other members include nonspecific interstitial pneumonia, cryptogenic organizing pneumonia, acute interstitial pneumonia, respiratory bronchiolitis-associated interstitial lung disease, desquamative interstitial pneumonia, and lymphocytic interstitial pneumonia. Usual interstitial pneumonia (UIP) is the pathological equivalent of idiopathic pulmonary fibrosis. Prior to 2011, the diagnosis was based on major and minor criteria, but because these criteria were not evidence based, the criteria were modified by consensus from the American Thoracic Society, the European Respiratory Society, the Japanese Respiratory Society, and the Latin American Thoracic Association. These new criteria now include satisfying three core requirements, including exclusion of other possible cause of interstitial lung disease, specific findings of usual interstitial pneumonia on high resolution computed tomography, and a combination of “possible UIP” findings on high resolution computed tomography and UIP findings on lung biopsy. Idiopathic pulmonary fibrosis is a severe, progressive disease with limited treatment options, and exacerbations are associated with a high degree of morbidity and mortality.     

Lung protection by thiol-containing antioxidants

It is becoming increasingly clear that certain types of pulmonary injury may be closely related to oxidant-antioxidant imbalance in the lung, resulting from the production of reactive oxygen species within the lung during endogenous metabolism and xenobiotic insult. We have investigated the role of glutathione in pneumoprotection from such reactive species and, in particular, methods of manipulating the resident antioxidant capacity of lung glutathione. One such approach has been the use of the thiol-containing drug N-acetylcysteine. We have shown that N-acetylcysteine is able to both support intracellular glutathione biosynthesis and act as a 'scavenger' of reactive electrophilic species through the chemical reactivity of its thiol group. N-acetylcysteine reduces hydrogen peroxide to water, with the commensurate formation of N-acetylcysteine disulphide both when the peroxide was supplied directly or generated enzymatically. This basal reduction of hydrogen peroxide by N-acetylcysteine was greatly enhanced by the inclusion of catalytic amounts of the selenium-containing heterocycle, Ebselen, in the incubations. Thus, Ebselen mimics the activity of glutathione peroxidase but, unlike the enzyme, is able to use N-acetylcysteine as a co-substrate. Thus, the combination of N-acetylcysteine and Ebselen may provide a useful therapeutic tool in conditions of pulmonary toxicity associated with oxidant insult.     

Idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis is a rapidly progressive illness of unknown cause characterized by sequential acute lung injury with subsequent scarring and end-stage lung disease. Treatment at present remains largely supportive, with evidence that patients' satisfaction and survival may be improved by referral to centers specializing in the evaluation of interstitial lung diseases. Although no drug therapy has clearly been demonstrated to benefit patients with idiopathic pulmonary fibrosis, a number of novel investigational agents hold promise for future study. Given the poor prognosis associated with idiopathic pulmonary fibrosis, patients should be referred to regional centers of expertise for enrollment in therapeutic clinical trials or for lung transplantation.     

Oxidative stress in allergic respiratory diseases

There is ample evidence that allergic disorders, such as asthma, rhinitis, and atopic dermatitis, are mediated by oxidative stress. Excessive exposure to reactive oxygen and nitrogen species is the hallmark of oxidative stress and leads to damage of proteins, lipids, and DNA. Oxidative stress occurs not only as a result of inflammation but also from environmental exposure to air pollution and cigarette smoke. The specific localization of antioxidant enzymes in the lung and the rapid reaction of nitric oxide with reactive oxygen species, such as superoxide, suggest that antioxidant enzymes might also function as cell-signaling agents or regulators of cell signaling. Therapeutic interventions that decrease exposure to environmental reactive oxygen species or augment endogenous antioxidant defenses might be beneficial as adjunctive therapies for allergic respiratory disorders.     

