Inhibitory effects of interferon-γ on the heterologous desensitization of β-adrenoceptors by transforming growth factor-β1 in tracheal smooth muscle

BACKGROUND: Transforming growth factor-beta1 (TGF-beta 1) is generally considered to play an important role in the pathogenesis of chronic inflammation and fibrosis. OBJECTIVE AND METHODS: This study was designed to determine mechanisms of reduced responsiveness of guinea-pig tracheal smooth muscle to beta-adrenoceptor agonists by TGF-beta 1, using isometric tension records and tissue cAMP measurement. Moreover, we examined the involvement of the signal transduction processes of TGF-beta superfamily in the desensitization of beta-adrenoceptors. RESULTS: After exposure to 0.2-2000 pm TGF-beta 1 for 4-8 h, the inhibitory effects of 1 microm isoprenaline (ISO) and 10 microm forskolin on 1 microm MCh-induced contraction were markedly reduced in a concentration-dependent fashion. The desensitization by TGF-beta 1 was greater against ISO than for forskolin. The values of EC75 for the curves for ISO after exposure to the normal bathing solution and TGF-beta 1 were 0.039 +/- 0.02 and 0.38 +/- 0.28 microm, respectively. The values of EC50 for the curves for forskolin under these conditions were 0.50 +/- 0.12 and 0.89 +/- 0.21 microm, respectively. On the other hand, the inhibitory effects of phosphodiesterase inhibitors such as theophylline and rolipram were not attenuated after exposure to TGF-beta 1. Concentration-inhibition curve for ISO was shifted to the right after exposure to 2000 pm TGF-beta 1 for 8 h more than that curve for forskolin. In contrast, the curve for theophylline was not shifted to the right by TGF-beta 1. When the tissues were incubated with TGF-beta 1 in the presence of IFN-gamma, an intracellular antagonist of TGF-beta signalling, IFN-gamma inhibited the reduced response to ISO and forskolin after exposure to TGF-beta 1 in a concentration-dependent fashion. After exposure to TGF-beta 1, the effects of cAMP accumulation of ISO was significantly reduced, however, neither forskolin-nor theophylline-induced cAMP accumulation was affected. IFN-gamma had no significant effect on cAMP accumulation either to ISO or forskolin. CONCLUSIONS: Impairment of the beta-adrenoceptors/adenylyl cyclase pathway are involved in heterologous desensitization of beta-adrenoceptors induced by TGF-beta 1 in airway smooth muscle. IFN-gamma functionally suppresses this phenomenon via cAMP-independent processes. Phosphodiesterase is still intact under this condition.     

Altered compartimentalization of transforming growth factor-β in asthmatic airways

Background Asthma is characterized by alterations of the bronchial epithelium associated with inflammatory cell infiltrates and sub-epithelial fibrosis. Transforming Growth Factor-β (TGF-β) is an anti-inflammatory and fibrosing cytokine normally present in bronchial epithelial cells and also potentially produced by inflammatory cells. Thus, TGF-β could play a role in the asthmatic process, and its expression could be modified in asthmatic airways. Objective To test this latter hypothesis, we studied the bronchial distribution of TGF-β in asthmatic patients. Methods TGF-β1, 2, 3 distribution was studied by immunohistochemistry in bronchial biopsies from 12 asthmatic patients and 10 non-asthmatic subjects. Results Bronchial epithelial cells from asthmatics were negative or faintly positive while a bright staining was detected in these from non-asthmatics (P < 0.0l). In both groups, when inflammatory cells were present beneath the basement membrane, they were stained by the anti-TGF-β antibody. Conclusion This study shows an altered compartimentalization of TGF-β in asthma. (a) TGF-β is scarse in asthmatic bronchial epithelial cells, which could favour the perennization of the bronchial inflammation, and (b) TGF-β is present in inflammatory cells beneath the basement membrane, where it could be involved in the frequent sub-epithelial fibrosis.     

Fibroblast growth factor 9 signaling inhibits airway smooth muscle differentiation in mouse lung

In mammalian lungs, airway smooth muscle cells (airway SMCs) are present in the proximal lung adjacent to bronchi and bronchioles, but are absent in the distal lung adjacent to terminal sacs that expand during gas exchange. Evidence suggests that this distribution is essential for the formation of a functional respiratory tree, but the underlying genetic mechanism has not been elucidated. In this study, we test the hypothesis that fibroblast growth factor 9 (Fgf9) signaling is essential to restrict SMC differentiation to the proximal lung. We show that loss of Fgf9 or conditional inactivation of Fgf receptors (Fgfr) 1 and 2 in mouse lung mesenchyme results in ectopic SMCs. Our data support a model where FGF9 maintains a SMC progenitor population by suppressing differentiation and promoting growth. This model also represents our findings on the genetic relationship between FGF9 and sonic hedgehog (SHH) in the establishment of airway SMC pattern. Developmental Dynamics 238:123–137, 2009. © 2008 Wiley‐Liss, Inc.     

