Toshitaka Maeno

Increased Connective Tissue Growth Factor Relative to Brain Natriuretic Peptide as a Determinant of Myocardial Fibrosis

Excessive fibrosis contributes to an increase in left ventricular stiffness. The goal of the present study was to investigate the role of connective tissue growth factor (CCN2/CTGF), a profibrotic cytokine of the CCN (Cyr61, CTGF, and Nov) family, and its functional interactions with brain natriuretic peptide (BNP), an antifibrotic peptide, in the development of myocardial fibrosis and diastolic heart failure. Histological examination on endomyocardial biopsy samples from patients without systolic dysfunction revealed that the abundance of CTGF-immunopositive cardiac myocytes was correlated with the excessive interstitial fibrosis and a clinical history of acute pulmonary congestion. In a rat pressure overload cardiac hypertrophy model, CTGF mRNA levels and BNP mRNA were increased in proportion to one another in the myocardium. Interestingly, relative abundance of mRNA for CTGF compared with BNP was positively correlated with diastolic dysfunction, myocardial fibrosis area, and procollagen type 1 mRNA expression. Investigation with conditioned medium and subsequent neutralization experiments using primary cultured cells demonstrated that CTGF secreted by cardiac myocytes induced collagen production in cardiac fibroblasts. Further, G protein-coupled receptor ligands induced expression of the CTGF and BNP genes in cardiac myocytes, whereas aldosterone and transforming growth factor-&bgr; preferentially induced expression of the CTGF gene. Finally, exogenous BNP prevented the production of CTGF in cardiac myocytes. These data suggest that a disproportionate increase in CTGF relative to BNP in cardiac myocytes plays a central role in the induction of excessive myocardial fibrosis and diastolic heart failure.

HERP1 Inhibits Myocardin-Induced Vascular Smooth Muscle Cell Differentiation by Interfering With SRF Binding to CArG Box

Objective—Myocardin is a coactivator of serum response factor (SRF) required for vascular smooth muscle cell (VSMC) differentiation. HERP1 is a transcriptional repressor, which is abundantly expressed in vascular system and is known to function as a target gene of Notch. However, the role of HERP1 in the pathogenesis of vascular lesions remains unknown. The present study characterizes the expression of HERP1 in normal and diseased vessels, and tests the hypothesis that HERP1 inhibits SRF/myocardin-dependent SMC gene expression. Methods and Results—Immunohistochemistry revealed that HERP1 and myocardin expression was localized to SMC in the neointima of balloon-injured rat aorta and in human coronary atherosclerotic lesions. Expression of both HERP1 and myocardin was elevated in cultured VSMCs compared with medial SMC. Overexpressed HERP1 inhibited the myocardin-induced SMC marker gene expression in 10T1/2 cells. HERP1 protein interfered with the SRF/CArG–box interaction in vivo and in vitro. Immunoprecipitation assays showed that HERP1 physically interacts with SRF. Conclusions—HERP1 expression was associated with the SMC proliferation and dedifferentiation in vitro and in vivo. HERP1 may play a role in promoting the phenotypic modulation of VSMCs during vascular injury and atherosclerotic process by interfering with SRF binding to CArG-box through physical association between HERP1 and SRF.

Pioglitazone, a Peroxisome Proliferator-Activated Receptor Gamma Ligand, Suppresses Bleomycin-Induced Acute Lung Injury and Fibrosis

Background: Peroxisome proliferator-activated receptor-γ (PPARγ) ligands have been shown to possess potent anti-inflammatory actions. Idiopathic interstitial pneumonia is defined as a specific form of chronic fibrosing lung disease characterized by progressive fibrosis which leads to deterioration and destruction of the lungs. Objective: To investigate whether the PPARγ ligand pioglitazone (PGZ) inhibited bleomycin (BLM)-induced acute lung injury and subsequent fibrosis. Methods: BLM was administered intratracheally to Wistar rats which were then treated with PGZ. Rat alveolar macrophages were stimulated with BLM for 6 h with or without PGZ pretreatment for 18 h. MRC-5 cells (human lung fibroblasts) were treated with PGZ for 18 h. After the treatment, the cells were stimulated with transforming growth factor- β (TGF-β) for 6 h. Results: PGZ inhibited BLM-induced acute lung injury and subsequent lung fibrosis when it was administered from day –7. PGZ treatment suppressed the accumulation of inflammatory cells in lungs and the concentration of tumor necrosis factor-α (TNF-α) in bronchoalveolar lavage fluid on day 3. PGZ also inhibited BLM-induced TNF-α production in alveolar macrophages. Furthermore, PGZ inhibited fibrotic changes and an increase in hydroxyproline content in lungs after instillation of BLM, even when PGZ was administered in the period from day 7 to day 28. Northern blot analyses revealed that PGZ inhibited TGF-β-induced procollagen I and connective tissue growth factor (CTGF) expression in MRC-5 cells. Conclusion: These results suggest that activation of PPARγ ameliorates BLM-induced acute inflammatory responses and fibrotic changes at least partly through suppression of TNF-α, procollagen I and CTGF expression. Beneficial effects of this PPARγ ligand on inflammatory and fibrotic processes open new perspectives for a potential role of PPARγ as a molecular target in fibroproliferative lung diseases.