Evidence for Calreticulin Attenuation of Cardiac Hypertrophy Induced by Pressure Overload and Soluble Agonists

Bleomycin has potent anti-oncogenic properties for several neoplasms, but drug administration is limited by bleomycin-induced lung fibrosis. Inhibition of the renin-angiotensin system has been suggested to decrease bleomycin toxicity, but the efficacy of such strategies remains uncertain and somewhat contradictory. Our hypothesis is that, besides angiotensin II, other substrates of angiotensin-converting enzyme (ACE), such as the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), play a significant role in controlling fibrosis. We studied bleomycin-induced lung injury in normotensive mice, termed N-KO and C-KO, which have point mutations inactivating either the N- or C-terminal catalytic sites of ACE, respectively. N-KO, but not C-KO mice, have a marked resistance to bleomycin lung injury as assessed by lung histology and hydroxyproline content. To determine the importance of the ACE N-terminal peptide substrate AcSDKP in the resistance to bleomycin injury, N-KO mice were treated with S-17092, a prolyl-oligopeptidase inhibitor that inhibits the formation of AcSDKP. In response to bleomycin injection, S-17092-treated N-KO mice developed lung fibrosis similar to wild-type mice. In contrast, the administration of AcSDKP to wild-type mice reduced lung fibrosis due to bleomycin administration. This study shows that the inactivation of the N-terminal catalytic site of ACE significantly reduced bleomycin-induced lung fibrosis and implicates AcSDKP in the mechanism of protection. These data suggest a possible means to increase tolerance to bleomycin and to treat fibrosing lung diseases.Pulmonary fibrosis is a serious medical problem affecting more than 200,000 patients in the United States and is responsible for more than 40,000 deaths annually. At present, few treatments are available to counteract pulmonary fibrosis. Modulation of the renin-angiotensin system has been suggested to alter the evolution of this ailment, but the efficacy of such strategies remains uncertain and the conclusions of previous reports are somewhat contradictory.^1,2,3,4The renin-angiotensin system controls blood pressure and renal homeostasis in mammals. Angiotensin-converting enzyme (ACE) is the last enzyme of a cascade generating angiotensin II. The complete inactivation of ACE in mice results in a complex phenotype, including low blood pressure, anemia, male sterility, and kidney malformations.^5,6 These knockout mice, as well as data from several different experimental approaches, demonstrate physiological roles for ACE beyond simple blood pressure control. For example, angiotensin II has been implicated in promotion of fibrosis.^7,8,9Although a single polypeptide chain, ACE is composed of two homologous but independent catalytic domains. These domains, termed the N- and C-domains, each bind a single zinc atom, which is required for catalysis.¹⁰ Both domains can cleave angiotensin I, and both domains can degrade bradykinin; however, the two domains of ACE have some important differences. For example, only the N-domain can use the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) as a substrate, cleaving this peptide into inactive fragments.¹¹ Initially, AcSDKP was presented as a suppressor of bone marrow proliferation.¹² However, several in vivo studies have challenged this assertion and suggested that AcSDKP may suppress fibrosis in models of tissue injury.¹³ ACE is the primary enzyme responsible for the degradation of AcSDKP; its actions in producing the profibrotic molecule angiotensin II and in degrading the antifibrotic molecule AcSDKP suggest that ACE and the renin-angiotensin system may be important in some fibrotic diseases.To further investigate the role of ACE in lung fibrosis, we used bleomycin to induce lung fibrosis in two new strains of genetically engineered mice with point mutations eliminating zinc binding in one of the two catalytic domains of ACE. The absence of zinc catalytically inactivates either the N-domain or the C-domain, and these mice are termed N-KO and C-KO, respectively.^14,15 Because we only introduced point mutations into the ACE gene, both lines of mice have normal serum and tissue levels of ACE protein. Because each line preserves catalytic function in one of the two ACE catalytic domains, both lines have serum levels of angiotensin II and blood pressures equivalent to those of wild-type mice. Thus, these mice allow investigation of ACE domain-specific functions independent of any secondary effects of blood pressure changes, a typical bias introduced with either ACE inhibitors (which inhibit both domains of ACE) or the genetic elimination of all ACE expression (use of ACE knockout animals).Bleomycin-induced lung injury is a well accepted model of lung fibrosis,¹⁶ which has direct clinical correlates, because the antineoplastic use of bleomycin is limited by its lung toxicity. Meta-analysis suggests that 10 to 50% of patients treated with bleomycin develop sufficient lung injury to necessitate cessation of treatment.¹⁷ Of these, 1 to 3% die of lung injury.¹⁸ Here, we show that N-KO mice are markedly resistant to the toxicity of bleomycin and that the accumulation of the ACE N-terminal peptide substrate AcSDKP ameliorates bleomycin-induced lung fibrosis. These data suggest a means to increase tolerance to bleomycin and perhaps a new approach for treating other fibrosing lung diseases.     