Leukotriene D4-induced, epithelial cell-derived transforming growth factor β1 in human bronchial smooth muscle cell proliferation

Background Cysteinyl‐leukotrienes (cys‐LTs) orchestrate many pathognomonic features of asthma in animal models of allergic airway inflammation, including bronchial smooth muscle cell (BSMC) hyperplasia. However, because cys‐LTs alone do not induce mitogenesis in monocultures of human BSMC, the effect observed in vivo seemingly involves indirect mechanisms, which are still undefined. Objective This study aims to investigate the regulatory role of leukotriene (LT)D₄ on TGF‐β1 expression in airway epithelial cells and the consequence of this interplay on BSMC proliferation. Methods HEK293 cells stably transfected with cys‐LT receptor 1 (CysLT1) (293LT1) were stimulated with LTD₄ and TGF‐β1 mRNA and protein expression was measured using Northern blot and ELISA, respectively. Conditioned medium (CM) harvested from LTD₄‐treated cells was then assayed for its proliferative effect on primary human BSMC. TGF‐β1 mRNA expression was also determined in tumoural type II pneumocytes A549 and in normal human bronchial epithelial cells (NHBE) following LTD₄ stimulation. Results The results demonstrated that LTD₄‐induced TGF‐β1 mRNA production in a time‐ and concentration‐dependent manner in 293LT1. TGF‐β1 secretion was also up‐regulated and CM from LTD₄‐treated 293LT1 was shown to increase BSMC proliferation in a TGF‐β1‐dependent manner. The increased expression of TGF‐β1 mRNA by LTD₄ also occured in A549 and NHBE cells via a CysLT1‐dependent mechanism. Conclusion In conclusion, elevated expression of cys‐LTs in asthmatic airways might contribute to BSMC hyperplasia and concomitant clinical features of asthma such as airway hyperresponsiveness via a paracrine loop involving TGF‐β1 production by airway epithelial cells.     

Proliferative effects of eosinophil lysates on cultured human airway smooth muscle cells

BACKGROUND: The hypertrophy/hyperplasia of airway smooth muscle (ASM) cells is one of the characteristic features of bronchial asthma. This structural change leads to the thickening of airway walls resulting in the amplification of airway narrowing. However, the pathogenesis of this structural change has not yet been determined. Eosinophils, which play a pathogenic role in asthma, have been demonstrated to have proliferative effects on fibroblasts and vascular smooth muscle cells. OBJECTIVE: We attempted to investigate the potential of eosinophils to induce the proliferation of ASM cells. METHODS: We examined the effect of lysates of eosinophils purified from peripheral blood of healthy donors on cultured human ASM cell proliferation. RESULTS: Eosinophil lysates significantly induced ASM cell proliferation in time- and dose-dependent manners, reaching a maximum on day 6 at 50% of eosinophil lysates (6.0 +/- 0.7 x 104 [mean +/- SD] /well, n = 5 vs. 4.5 +/- 1.1 x 104/well, n = 5; P < 0.05). This proliferative activity was heat-sensitive and recovered in the soluble fraction of the eosinophil lysates. Furthermore, the molecular weight of the mitogenic activity in the soluble fraction was identified as lower than 10 kDa. The inhibitory activity to ASM cell proliferation was also found in the insoluble fraction of the lysates. CONCLUSION: These results indicate that circulating eosinophils store mitogenic activity for ASM cells, suggesting that eosinophils might contribute to the development of the hyperplasia of ASM cells in asthmatics through the release of the stored mitogenic activity upon stimulation at the site of inflammation.     

Collagen synthesis of human arterial smooth muscle cells: Effects of platelet-derived growth factor,transforming growth factor-β1 and interleukin-1

