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Increased numbers of T lymphocytes are observed in the lungs of patients with chronic obstructive pulmonary disease, but their role in the disease process is not known. We investigated the role of CD8+ T cells in inflammatory cell recruitment and lung destruction in a cigarette smoke-induced murine model of emphysema. In contrast to wild-type C57BL/6J mice that displayed macrophage, lymphocyte, and neutrophil recruitment to the lung followed by emphysema in response to cigarette smoke, CD8+ T cell-deficient (CD8−/−) mice had a blunted inflammatory response and did not develop emphysema when exposed to long-term cigarette smoke. Further studies supported a pathogenetic pathway whereby the CD8+ T cell product, IFN-γ-inducible protein-10, induces production of macrophage elastase (matrix metalloproteinase 12) that degrades elastin, both causing lung destruction directly and generating elastin fragments that serve as monocyte chemokines augmenting macrophage-mediated lung destruction. These studies demonstrate a requirement for CD8+ T cells for the development of cigarette smoke-induced emphysema and they provide a unifying pathway whereby CD8+ T cells are a central regulator of the inflammatory network in chronic obstructive pulmonary disease.

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. Key Words: HERP1 Ⅲ myocardin Ⅲ serum response factor Ⅲ smooth muscle cells P henotypic modulation of vascular smooth muscle cells (VSMCs) from contractile to synthetic forms plays a pivotal role in the pathogenesis of vascular diseases including atherosclerosis and restenosis after angioplasty. 1 It is wellestablished that VSMC phenotype is regulated by a complex array of local environmental cues including humoral factors, cell-cell and cell-matrix interactions, inflammatory stimuli, and mechanical stresses. Such complex stimuli downregulate a number of genes required for the contractile phenotype in synthetic VSMCs. These include smooth muscle myosin heavy chain (SM-MHC), SM22␣, caldesmon, and calponin. Because the genes encoding these proteins are differentially expressed depending on the proliferative state of VSMCs, transcription factors regulated by numerous stimuli are responsible at least in part for the distinct pattern of gene expression seen in synthetic VSMCs.There is mounting evidence that most SMC marker proteins such as SM-MHC and SM22␣ are controlled by serum response factor (SRF), which binds to a sequence known as a CArG box and recruits a potent coactivator, myocardin, for SMC differentiation. 1 When myocardin is ectopically expressed in nonmuscle cells, it can induce SMC differentiation. 2,3 Most importantly, mouse embryos deficient for myocardin show no evidence of vascular SMC, indicating myocardin as a necessary and sufficient factor for SMC differentiation in vivo. 4 These observations, in conjunction with downregulation of SMC marker genes in synthetic VSMC, led us to speculate that myocardin express...

Abstract-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-␤ 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. Key Words: extracellular matrix Ⅲ hypertrophy Ⅲ cardiac function Ⅲ connective tissue growth factor Ⅲ natriuretic peptide E pidemiological studies have established that 40% to 50% of patients with heart failure have normal or minimally impaired left ventricular (LV) ejection fraction, a clinical syndrome that is commonly referred to as diastolic heart failure (DHF). These patients typically have cardiac hypertrophy that is induced by long-standing hypertension or by primary hypertrophic cardiomyopathy, as well as increased passive LV stiffness. 1 Among various molecular mechanisms that regulate LV stiffness, 2 abnormalities in the transcriptional or posttranscriptional regulation of the collagen gene can result in the disproportionate accumulation of fibrous tissue and elevation of stiffness in the hypertrophied heart. 2,3 Recent studies have shown that, in addition to mechanical load, autocrine, paracrine, and endocrine factors, such as angiotensin II, aldosterone (Aldo), endothelin-1 (ET1), natriuretic peptides, osteopontin, and transforming growth factor-␤1 (TGF-␤), play important roles in the development of myocardial hypertrophy and fibrosis. 4,5 However, the precise molecular mechanisms that initiate and promote myocardial fibrosis and increases in ventricular stiffness remain largely unknown.Connec...

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.

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 À / À ) 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 À / À mice, resulting in induction of apoptosis, TGF-b1 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.

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.

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