Smoking-related interstitial fibrosis (SRIF), pathogenesis and treatment of usual interstitial pneumonia (UIP), and transbronchial biopsy in UIP

This review focuses on three selected topics of current interest that are related to chronic fibrosing lung disorders and are important for pathologists. First, the clinical and pathologic features of smoking-related interstitial fibrosis (SRIF) are highlighted. SRIF is a common finding in smokers that has striking histologic changes but only mild associated clinical manifestations. It is characterized by marked alveolar septal fibrosis composed of a distinct form of hyalinized collagen deposition. The process is present mainly in subpleural and centrilobular parenchyma and is associated with emphysema and respiratory bronchiolitis. Second, important aspects of the pathogenesis and treatment of usual interstitial pneumonia (UIP) are reviewed. The current theory proposes that UIP is caused by tiny foci of acute lung injury (manifest pathologically by fibroblast foci) that occur and recur in the interstitium over many years. Inflammation may be present as a secondary phenomenon, but is not the primary cause, and therefore anti-inflammatory agents have little effect. The recurrent injury leads to permanent fibrosis, through a process that is considered to represent a form of abnormal wound healing. Multiple therapies have been attempted that are aimed largely at interrupting the fibrosing process, but none have been successful. The cause of the injury is unknown, but a role for aspiration due to gastroesophageal reflux is a popular current theory, and there is some evidence that anti-reflux therapy may be beneficial. Genetic predisposition has been implicated in the etiology of familial cases, and there is evidence that telomere shortening may be important in sporadic cases. Third, the use of transbronchial biopsy (TBB) in diagnosing UIP is reviewed. TBB can provide a surprising amount of information and is especially useful in certain situations, such as elderly or very sick patients in whom surgical lung biopsy carries increased morbidity and mortality.     

Radiation-Induced Lung Injury Is Mitigated by Blockade of Gastrin-Releasing Peptide

Gastrin-releasing peptide (GRP), secreted by pulmonary neuroendocrine cells, mediates oxidant-induced lung injury in animal models. Considering that GRP blockade abrogates pulmonary inflammation and fibrosis in hyperoxic baboons, we hypothesized that ionizing radiation triggers GRP secretion, contributing to inflammatory and fibrotic phases of radiation-induced lung injury (RiLI). Using C57BL/6 mouse model of pulmonary fibrosis developing ≥20 weeks after high-dose thoracic radiation (15 Gy), we injected small molecule 77427 i.p. approximately 1 hour after radiation then twice weekly for up to 20 weeks. Sham controls were anesthetized and placed in the irradiator without radiation. Lung paraffin sections were immunostained and quantitative image analyses performed. Mice exposed to radiation plus PBS had increased interstitial CD68⁺ macrophages 4 weeks after radiation and pulmonary neuroendocrine cells hyperplasia 6 weeks after radiation. Ten weeks later radiation plus PBS controls had significantly increased pSmad2/3⁺ nuclei/cm². GRP blockade with 77427 treatment diminished CD68⁺, GRP⁺, and pSmad2/3⁺ cells. Finally, interstitial fibrosis was evident 20 weeks after radiation by immunostaining for α-smooth muscle actin and collagen deposition. Treatment with 77427 abrogated interstitial α-smooth muscle actin and collagen. Sham mice given 77427 did not differ significantly from PBS controls. Our data are the first to show that GRP blockade decreases inflammatory and fibrotic responses to radiation in mice. GRP blockade is a novel radiation fibrosis mitigating agent that could be clinically useful in humans exposed to radiation therapeutically or unintentionally.Radiation fibrosis is a serious complication that affects normal lung following unintentional exposure or due to therapeutic ionizing radiation of thoracic tumors. Despite advances in radiobiology, precise mechanisms by which radiation induces lung injury remain controversial.¹ Classically, radiation-induced lung injury (RiLI) is characterized by a latent period that can last for weeks to months after radiation exposure, followed by 2 stages of overt lung injury that can lead to life-threatening and debilitating pulmonary toxic effects.^2,3 Acute inflammatory lung injury arises 1 to 6 months after radiation exposure, with diffuse alveolar damage, similar to acute respiratory distress syndrome. Later, chronic interstitial and intra-alveolar fibrosis develops, predominantly in irradiated segments, with myofibroblast proliferation and collagen deposition. It is unclear why only approximately 15% of radiation-exposed patients develop RiLI.^1,4 General cytoprotective agents, such as a catalytic antioxidant metalloporphyrin (AEOL10113), can reduce the severity of RiLI by decreasing free radical injury after radiation.⁵Our novel paradigm links gastrin-releasing peptide (GRP) to radiation lung injury. We hypothesized that GRP is a mediator of RiLI: promoting both macrophage accumulation and fibrosis. We propose that ionizing radiation triggers pulmonary neuroendocrine cell (PNEC) hyperplasia, leading to GRP secretion, which then mediates chronic lung injury. GRP receptor (GRPR) gene expression is detected and functional in pulmonary epithelial cells, fibroblasts, endothelial cells, and macrophages.^6–10 GRP is a proinflammatory neuropeptide that functions as an inflammatory cell activator, mitogen, and cell differentiation factor.^8,10,11 GRP is expressed at the highest levels in PNEC in fetal lung,¹² where it can promote lung development.¹³ After birth, GRP production normally decreases, but elevated levels are associated with many inflammatory lung conditions, including chronic lung disease of newborns (bronchopulmonary dysplasia).^14–17 PNEC hyperplasia can be triggered by inflammation or exposure to oxygen or ozone^10,16,18 and can take weeks to reach peak levels.¹⁹The present investigation tests the hypothesis that GRP contributes to radiation-induced pulmonary fibrosis in C57BL/6 mice. One hour post exposure to thoracic radiation (15 Gy), we treated mice i.p. with either PBS or GRP blockade by using small molecule 77427. We have quantified results of immunohistochemistry (IHC) by using image analysis with ImageJ version 1.46e (NIH, Bethesda, MD) to determine whether GRP contributes to radiation-induced inflammatory responses and/or fibrosis, specifically including assessment of active transforming growth factor (TGF)-β signaling.     