The effects of platelet-derived growth factor (PDGF), transforming growth factor-β1 (TGF-β1) and interleukin-1 (IL-1) on collagen synthesis of cultured human arterial smooth muscle cells in a confluent state were investigated. Synthetic activity of collagenous protein was determined with [³H]-proline uptake, and subsequent analysis of collagen types by sodium dodecylsulfte-polyacrylmide gel electrophoresis (SDS-PAGE) followed by fluorography. Although PDGF (0.5 U/mL and 5.0 U/mL) enhanced total collagen synthesis per dish, it suppressed total collagen synthesis per DNA (DNA content in a dish). TGF-β1 (10 pmol/L and 100 pmol/L) enhanced total collagen synthesis both per dish and per DNA. IL-1 (0.1 U/mL and 1.0 U/mL) suppressed total collagen synthesis both per dish and per DNA. A fluorogram revealed that human arterial smooth muscle cells synthesize types I, III, IV and V collagen. Densitometric analysis showed PDGF suppressed the proportion of type V collagen. TGF-β1 increased the proportions of types IV and V collagen. IL-1 elicited un-remarkable change in the proportion of collagen types. These results suggest that, in the event of human atherosclerosis, TGS-β1 is most effective in enhancing collagen synthesis, and PDGF modulates collagen metabolism by stimulating a cell division of smooth muscle cells with a resultant increase of collagenous protein, especially of type V collagen.     

哮喘气道平滑肌细胞表观遗传学调控的研究进展

哮喘气道重塑中一个主要方面即是平滑肌细胞的增生肥大,近些年,人们逐渐发现表观遗传学对气道平滑肌细胞的增殖和分泌炎性因子方面有着重要的调节作用,其中包括DNA甲基转移酶抑制剂可以抑制其表型转换;组蛋白乙酰化与其增生肥大相关;另外,microRNA可以调控哮喘模型中气道平滑肌细胞的各种生理功能,包括抑制其增殖及炎性因子的释放。希望表观遗传学能够成为治疗哮喘的新型靶点。     

Kinetics of transforming growth factor-β1 and extracellular matrix in renal tubulointerstitial lesions of mercuric chloride-treated Brown Norway rats

Renal tubulointerstitial lesions in mercuric chloride(HgCl2)-treated Brown Norway rats were investigated focusing on the kinetics of transforming growth factor-beta1(TGF-beta1) and extracellular matrix (ECM). Rats were injected with 1 mg/kg b.w. of HgCl2 at days 0, 2, and 4, and 5 rats were killed at days 2, 4, 6, 8, 10, and 20, respectively. TGF-beta1 mRNA expression in the renal cortex measured by competitive RT-PCR method reached a peak at day 6, mildly decreased at days 8 and 10, and increased again toward day 20. Signals of TGF-beta1 mRNA examined by in situ hybridization method were recognized in the regenerative tubular epithelium at day 6, and in both tubular epithelium and infiltrated mononuclear cells at day 20. After tubular injury, strong immunoreactivity to TGF-beta1 protein was found in desquamated tubular epithelial cells. Then, positive staining was found in the regenerative tubular epithelial cells. Later, infiltrated mononuclear cells also became positive for TGF-beta1 protein. In the ECM, deposition of fibronectin was prominent throughout the experimental period. In conclusion, this strongly suggests that TGF-beta1 derived from tubular epithelial cells and some macrophages might be related to the development of renal interstitial fibrosis in HgCl2-treated BN rats.     

'Yin–Yang' functions of transforming growth factor-β and T regulatory cells in immune regulation

Summary: Transforming growth factor-β (TGF-β) and forkhead box p3-expressing T-regulatory (Treg) cells are critical in maintaining self-tolerance and immune homeostasis. The immune suppressive functions of TGF-β and Treg cells are widely acknowledged and extensively studied. Nonetheless, recent studies revealed the positive roles of TGF-β and Treg cells in shaping the immune system and the inflammatory responses. This review discusses our and other's efforts in understanding the negative (Yin) as well as the positive (Yang) roles for TGF-β and Treg cells in immune regulation.     

Heterogeneous response of nasal and lung fibroblasts to transforming growth factor-β1

Background Nasal polyposis is characterized by marked oedema, sparse extracellular matrix (ECM) and proliferating blood vessels. Pulmonary fibrosis is characterized by inflammatory cells accumulation, considerable ECM deposition and vascular abnormalities. Although lung fibrosis is not only and necessarily an inflammatory disorder, we hypothesized that the difference between nasal polyposis and pulmonary fibrosis may, in part, be due to the heterogeneity between nasal and lung fibroblasts. Fibroblasts participate in the inflammatory response by releasing ECM proteins and cytokines. TGF‐β is thought to participate in chronic inflammation and fibrosis. Myofibroblasts are the activated form of fibroblasts. A phenotypic hallmark of myofibroblasts is the expression of smooth muscle α‐actin (SMA). Objective We examined whether there is any heterogeneity between nasal and lung fibroblasts upon stimulation with TGF‐β₁ with regard to the synthesis of SMA, pro‐collagen type I and vascular endothelial growth factor (VEGF) as well as translocation of Smad proteins. Methods Fibroblasts lines were established from human biopsy tissue. The expression of SMA, pro‐collagen type I, VEGF mRNA was evaluated by reverse transciptase RT‐PCR. The amount of pro‐collagen type I and VEGF was measured by ELISA. By immunocytochemistry, we analysed the expression of SMA and Smad2, 3, 4 in cultured fibroblasts. Results TGF‐β₁ induced SMA and pro‐collagen type I synthesis in lung, but not in nasal fibroblasts. By contrast, TGF‐β₁ induced VEGF synthesis in both lung and nasal fibroblasts. After stimulation with TGF‐β₁, Smad2, 3, 4 were translocated from the cytoplasm to the nucleus in lung fibroblasts, whereas only Smad3 was translocated in nasal fibroblasts. Conclusion These results establish the heterogeneous responsiveness of fibroblast populations in the airways to TGF‐β₁ and that such a heterogeneity may contribute, at least in part, to the different pathological outcomes of inflammation in the upper and lower airways.     

