RESUMO
Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant activation of the alveolar epithelium, the expansion of the fibroblast population, and the accumulation of extracellular matrix. Global gene expression of human lung fibroblasts stimulated with TGFß-1, a strong fibrotic mediator revealed the overexpression of ZNF365, a zinc finger protein implicated in cell cycle control and telomere stabilization. We evaluated the expression and localization of ZNF365 in IPF lungs and in the fibrotic response induced by bleomycin in WT and deficient mice of the orthologous gene Zfp365. In IPF, ZNF365 was overexpressed and localized in fibroblasts/myofibroblasts and alveolar epithelium. Bleomycin-induced lung fibrosis showed an upregulation of Zfp365 localized in lung epithelium and stromal cell populations. Zfp365 KO mice developed a significantly higher fibrotic response compared with WT mice by morphology and hydroxyproline content. Silencing ZNF365 in human lung fibroblasts and alveolar epithelial cells induced a significant reduction of growth rate and increased senescence markers, including Senescence Associated ß Galactosidase activity, p53, p21, and the histone variant γH2AX. Our findings demonstrate that ZNF365 is upregulated in IPF and experimental lung fibrosis and suggest a protective role since its absence increases experimental lung fibrosis mechanistically associated with the induction of cell senescence.
Assuntos
Proteínas de Ligação a DNA , Fibrose Pulmonar Idiopática , Fatores de Transcrição , Animais , Bleomicina/toxicidade , Senescência Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibrose , Histonas , Humanos , Hidroxiprolina , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Camundongos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53 , beta-Galactosidase/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is a lethal age-related lung disease whose pathogenesis involves an aberrant response of alveolar epithelial cells (AEC). Activated epithelial cells secrete mediators that participate in the activation of fibroblasts and the excessive deposition of extracellular matrix proteins. Previous studies indicate that matrix metalloproteinase 14 (MMP14) is increased in the lung epithelium in patients with IPF, however, the role of this membrane-type matrix metalloproteinase has not been elucidated. In this study, the role of Mmp14 was explored in experimental lung fibrosis induced with bleomycin in a conditional mouse model of lung epithelial MMP14-specific genetic deletion. Our results show that epithelial Mmp14 deficiency in mice increases the severity and extension of fibrotic injury and affects the resolution of the lesions. Gain-and loss-of-function experiments with human epithelial cell line A549 demonstrated that cells with a deficiency of MMP14 exhibited increased senescence-associated markers. Moreover, conditioned medium from these cells increased fibroblast expression of fibrotic molecules. These findings suggest a new anti-fibrotic mechanism of MMP14 associated with anti-senescent activity, and consequently, its absence results in impaired lung repair. Increased MMP14 in IPF may represent an anti-fibrotic mechanism that is overwhelmed by the strong profibrotic microenvironment that characterizes this disease.
Assuntos
Células Epiteliais/patologia , Fibrose Pulmonar Idiopática/genética , Metaloproteinase 14 da Matriz/genética , Alvéolos Pulmonares/metabolismo , Células A549 , Actinas/genética , Actinas/metabolismo , Animais , Bleomicina/administração & dosagem , Senescência Celular/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Humanos , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Metaloproteinase 14 da Matriz/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/patologia , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is characterized by the expansion of the myofibroblast population, excessive extracellular matrix accumulation, and destruction of the lung parenchyma. The R-spondin family (RSPO) comprises a group of proteins essential for development. Among them, RSPO2 is expressed primarily in the lungs, and its mutations cause severe defects in the respiratory tract. Interestingly, RSPO2 participates in the canonical Wingless/int1 pathway, a critical route in the pathogenesis of IPF. Thus, the aim of this study was to examine the expression and putative role of RSPO2 in this disease. We found that RSPO2 and its receptor leucine-rich G protein-coupled receptor 6 were upregulated in IPF lungs, where they localized primarily in fibroblasts and epithelial cells. Stimulation of IPF and normal lung fibroblasts with recombinant human RSPO2 resulted in the deregulation of numerous genes, although the transcriptional response was essentially distinct. In IPF fibroblasts, RSPO2 stimulation induced the up- or downregulation of several genes involved in the Wingless/int1 pathway (mainly from noncanonical signaling). In both normal and IPF fibroblasts, RSPO2 modifies the expression of genes implicated in several pathways, including the cell cycle and apoptosis. In accordance with gene expression, the stimulation of normal and IPF fibroblasts with RSPO2 significantly reduced cell proliferation and induced cell death. RSPO2 also inhibited collagen production and increased the expression of matrix metalloproteinase 1. Silencing RSPO2 with shRNA induced the opposite effects. Our findings demonstrate, for the first time to our knowledge, that RSPO2 is upregulated in IPF, where it appears to have an antifibrotic role.