Oxidative injury and its defense system in vivo

We and other researchers verified that excessively produced free radicals by neutrophils induce various diseases such as Beh?et's disease, MCLS, SLE (neutrophil-stimulated lymphocytes), RA (synovial fluid neutrophils), Crohn's disease, colitis ulcerosa, and dermatitis herpetiformis (Dühring). Recently, it was reported that environmental toxic agents including herbicides such as paraquat, insecticides, nitrogen oxide, and ultraviolet radiation produce free radicals. Nitrogen oxide, a main product of the combustion of petroleum, is a prominent component of exhaust from automobiles. Generation of 1O2 by ultraviolet radiation has also increased by breaks in the earth's ozone layer induced by halogenated hydrocarbon gas. Various diseases have been induced by these agents such as malignancies, severe adult atopic dermatitis, complication of cataract and retinolysis with atopic dermatitis, skin cancer, male infertility, severe collagen diseases, and lung fibrosis. It was also found that PCB, methyl-Hg and Mn, Cd induce neuropathic diseases through the increase in free radical production. On the other hand, a self-defense system exists against oxidative injuries; high-molecular-weight antioxidants such as SOD, catalase, and GSH-Px, and low-molecular-weight antioxidants such as vitamin C, E, A, polyphenols, flavonoids, and catechin. As protection from oxidative injury, various antioxidant products have been developed, however, the development of SOD injection was given up by all the pharmaceutical companies in the world on account of ineffectiveness due to rapid excretion from the kidney, low affinity to the receptor and weak penetration into the cell. A.M. Michelson, a French biochemist succeeded in developing an effective bovine liposomal-encapsulated SOD solving these problems, however, he also gave it up since French government prohibited bovine products due to the prion virus. Regarding low-molecular-weight antioxidants, synthetic products generally show low affinity to human cell receptors, however, natural products also has demerits that abundant low-molecular-weight compounds with antioxidant activity that exist in the native materials as repeating subunits of high-molecular-weight polymers resulting in inactive forms. We have developed effective natural antioxidant by a unique treating procedure that includes heating with far infrared radiation of 4-14 microns wavelength, brewing with the fungus Aspergillus oryzae, and emulsification with similarly heated sesame oil. This formulation is based on our hypothesis that treatment of natural plant products with heating and brewing results in the liberation of abundant low-molecular-weight compounds with antioxidant activity that exist in an inactive form as repeating polymers as described above.     

Development of a bleomycin hamster model of subchronic lung fibrosis.