The pro-fibrogenic effect of nerve growth factor on conjunctival fibroblasts is mediated by transforming growth factor-β

BACKGROUND: Nerve growth factor (NGF) and nerve growth factor receptor (NGFR) expressions have been found to be increased in sub-conjunctival scarring. OBJECTIVE: The aim of this study was to investigate the in vitro effects of NGF on some pro-fibrogenic properties of human conjunctival fibroblasts. METHODS: Expression of NGF, trkA(NGFR) and p75NTR on human fibroblasts grown from conjunctival biopsies and incubated for 2 or 6 days with NGF were evaluated by immunofluorescence, RT-PCR, flow cytometry and ELISA. The fibrogenic effect of NGF on conjunctival fibroblasts was investigated by evaluating their migration (wound model), proliferation ([3H]-thymidine incorporation), collagen production (3H]-proline incorporation), expression of alpha-smooth muscle actin (alpha-SMA) (cell surface ELISA) and contraction of 3D collagen gels. RESULTS: NGF induced the expression of p75NTR in the fibroblasts that constitutively expressed only trkA(NGF) and increased the migration of wounded fibroblasts, but not their proliferation and collagen production. NGF induced the conversion of fibroblasts into myofibroblasts expressing alpha-SMA, and enhanced their contraction of a collagen matrix. Interestingly, chronic NGF treatment induced transforming growth factor-beta1 (TGF-beta1) production by fibroblasts, and following specific TGF-beta neutralization, all the NGF-induced effects were completely abrogated. CONCLUSION: Our findings indicate that NGF, via TGF-beta induction, is likely to be involved in the healing or fibrotic processes occurring in conjunctiva during some pathological conditions.     

MAPK regulation of gene expression in airway smooth muscle

Mitogen-activated protein kinases (MAPK) are important components of signaling modules activated by neurotransmitters, cytokines, and growth factors, as well as chemical and mechanical stressors. In the airway, these external signals produce acute responses that modify smooth muscle contraction and may also induce chronic responses that modify airway structure. Both acute and chronic events in airway remodeling result from altered expression of multiple genes encoding protein mediators of cell-cell signaling, extracellular matrix remodeling, cell cycle control and intracellular signaling pathways. This review will focus on inflammatory and growth factor mediators of cell-cell signaling regulated by the ERK and p38 MAPK pathways in airway smooth muscle (ASM). These signaling mediators affect ASM tissue mechanics, cell migration, and gene expression patterns in a paracrine and autocrine fashion, although the relative importance of each MAPK pathway varies with the stimulus. These events thereby contribute to normal airway function and participate in pathological changes in ASM that accompany symptoms of asthma.     

Role of licochalcone A in VEGF-induced proliferation of human airway smooth muscle cells: implications for asthma

Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodeling, which is associated with increased airway smooth muscle (ASM) mass. Licochalcone A is the predominant characteristic chalcone in licorice root. We found that licochalcone A inhibited vascular endothelial growth factor (VEGF)-induced ASM cell proliferation and induced cell cycle arrest. Additionally, VEGF-induced ASM cell proliferation was suppressed via inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) activity, but not that of Akt. Furthermore, licochalcone A treatment inhibited VEGF-induced activation of VEGF receptor 2 (VEGFR2) and ERK and blocked the downregulation of caveolin-1 in a concentration-dependent manner. Collectively, our findings suggested that licochalcone A inhibited VEGF-induced ASM cell proliferation by suppressing VEGFR2 and ERK1/2 activation and downregulating caveolin-1. Further studies of these mechanisms are needed to facilitate the development of treatments for smooth muscle hyperplasia-associated diseases of the airway, such as asthma.