Assuntos
Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Regulação para Cima/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Colágeno/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibroblastos/efeitos dos fármacos , Inativação Gênica , Genoma Humano , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Pulmão/metabolismo , Pulmão/patologia , Metaloproteinase 1 da Matriz/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/farmacologia , Regulação para Cima/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genéticaRESUMO
Acute kidney injury (AKI) is often associated to acute respiratory distress syndrome (ARDS) due to influenza A/H1N1 virus infection. The profile of angiogenic and inflammatory factors in ARDS patients may be relevant for AKI. We analyzed the serum levels of several angiogenic factors, cytokines, and chemokines in 32 patients with A/H1N1 virus infection (17 with ARDS/AKI and 15 ARDS patients who did not developed AKI) and in 18 healthy controls. Significantly higher levels of VEGF, MCP-1, IL-6, IL-8 and IP-10 in ARDS/AKI patients were detected. Adjusting by confusing variables, levels of MCP-1 ≥150 pg/mL (OR=12.0, p=0.04) and VEGF ≥225 pg/mL (OR=6.4, p=0.03) were associated with the development of AKI in ARDS patients. Higher levels of MCP-1 and IP-10 were significantly associated with a higher risk of death in patients with ARDS (hazard ratio (HR)=10.0, p=0.02; HR=25.5, p=0.03, respectively) even taking into account AKI. Patients with influenza A/H1N1 infection and ARDS/AKI have an over-production of MCP-1, VEGF and IP-10 possibly contributing to kidney injury and are associated to a higher risk of death.
Assuntos
Injúria Renal Aguda/metabolismo , Proteínas Angiogênicas/metabolismo , Inflamação/metabolismo , Influenza Humana/metabolismo , Neovascularização Patológica/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Injúria Renal Aguda/mortalidade , Injúria Renal Aguda/virologia , Adulto , Biomarcadores/metabolismo , Quimiocina CCL2/metabolismo , Quimiocina CXCL10/metabolismo , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/complicações , Influenza Humana/mortalidade , Masculino , México/epidemiologia , Síndrome do Desconforto Respiratório/mortalidade , Síndrome do Desconforto Respiratório/virologia , Taxa de Sobrevida , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disease of unknown etiology and uncertain pathogenic mechanisms. Recent studies indicate that the pathogenesis of the disease may involve the abnormal expression of certain developmental pathways. Here we evaluated the expression of Sonic Hedgehog (SHH), Patched-1, Smoothened, and transcription factors glioma-associated oncogene homolog (GLI)1 and GLI2 by RT-PCR, as well as their localization in IPF and normal lungs by immunohistochemistry. The effects of SHH on fibroblast proliferation, migration, collagen and fibronectin production, and apoptosis were analyzed by WST-1, Boyden chamber chemotaxis, RT-PCR, Sircol, and annexin V-propidium iodide binding assays, respectively. Our results showed that all the main components of the Sonic signaling pathway were overexpressed in IPF lungs. With the exception of Smoothened, they were also upregulated in IPF fibroblasts. SHH and GLI2 localized to epithelial cells, whereas Patched-1, Smoothened, and GLI1 were observed mainly in fibroblasts and inflammatory cells. No staining was detected in normal lungs. Recombinant SHH increased fibroblast proliferation (P < 0.05), collagen synthesis, (2.5 ± 0.2 vs. 4.5 ± 1.0 µg of collagen/ml; P < 0.05), fibronectin expression (2-3-fold over control), and migration (190.3 ± 12.4% over control, P < 0.05). No effect was observed on α-smooth muscle actin expression. SHH protected lung fibroblasts from TNF-α/IFN-γ/Fas-induced apoptosis (14.5 ± 3.2% vs. 37.3 ± 7.2%, P < 0.0001). This protection was accompanied by modifications in several apoptosis-related proteins, including increased expression of X-linked inhibitor of apoptosis. These findings indicate that the SHH pathway is activated in IPF lungs and that SHH may contribute to IPF pathogenesis by increasing the proliferation, migration, extracellular matrix production, and survival of fibroblasts.
