The role of the bronchial microvasculature in the airway remodelling in asthma and COPD

In recent years, there has been increased interest in the vascular component of airway remodelling in chronic bronchial inflammation, such as asthma and COPD, and in its role in the progression of disease. In particular, the bronchial mucosa in asthmatics is more vascularised, showing a higher number and dimension of vessels and vascular area. Recently, insight has been obtained regarding the pivotal role of vascular endothelial growth factor (VEGF) in promoting vascular remodelling and angiogenesis. Many studies, conducted on biopsies, induced sputum or BAL, have shown the involvement of VEGF and its receptors in the vascular remodelling processes. Presumably, the vascular component of airway remodelling is a complex multi-step phenomenon involving several mediators. Among the common asthma and COPD medications, only inhaled corticosteroids have demonstrated a real ability to reverse all aspects of vascular remodelling. The aim of this review was to analyze the morphological aspects of the vascular component of airway remodelling and the possible mechanisms involved in asthma and COPD. We also focused on the functional and therapeutic implications of the bronchial microvascular changes in asthma and COPD.     

Impaired TNFα-induced VEGF Expression in Human Airway Smooth Muscle Cells from Smokers with COPD: Role of MAPkinases and Histone Acetylation—Effect of Dexamethasone

The cytokine and potent angiogenic factor vascular endothelial growth factor (VEGF) plays an important role in airway remodelling in various airway diseases such as idiopathic pulmonary fibrosis, pulmonary hypertension, lung cancer, asthma and chronic obstructive pulmonary disease (COPD). The effect of cigarette-smoking on VEGF expression, the modulatory role of extracellular signal-regulated kinase (ERK)-1,-2, p38mitogen-activated protein kinase (MAPK), histone acetylation and the anti-inflammatory effect of dexamethasone on TNFα-induced VEGF expression were examined in human airway smooth muscle cells (HASMC) of five non-smokers, 17 smokers without airflow limitation and 15 smokers with COPD. TNFα increased VEGF expression 5.4-fold and 4.0-fold in HASMC from non-smokers and smokers without airflow limitation, respectively, but only 2.5-fold in HASMC from smokers with COPD compared with non-stimulated HASMC. VEGF production was dependent on phosphorylation of ERK-1,-2 and p38^MAPK, as was shown by examining the effects of PD 098059 (10 μM), an inhibitor of the upstream activator of MAPKkinase (MKK)-1, and SB 203580 (10 μM), an inhibitor of p38^MAPK; there were no differences between non-smokers, smokers without airflow limitation and smokers with COPD in this respect. Dexamethasone (DEX; 10⁻¹²–10⁻⁴ M) reduced TNFα-induced phosphorylation of ERK-1/-2 and prevented TNFα-induced VEGF generation without differences between non-smokers, smokers with and without COPD. There was an additional inhibitory effect of DEX (10⁻¹² M) on VEGF-release when PD 098059 was added. The basal and TNFα-induced acetylation status of the VEGF-promoter (chromatin immunoprecipitation [ChIP] assay) was increased in HASMC from smokers with COPD compared with smokers without airflow limitation and non-smokers. In comparison to non-stimulated HASMC, TNFα decreased the acetylation status of the VEGF-promoter by ∼46% and ∼43% in HASMC from non-smokers and smokers without COPD compared with ∼68% in HASMC from smokers with COPD. The data suggest that HASMC express VEGF in response to TNFα and that this may be reduced in HASMC of smokers with COPD in a smoking-independent manner. VEGF expression is directly modulated by phosphorylation of ERK-1,-2 and p38^MAPK and by histone acetylation and the acetylation status of the VEGF gene is increased in HASMC of smokers with COPD in a smoking-independent manner. TNFα reduced the acetylation status of the VEGF promoter in HASMC.     