Hypoxia-inducible factor-1α mediates TGF-β-induced PAI-1 production in alveolar macrophages in pulmonary fibrosis

Hypoxia-inducible factor-1α (HIF-1α), a transcription factor that functions as a master regulator of oxygen homeostasis, has been implicated in fibrinogenesis. Here, we explore the role of HIF-1α in transforming growth factor-β (TGF-β) signaling by examining the effects of TGF-β(1) on the expression of plasminogen activator inhibitor-1 (PAI-1). Immunohistochemistry of lung tissue from a mouse bleomycin (BLM)-induced pulmonary fibrosis model revealed that expression of HIF-1α and PAI-1 was predominantly induced in alveolar macrophages. Real-time RT-PCR and ELISA analysis showed that PAI-1 mRNA and activated PAI-1 protein level were strongly induced 7 days after BLM instillation. Stimulation of cultured mouse alveolar macrophages (MH-S cells) with TGF-β(1) induced PAI-1 production, which was associated with HIF-1α protein accumulation. This accumulation of HIF-1α protein was inhibited by SB431542 (type I TGF-β receptor/ALK receptor inhibitor) but not by PD98059 (MEK1 inhibitor) and SB203580 (p38 MAP kinase inhibitor). Expression of prolyl-hydroxylase domain (PHD)-2, which is essential for HIF-1α degradation, was inhibited by TGF-β(1), and this decrease was abolished by SB431542. TGF-β(1) induction of PAI-1 mRNA and its protein expression were significantly attenuated by HIF-1α silencing. Transcriptome analysis by cDNA microarray of MH-S cells after HIF-1α silencing uncovered several pro-fibrotic genes whose regulation by TGF-β(1) required HIF-1α, including platelet-derived growth factor-A. Taken together, these findings expand our concept of the role of HIF-1α in pulmonary fibrosis in mediating the effects of TGF-β(1) on the expression of the pro-fibrotic genes in activated alveolar macrophages.

A single dose of lipopolysaccharide into mice with emphysema mimics human Chronic obstructive pulmonary disease exacerbation as assessed by micro-computed tomography

Chronic obstructive pulmonary disease (COPD), manifested as emphysema and chronic airway obstruction, can be exacerbated by bacterial and viral infections. Although the frequency of exacerbations increases as the disease progresses, the mechanisms underlying this phenomenon are largely unknown, and there is a need for a simple in vivo exacerbation model. In this study, we compared four groups of mice treated with PBS alone, elastase alone, LPS alone, and elastase plus LPS. A single intratracheal administration of LPS to mice with elastase-induced emphysema provoked infiltration of inflammatory cells, especially CD8(+) T cells, into alveolar spaces and increased matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, and perforin production in bronchoalveolar lavage fluid at the acute inflammatory phase compared with the other groups. We also measured the percentage of low-attenuation area (LAA%) in the above mice using micro-computed X-ray tomography. The LAA% was the most sensitive parameter for quantitative assessments of emphysema among all the parameters evaluated. Using the parameter of LAA%, we found significantly more severe alveolar destruction in the group treated with elastase plus LPS compared with the other groups during long-term longitudinal observations. We built three-dimensional images of the emphysema and confirmed that the lungs of elastase plus LPS-treated mice contained larger emphysematous areas than mice treated with elastase alone. Although human exacerbation of COPD is clinically and pathologically complicated, this simple mouse model mimics human cases to some extent and will be useful for elucidating its mechanism and developing therapeutic strategies.

Sensitivity of heterozygous α1,6-fucosyltransferase knock-out mice to cigarette smoke-induced emphysema: Implication of aberrant transforming growth factor-β signaling and matrix metalloproteinase gene expression

Background: Fut8−/− mice show emphysematous lesions, the major risk factor for which is exposure to cigarette smoke (CS). Results: Fut8+/− mice developed CS-induced emphysematous lesions, which are associated with an aberrant Smad7-Smad2-matrix metalloproteinase signaling pathway. Conclusion: Genetic ablation of Fut8 increases sensitivity to CS-induced emphysema. Significance: Core fucosylation appears to be involved in the development of chronic obstructive pulmonary disease. We previously demonstrated that a deficiency in core fucosylation caused by the genetic disruption of α1,6-fucosyltransferase (Fut8) leads to lethal abnormalities and the development of emphysematous lesions in the lung by attenuation of TGF-β1 receptor signaling. Herein, we investigated the physiological relevance of core fucosylation in the pathogenesis of emphysema using viable heterozygous knock-out mice (Fut8+/−) that were exposed to cigarette smoke (CS). The Fut8+/− mice exhibited a marked decrease in FUT8 activity, and matrix metalloproteinase (MMP)-9 activities were elevated in the lung at an early stage of exposure. Emphysema developed after a 3-month CS exposure, accompanied by the recruitment of large numbers of macrophages to the lung. CS exposure substantially and persistently elevated the expression level of Smad7, resulting in a significant reduction of Smad2 phosphorylation (which controls MMP-9 expression) in Fut8+/− mice and Fut8-deficient embryonic fibroblast cells. These in vivo and in vitro studies show that impaired core fucosylation enhances the susceptibility to CS and constitutes at least part of the disease process of emphysema, in which TGF-β-Smad signaling is impaired and the MMP-mediated destruction of lung parenchyma is up-regulated.