Assuntos
Proteínas Hedgehog/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Apoptose , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Fibronectinas/genética , Fibronectinas/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/fisiologia , Humanos , Fibrose Pulmonar Idiopática/patologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores Patched , Receptor Patched-1 , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Receptor Smoothened , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de ZincoRESUMO
RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by epithelial phenotypic changes and fibroblast activation. Based on the temporal heterogeneity of IPF, we hypothesized that hyperplastic alveolar epithelial cells regulate the fibrotic response. OBJECTIVES: To identify novel mediators of fibrosis comparing the transcriptional signature of hyperplastic epithelial cells and conserved epithelial cells in the same lung. METHODS: Laser capture microscope and microarrays analysis were used to identify differentially expressed genes in IPF lungs. Bleomycin-induced lung fibrosis was evaluated in Mmp19-deficient and wild-type (WT) mice. The role of matrix metalloproteinase (MMP)-19 was additionally studied by transfecting the human MMP19 in alveolar epithelial cells. MEASUREMENTS AND MAIN RESULTS: Laser capture microscope followed by microarray analysis revealed a novel mediator, MMP-19, in hyperplastic epithelial cells adjacent to fibrotic regions. Mmp19(-/-) mice showed a significantly increased lung fibrotic response to bleomycin compared with WT mice. A549 epithelial cells transfected with human MMP19 stimulated wound healing and cell migration, whereas silencing MMP19 had the opposite effect. Gene expression microarray of transfected A549 cells showed that PTGS2 (prostaglandin-endoperoxide synthase 2) was one of the highly induced genes. PTGS2 was overexpressed in IPF lungs and colocalized with MMP-19 in hyperplastic epithelial cells. In WT mice, PTGS2 was significantly increased in bronchoalveolar lavage and lung tissues after bleomycin-induced fibrosis, but not in Mmp19(-/-) mice. Inhibition of Mmp-19 by siRNA resulted in inhibition of Ptgs2 at mRNA and protein levels. CONCLUSIONS: Up-regulation of MMP19 induced by lung injury may play a protective role in the development of fibrosis through the induction of PTGS2.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Fibrose Pulmonar Idiopática/enzimologia , Metaloproteinases da Matriz Secretadas/metabolismo , Animais , Bleomicina , Células Cultivadas , Células Epiteliais/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Microdissecção e Captura a Laser , Metaloproteinases da Matriz Secretadas/genética , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Alvéolos Pulmonares/metabolismo , Regulação para CimaRESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disorder of unknown etiology. IPF is likely the result of complex interrelationships between environmental and host factors, although the genetic risk factors are presently uncertain. Because we have found that some MHC polymorphisms confer susceptibility to IPF, in the present study we aimed to evaluate the role of the MHC class I chain-related gene A (MICA) in the risk of developing the disease. MICA molecular typing was done by reference strand mediated conformation analysis in a cohort of 80 IPF patients and 201 controls. In addition, the lung cellular source of the protein was examined by immunohistochemistry, the expression of the MICA receptor NKG2D in lung cells by flow cytometry and soluble MICA by ELISA. A significant increase of MICA*001 was observed in the IPF cohort (OR = 2.91, 95% CI = 1.04-8.25; pC = 0.03). Likewise, the frequency of the MICA*001/*00201 genotype was significantly increased in patients with IPF compared with the healthy controls (OR = 4.72, 95% CI = 1.15-22.51; pC = 0.01). Strong immunoreactive MICA staining was localized in alveolar epithelial cells and fibroblasts from IPF lungs while control lungs were negative. Soluble MICA was detected in 35% of IPF patients compared with 12% of control subjects (P = 0.0007). The expression of NKG2D was significantly decreased in gammadelta T cells and natural killer cells obtained from IPF lungs. These findings indicate that MICA polymorphisms and abnormal expression of the MICA receptor NKG2D might contribute to IPF susceptibility.
Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Fibrose Pulmonar Idiopática/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Polimorfismo Genético , Adulto , Idoso , Estudos de Casos e Controles , Células Cultivadas , Feminino , Expressão Gênica , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Antígenos HLA-B/genética , Antígenos de Histocompatibilidade Classe I/sangue , Humanos , Células Matadoras Naturais/metabolismo , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Análise de Sequência de DNA , Subpopulações de Linfócitos T/metabolismoRESUMO
RATIONALE: Hypersensitivity pneumonitis (HP) exhibits a diverse outcome. Patients with acute/subacute HP usually improve, whereas patients with chronic disease often progress to fibrosis. However, the mechanisms underlying this difference are unknown. OBJECTIVES: To examine the T-cell profile from patients with subacute HP and chronic HP. METHODS: T cells were obtained by bronchoalveolar lavage from 25 patients with subacute HP, 30 patients with chronic HP, and 8 control subjects. T-cell phenotype and functional profile were evaluated by flow cytometry, cytometric bead array, and immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: Patients with chronic HP showed higher CD4+:CD8+ ratio (median, 3.05; range, 0.3-15; subacute HP: median, 1.3; range, 0.1-10; control: median, 1.3; range, 0.7-2.0; P < 0.01), and a decrease of gammadeltaT cells (median, 2.0; range, 0.5-3.4; subacute HP: median, 10; range, 4.8-17; control: median, 15; range, 5-19; P < 0.01). Patients with chronic HP exhibited an increase in the terminally differentiated memory CD4+ and CD8+ T-cell subsets compared with patients with subacute HP (P < 0.05). However, memory cells from chronic HP showed lower IFN-gamma production and decreased cytotoxic activity by CD8+ T lymphocytes. Chronic HP displayed a Th2-like phenotype with increased CXCR4 expression (median, 6%; range, 1.7-36, vs. control subjects: median, 0.7%; range, 0.2-1.4; and subacute HP: median, 2.2%; range, 0.1-5.3; P < 0.01), and decreased CXCR3 expression (median, 4.3%; range, 1.4-25%, vs. subacute HP: median, 37%; range, 4.9-78%; P < 0.01). Likewise, supernatants from antigen-specific-stimulated cells from chronic HP produced higher levels of IL-4 (80 +/- 63 pg/ml vs. 25 +/- 7 pg/ml; P < 0.01), and lower levels of IFN-gamma (3,818 +/- 1671 pg/ml vs. 100 +/- 61 pg/ml; P < 0.01) compared with subacute HP. CONCLUSIONS: Our findings indicate that patients with chronic HP lose effector T-cell function and exhibit skewing toward Th2 activity, which may be implicated in the fibrotic response that characterizes this clinical form.