Vasoactive peptides upregulate mRNA expression and secretion of vascular endothelial growth factor in human airway smooth muscle cells

Airway remodeling and associated angiogenesis are documented features of asthma, of which the molecular mechanisms are not fully understood. Angiotensin (ANG)II and endothelin (ET)-1 are potent vasoconstricting circulatory hormones implicated in asthma. We investigated the effects of ANG II and ET-1 on human airway smooth muscle (ASM) cells proliferation and growth and examined the mRNA expression and release of the angiogenic peptide, vascular endothelial growth factor (VEGF). Serum deprived (48 h) human ASM cells were incubated with ANG II (100 nM) or ET-1 (10nM) for 30 min, 1, 2, 4, 8, 16, and 24 h and the endogenous synthesis of VEGF was examined in relation to control cells receiving serum free culture medium. ET-1 induced time dependent DNA biosynthesis as determined by [³H]-thymidine incorporation assay. Using northern blot hybridization, we detected two mRNA species of 3.9 and 1.7 kb encoding VEGF in the cultured smooth muscle cells. Both ANG II and ET-1 induced the mRNA expression (two-to threefold) and secretion (1.8-to 2.8-fold) of VEGF reaching maximal levels between 4–8 h of incubation. Induced expression and release of VEGF declined after 8 h of ANG II incubation while levels remained elevated in the case of ET-1. The conditioned medium derived from ET-1-treated ASM cells induced [³H]-thymidine incorporation and cell number in porcine pulmonary artery endothelial as well as human umbilical vein endothelial cells. Moreover, the VEGF tyrosine kinase receptor inhibitor blocked the conditioned medium induced mitogenesis in endothelial cells. Our results suggest a potential role for ANG II and ET-1 in ASM cell growth and upregulation of VEGF that may participate in endothelial cell proliferation via paracrine mechanisms and thus causing pathological angiogenesis and vascular remodelling seen during asthma.     

Angiogenesis and Vascular Remodeling in Chronic Airway Diseases

Asthma and chronic obstructive pulmonary disease remain a global health problem, with increasing morbidity and mortality. Despite differences in the causal agents, both diseases exhibit various degrees of inflammatory changes, structural alterations of the airways leading to airflow limitation. The existence of transient disease phenotypes which overlap both diseases and which progressively decline the lung function has complicated the search for an effective therapy. Important characteristics of chronic airway diseases include airway and vascular remodeling, of which the molecular mechanisms are complex and poorly understood. Recently, we and others have shown that airway smooth muscle (ASM) cells are not only structural and contractile components of airways, rather they bear capabilities of producing large number of pro-inflammatory and mitogenic factors. Increase in size and number of blood vessels both inside and outside the smooth muscle layer as well as hyperemia of bronchial vasculature are contributing factors in airway wall remodeling in patients with chronic airway diseases, proposing for the ongoing mechanisms like angiogenesis and vascular dilatation. We believe that vascular changes directly add to the airway narrowing and hyper-responsiveness by exudation and transudation of proinflammatory mediators, cytokines and growth factors; facilitating trafficking of inflammatory cells; causing oedema of the airway wall and promoting ASM accumulation. One of the key regulators of angiogenesis, vascular endothelial growth factor in concerted action with other endothelial mitogens play pivotal role in regulating bronchial angiogenesis. In this review article we address recent advances in pulmonary angiogenesis and remodelling that contribute in the pathogenesis of chronic airway diseases.     