The existing bleomycin (BLM)-rodent model of lung fibrosis requires large doses and is often associated with morbidity and high mortality. We have developed an intratracheal multiple-dose BLM-hamster model of lung fibrosis. In this model, 3 consecutive doses of BLM (2.5 U, 2.0 U and 1.5 U/5mL/kg) were instilled intratracheally, one dose per week. The hamsters were killed at 10, 20, 30, 60 and 90 days after the last IT instillation and the lungs were lavaged or perfused with saline. This regimen of BLM administration was devoid of morbidity and caused only 6% overall mortality. Lung prolyl hydroxylase activity at 10 days and hydroxyproline content at 20, 30, 60 and 90 days were significantly higher than noted for the controls. Bronchoalveolar lavage fluid-supernatant protein and the total number of recovered cells of all types were significantly higher than observed for the controls at all times, except at 90 days. Lungs showed a multifocal mixed mononuclear infiltrate at 10 and 20 days and septal fibrosis, which was most severe and organized at 30 days and less severe at 60 and 90 days. The parenchymal lesions were significantly greater than those of the controls at all times, except at 10 days. This model, which required only 6 U BLM/kg, induced a moderate level of lung fibrosis. It has been concluded, therefore, that this model, inasmuch as it is not associated with an overwhelmingly acute inflammation, would be more applicable for screening potential antifibrotic agents than existing models of lung fibrosis.     

Oxidative stress and cerebrovascular dysfunction in mouse models of Alzheimer's disease

Several factors have been implicated in Alzheimer's disease (AD) but there is no definite conclusion as to the main pathogenic agents. Mutations in the amyloid precursor protein (APP) that lead to increased production of amyloid beta peptide (A beta) are associated with the early-onset, familial forms of AD. However, in addition to ageing, the most common risk factors for the sporadic, prevalent form of AD are hypertension, hypercholesterolaemia, ischaemic stroke, the ApoE4 allele and diabetes, all characterized by a vascular pathology. In AD, the vascular pathology includes accumulation of A beta in the vessel wall, vascular fibrosis, and other ultrastructural changes in constituent endothelial and smooth muscle cells. Moreover, the ensuing chronic cerebral hypoperfusion has been proposed as a determinant factor in the accompanying cognitive deficits. In transgenic mice that overexpress mutated forms of the human APP (APP mice), the increased production of A beta results in vascular oxidative stress and loss of vasodilatory function. The culprit molecule, superoxide, triggers the synthesis of other reactive oxygen species and the sequestration of nitric oxide (NO), thus impairing resting cerebrovascular tone and NO-dependent dilatations. The A beta-induced cerebrovascular dysfunction can be completely abrogated in aged APP mice with antioxidant therapy. In contrast, in mice that overproduce an active form of the cytokine transforming growth factor-beta1 and recapitulate the vascular structural changes seen in AD, antioxidants have no beneficial effect on the accompanying cerebrovascular deficits. This review discusses the beneficial role and limitations of antioxidant therapy in AD cerebrovascular pathology.     

Effects of gefitinib on radiation-induced lung injury in mice.

Clinical studies have demonstrated that gefitinib, an epidermal growth factor receptor inhibitor, is an effective treatment for some patients with advanced non-small cell lung cancer and is generally well-tolerated. However, several reports have also suggested that gefitinib is associated with acute lung injury and subsequent fibrosis. One hypothesis is that gefitinib exacerbates lung injury induced by radiation therapy. It is important to confirm the safety of gefitinib in radiotherapy for patients with lung cancer. In this preclinical study we aimed to clarify the effect of gefitinib on thoracic radiotherapy. Six-week-old female C57BL/6 mice were immobilized in a plastic frame, and the thorax was irradiated once with a dose of 12 Gy on day 0. Gefitinib (20, 90 and 200 mg/kg/day) was administered on days 0 to 5 (acute phase) or days 14 to 19 (late phase) postirradiation. Thoracic irradiation induced lung injury and subsequent fibrosis 5 months later. Gefitinib, administered in the acute phase, had no effect on lung fibrosis or collagen levels induced by irradiation. A high dose of gefitinib (200 mg/kg/day) administered during the late phase significantly reduced fibrosis scores and collagen levels. These results suggest that gefitinib does not exacerbate radiation-induced lung injury and fibrosis in this strain of mice. Therefore, thoracic irradiation is unlikely to be a risk factor for lung injury associated with gefitinib treatment.