Assuntos
Alveolite Alérgica Extrínseca/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Idoso , Líquido da Lavagem Broncoalveolar/imunologia , Relação CD4-CD8 , Quimiocinas/metabolismo , Doença Crônica , Citocinas/metabolismo , Testes Imunológicos de Citotoxicidade , Feminino , Citometria de Fluxo , Humanos , Pulmão/imunologia , Contagem de Linfócitos , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Masculino , Pessoa de Meia-Idade , Fibrose Pulmonar/imunologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Pulmonary fibrosis is a common response to a variety of lung injuries, characterized by fibroblast/myofibroblast expansion and abnormal accumulation of extracellular matrix. An increased expression of matrix metalloprotease 9 (MMP9) in human and experimental lung fibrosis has been documented, but its role in the fibrotic response is unclear. We studied the effect of MMP9 overexpression in bleomycin-driven lung fibrosis using transgenic mice expressing human MMP9 in alveolar macrophages (hMMP9-TG). At 8 weeks post-bleomycin, the extent of fibrotic lesions and OH-proline content were significantly decreased in the TG mice compared to the WT mice. The decreased fibrosis in hMMP9-TG mice was preceded by a significant reduction of neutrophils and lymphocytes in bronchoalveolar lavage (BAL) at 1 and 4 weeks post-bleomycin, respectively, as well as by significantly less TIMP-1 than the WT mice. From a variety of cytokines/chemokines investigated, we found that BAL levels of insulin-like growth factor binding protein-3 (IGFBP3) as well as the immunoreactive protein in the lungs were significantly lower in hMMP9-TG mice compared with WT mice despite similar levels of gene expression. Using IGFBP-3 substrate zymography we found that BAL from TG mice at 1 week after bleomycin cleaved IGFBP-3. Further, we demonstrated that MMP9 degraded IGFBP-3 into lower molecular mass fragments. These findings suggest that increased activity of MMP9 secreted by alveolar macrophages in the lung microenvironment may have an antifibrotic effect and provide a potential mechanism involving IGFBP3 degradation.
Assuntos
Bleomicina/toxicidade , Macrófagos Alveolares/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Fibrose Pulmonar/enzimologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Citocinas/análise , Citocinas/metabolismo , Expressão Gênica , Humanos , Hidroxiprolina/metabolismo , Imuno-Histoquímica , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Pulmão/enzimologia , Pulmão/metabolismo , Pulmão/patologia , Linfócitos/citologia , Macrófagos Alveolares/citologia , Macrófagos Alveolares/metabolismo , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Neutrófilos/citologia , Peroxidase/análise , Peroxidase/metabolismo , Análise Serial de Proteínas , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
STUDY OBJECTIVE: To evaluate the effect of CP-471,474 (Pfizer Global Research and Development; Groton, CT), a broad-spectrum inhibitor of matrix metalloproteinases (MMPs) in an experimental model of emphysema. DESIGN: Randomized, double-blinded, controlled experiment. SETTING: Biochemistry and morphology laboratories and animal research facility. METHODS: Guinea pigs were exposed to cigarette smoke over 1 month, 2 months, and 4 months, and half of the animals received CP-471,474. Age-matched guinea pigs exposed to room air were used as control animals. After death, the lungs were lavaged with saline solution, and MMPs in the lavage fluid were determined by zymography and immunoblot. Lungs were fixed for histology, immunohistochemistry, and morphometry. RESULTS: Following a 1-month exposure to tobacco smoke, semiquantitative histologic assessment showed moderate lung inflammation, which progressed in extent and severity and reached a peak at 2 months. CP-471,474 significantly reduced both the extent (p < 0.002) and severity (p < 0.05) of inflammation at 2 months. At 4 months, a spontaneous reduction of the inflammatory response was observed in both treated and untreated animals, and consequently no difference was observed between both. Emphysematous changes, revealed by a significant increase in the average size of alveoli, were detected at 2 months and 4 months of tobacco smoke exposure. The inhibitor significantly decreased the destructive lesions mainly at 2 months (p < 0.0001) and also at 4 months (p < 0.02). Smoking increased MMP-9 and MMP-1 activities as shown by zymography and immunoblot. Immunoreactive MMP-9 was mainly localized in alveolar and bronchiolar epithelial cells, macrophages, and airways smooth-muscle cells. CONCLUSION: These findings support a role for MMPs in the early inflammatory response and in the emphysematous lesions provoked by cigarette smoking.