Upregulation of a disintegrin and metalloproteinase-33 by VEGF in 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. Features of airway remodeling include increased airway smooth muscle (ASM) mass. A disintegrin and metalloproteinase (ADAM)–33 has been identified as playing a role in the pathophysiology of asthma. ADAM-33 is expressed in ASM cells and is suggested to play a role in the function of these cells. However, the regulation of ADAM-33 is not fully understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodeling in asthmatics. Although VEGF was initially thought of as an endothelial-specific growth factor, recent reports have found that VEGF can promote proliferation of other cell types, including ASM cells. To investigate the precise mechanism of VEGF's effect on ASM cell proliferation, we tested the expression of ADAM-33, phospho-extracellularsignal-regulated kinase 1/2 (ERK1/2), and phospho-Akt in VEGF-stimulated ASM cells. We found that VEGF up-regulates ADAM-33 mRNA and protein levels in a dose- and time-dependent manner as well as phosphorylation of ERK1/2 and Akt. We also found that VEGF-induced ASM cell proliferation is inhibited by both ADAM-33 knockdown and a selective VEGF receptor 2 (VEGFR2) inhibitor (SU1498). Furthermore, VEGF-induced ADAM-33 expression and ASM cell proliferation were suppressed by inhibiting ERK1/2 activity, but not by inhibiting Akt activity. Collectively, our findings suggest that VEGF enhances ADAM-33 expression and ASM cell proliferation by activating the VEGFR2/ERK1/2 signaling pathway, which might be involved in the pathogenesis of airway remodeling. Further elucidation of the mechanisms underlying these observations might help develop therapeutic strategies for airway diseases associated with smooth muscle hyperplasia such as asthma.     

Abnormal histone methylation is responsible for increased vascular endothelial growth factor 165a secretion from airway smooth muscle cells in asthma

Vascular endothelial growth factor (VEGF), a key angiogenic molecule, is aberrantly expressed in several diseases including asthma where it contributes to bronchial vascular remodeling and chronic inflammation. Asthmatic human airway smooth muscle cells hypersecrete VEGF, but the mechanism is unclear. In this study, we defined the mechanism in human airway smooth muscle cells from nonasthmatic and asthmatic patients. We found that asthmatic cells lacked a repression complex at the VEGF promoter, which was present in nonasthmatic cells. Recruitment of G9A, trimethylation of histone H3 at lysine 9 (H3K9me3), and a resultant decrease in RNA polymerase II at the VEGF promoter was critical to repression of VEGF secretion in nonasthmatic cells. At the asthmatic promoter, H3K9me3 was absent because of failed recruitment of G9a; RNA polymerase II binding, in association with TATA-binding protein-associated factor 1, was increased; H3K4me3 was present; and Sp1 binding was exaggerated and sustained. In contrast, DNA methylation and histone acetylation were similar in asthmatic and nonasthmatic cells. This is the first study, to our knowledge, to show that airway cells in asthma have altered epigenetic regulation of remodeling gene(s). Histone methylation at genes such as VEGF may be an important new therapeutic target.     

VEGF induces proliferation, migration, and TGF-beta1 expression in mouse glomerular endothelial cells via mitogen-activated protein kinase and phosphatidylinositol 3-kinase

The role of glomerular endothelial cells in kidney fibrosis remains incompletely understood. While endothelia are indispensable for repair of acute damage, they can produce extracellular matrix proteins and profibrogenic cytokines that promote fibrogenesis. We used a murine cell line with all features of glomerular endothelial cells (glEND.2), which dissected the effects of vascular endothelial growth factor (VEGF) on cell migration, proliferation, and profibrogenic cytokine production. VEGF dose-dependently induced glEND.2 cell migration and proliferation, accompanied by up-regulation of VEGFR-2 phosphorylation and mRNA expression. VEGF induced a profibrogenic gene expression profile, including up-regulation of TGF-beta1 mRNA, enhanced TGF-beta1 secretion, and bioactivity. VEGF-induced endothelial cell migration and TGF-beta1 induction were mediated by the phosphatidyl-inositol-3 kinase pathway, while proliferation was dependent on the Erk1/2 MAP kinase pathway. This suggests that differential modulation of glomerular angiogenesis by selective inhibition of the two identified VEGF-induced signaling pathways could be a therapeutic approach to treat kidney fibrosis.     