c-Src and Hydrogen Peroxide Mediate Transforming Growth Factor-β1–Induced Smooth Muscle Cell–Gene Expression in 10T1/2 Cells

Objective— Transforming growth factor-β1 (TGF-β1) controls the expression of numerous genes, including smooth muscle cell (SMC)–specific genes and extracellular matrix protein genes. Here we investigated whether c-Src plays a role in TGF-β1 signaling in mouse embryonic fibroblast C3H10T1/2 cells. Methods and Results— TGF-β1 induction of the SMC contractile protein SM22α gene expression was inhibited by PP1 (an inhibitor of Src family kinases) or by C-terminal Src kinase (a negative regulator of c-Src). Induction of SM22α by TGF-β1 was markedly attenuated in SYF cells (c-Src−, Yes−, and Fyn−) compared with Src++ cells (c-Src++, Yes−, and Fyn−). PP1 also inhibited the TGF-β1–induced expression of serum response factor (SRF), a transcription factor regulating the SMC marker gene expression. Confocal immunofluorescence analysis showed that TGF-β1 stimulates production of hydrogen peroxide. Antioxidants such as catalase or NAD(P)H oxidase inhibitors such as apocynin inhibited the TGF-β1–induced expression of SM22α. Furthermore, we demonstrate that TGF-β1 induction of the plasminogen activator inhibitor-1 (PAI-1) gene, which is known to be dependent on Smad but not on SRF, is inhibited by PP1 and apocynin. Conclusion— Our results suggest that TGF-β1 activates c-Src and generates hydrogen peroxide through NAD(P)H oxidase, and these signaling pathways lead to the activation of specific sets of genes, including SM22α and PAI-1.

Deranged fatty acid composition causes pulmonary fibrosis in Elovl6 -deficient mice

Despite the established role of alveolar type II epithelial cells for the maintenance of pulmonary function, little is known about the deregulation of lipid composition in the pathogenesis of pulmonary fibrosis. The elongation of long-chain fatty acids family member 6 (Elovl6) is a rate-limiting enzyme catalysing the elongation of saturated and monounsaturated fatty acids. Here we show that Elovl6 expression is significantly downregulated after an intratracheal instillation of bleomycin (BLM) and in human lung with idiopathic pulmonary fibrosis. Elovl6-deficient (Elovl6⁻/⁻) mice treated with BLM exhibit severe fibroproliferative response and derangement of fatty acid profile compared with wild-type mice. Furthermore, Elovl6 knockdown induces a change in fatty acid composition similar to that in Elovl6⁻/⁻ mice, resulting in induction of apoptosis, TGF-β1 expression and reactive oxygen species generation. Our findings demonstrate a previously unappreciated role for Elovl6 in the regulation of lung homeostasis, and in pathogenesis and exacerbation of BLM-induced pulmonary fibrosis.

Alendronate inhalation ameliorates elastase-induced pulmonary emphysema in mice by induction of apoptosis of alveolar macrophages

Alveolar macrophages play a crucial role in the pathogenesis of emphysema, for which there is currently no effective treatment. Bisphosphonates are widely used to treat osteoclast-mediated bone diseases. Here we show that delivery of the nitrogen-containing bisphosphonate alendronate via aerosol inhalation ameliorates elastase-induced emphysema in mice. Inhaled, but not orally ingested, alendronate inhibits airspace enlargement after elastase instillation, and induces apoptosis of macrophages in bronchoalveolar fluid via caspase-3- and mevalonate-dependent pathways. Cytometric analysis indicates that the F4/80(+)CD11b(high)CD11c(mild) population characterizing inflammatory macrophages, and the F4/80(+)CD11b(mild)CD11c(high) population defining resident alveolar macrophages take up substantial amounts of the bisphosphonate imaging agent OsteoSense680 after aerosol inhalation. We further show that alendronate inhibits macrophage migratory and phagocytotic activities and blunts the inflammatory response of alveolar macrophages by inhibiting nuclear factor-κB signalling. Given that the alendronate inhalation effectively induces apoptosis in both recruited and resident alveolar macrophages, we suggest this strategy may have therapeutic potential for the treatment of emphysema.

Pleural amyloidosis in a patient with intractable pleural effusion and multiple myeloma

Pleural involvement of systemic amyloidosis has been rarely reported. We report a case with multiple myeloma presenting an intractable right pleural effusion, in which pleural amyloidosis was diagnosed through pleural biopsy using a Cope needle. The diagnosis of pleural amyloidosis is important, because its refractory pleural effusion should be treated with pleurodesis. Since closed pleural biopsy using a Cope needle is much less invasive than thoracoscopy, the former should be attempted first whenever pleural amyloidosis is suspected.