Epithelium-fibroblast interactions in response to airway inflammation

Dramatic changes to the architecture of the airway walls have been commonly described in the airways of patients with asthma, cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). Much research has focused on how airway inflammation drives these structural changes, particularly in terms of the mechanisms/mediators that are involved, and a number of parallels are observed between the disease phenotypes. For example, the increased deposition of extracellular matrix (ECM) at focal sites in the airway wall is seen in asthma and all interstitial lung diseases that involve fibrosis. In addition, increased expression of a number of well characterized cytokines and growth factors, such as TGF-beta and epidermal growth factor (EGF) have been demonstrated in these diseases. However, the role of the lesser-known cytokines, including the leukaemia inhibitory factor (LIF) and other members of the IL-6 family of cytokines in the pathogenesis of airway remodelling and fibrosis is largely unknown. However, the use of genetic manipulation in vivo and more specific inhibitors/antibodies in vitro has now provided increasing evidence to support the hypothesis that a complex interaction exists between these cytokines, ECM and integrins in regulating the function of both epithelial cells and fibroblasts.     

Immunomodulatory role of vascular endothelial growth factor and angiopoietin-1 in airway remodeling

The blood vessels formed in asthmatic airways are involved in inflammatory and airway remodeling processes in chronic asthma. Vascular endothelial cell growth factor (VEGF) and angiopoietin-1 (Ang-1) are primary angiogenic growth factors, involved in the formation of such blood vessels. VEGF has been reported to contribute to non-specific airway hyper-responsiveness, have chemotactic effects on eosinophils, and enhance airway smooth muscle cell proliferation. Furthermore, Th2 cells have receptors for VEGF, and Th2-associated cytokines increase VEGF production. There are reports that elevated levels of VEGF correlates with the severity of asthma. Ang-1 has been shown to induce pro-inflammatory effects such as eosinophil chemotaxis via tie-2 receptors. Reports indicate ang-1 contribution to increased secretion of matrix metalloproteinase-2 (MMP-2) and decreased secretion of tissue inhibitors of metalloproteinase-2 (TIMP-2). However, Ang-1 has also been shown to exhibit several anti-inflammatory properties such as suppressing expression of adhesion molecules, blocking vascular permeability and eosinophil chemotaxis induced by VEGF. These findings support the notion that apart from their roles in blood vessels formation, these angiogenic growth factors are directly involved in the pathogenesis of chronic asthma. This paper reviews individual and combined roles of VEGF and Ang-1. The potential therapeutic applications involving these factors are also discussed.     

Transforming growth factor-beta1 stimulates vascular endothelial growth factor 164 via mitogen-activated protein kinase kinase 3-p38alpha and p38delta mitogen-activated protein kinase-dependent pathway in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix synthesis leading to progressive glomerular fibrosis. The intracellular signaling mechanisms involved in this process remain incompletely understood. The p38 mitogen-activated protein kinase (MAPK) is a major stress signal transducing pathway that is rapidly activated by TGF-beta1 in mesangial cells. We have previously demonstrated MKK3 as the immediate upstream MAPK kinase required for selective activation of p38 MAPK isoforms, p38alpha and p38delta, and stimulation of pro-alpha1(I) collagen by TGF-beta1 in murine mesangial cells. In this study, we further sought to determine MAPK kinase 3 (MKK3)-dependent TGF-beta1 responses by gene expression profiling analysis utilizing mesangial cells isolated from Mkk3-/- mice compared with Mkk3+/+ controls. Interestingly, vascular endothelial growth factor (VEGF) was identified as a TGF-beta1-induced gene affected by deletion of Mkk3. VEGF is a well known endothelial mitogen, whose actions in nonendothelial cell types are still not well understood. We confirmed that TGF-beta1 increased VEGF mRNA and protein synthesis of VEGF164 and VEGF188 isoforms in wild-type mesangial cells. However, in the Mkk3-/- mesangial cells, both TGF-beta1-induced VEGF mRNA and VEGF164 protein expression were inhibited, whereas TGF-beta1-induced VEGF188 protein expression was unaffected. Furthermore, transfection of dominant negative mutants of p38alpha and p38delta resulted in marked inhibition of TGF-beta1-induced VEGF164 expression but not VEGF188, and treatment with recombinant mouse VEGF164 increased collagen and fibronectin mRNA expression in mesangial cells. Taken together, our findings suggest a critical role for the MKK3-p38alpha and p38delta MAPK pathway in mediating VEGF164 isoform-specific stimulation by TGF-beta1 in mesangial cells. Further, VEGF164 stimulates collagen and fibronectin expression in mesangial cells and thus in turn enhances TGF-beta1-induced extracellular matrix and may play an important role in progressive glomerular fibrosis.     

Smad3 mediates TGF-beta1 induction of VEGF production in lung fibroblasts

Transforming growth factor-beta1 (TGF-beta1) is a key factor in a variety of physiological and pathological processes. Vascular endothelial growth factor (VEGF) is a key angiogenic factor, and vascular change is one of the features of airway remodeling. We examined the effect of TGF-beta1 on VEGF production by fibroblasts from mice lacking expression of Smad2 or Smad3 as well as human lung fibroblasts treated with or without Smad2 or Smad3 siRNA. TGF-beta1 stimulated VEGF production by fibroblasts from Smad2 deficient animals and wildtype animals. In contrast, TGF-beta1 did not affect VEGF production by fibroblasts from Samd3 deficient mice. Similarly, TGF-beta1 failed to stimulate VEGF production by HFL-1 cells treated with Samd3 siRNA but significantly increased VEGF production by the cells treated with Smad2 siRNA. These result suggest that TGF-beta1 stimulation of VEGF production by fibroblasts is regulated by Smad3 but not by Smad2 signaling.     

Vascular endothelial growth factor-induced secretion of fibronectin is ERK dependent

In severe asthma, cytokines and growth factors contribute to the proliferation of smooth muscle cells and blood vessels, and to the increased extracellular matrix deposition that constitutes the process of airway remodeling. Vascular endothelial growth factor (VEGF), which regulates vascular permeability and angiogenesis, also modulates the function of nonendothelial cell types. In this study, we demonstrate that VEGF induces fibronectin secretion by human airway smooth muscle (ASM) cells. In addition, stimulation of ASM with VEGF activates ERK, but not p38MAPK, and fibronectin secretion is ERK dependent. Both ERK activation and fibronectin secretion appear to be mediated through the VEGF receptor flt-1, as evidenced by the effects of the flt-1-specific ligand placenta growth factor. Finally, we demonstrate that ASM cells constitutively secrete VEGF, which is increased in response to PDGF, transforming growth factor-beta, IL-1beta, and PGE(2). We conclude that ASM-derived VEGF, through modulation of the extracellular matrix, may play an important role in airway remodeling seen in asthma.     

Role of extracellular matrix and its regulators in human airway smooth muscle biology

Altered extracellular matrix (ECM) deposition contributing to airway wall remodeling is an important feature of asthma and chronic obstructive pulmonary disease (COPD). The molecular mechanisms of this process are poorly understood. One of the key pathological features of these diseases is thickening of airway walls. This thickening is largely to the result of airway smooth muscle (ASM) cell hyperplasia and hypertrophy as well as increased deposition of ECM proteins such as collagens, elastin, laminin, and proteoglycans around the smooth muscle. Many growth factors and cytokines, including fibroblast growth factor (FGF)-1, FGF-2, and transforming growth factor (TGF)-α₁, that are released from the airway wall have the potential to contribute to airway remodeling, revealed by enhanced ASM proliferation and increased ECM protein deposition. TGF-α₁ and FGF-1 stimulate mRNA expression of collagen I and III in ASM cells, suggesting their role in the deposition of extracellular matrix proteins by ASM cells in the airways of patients with chronic lung diseases. Focus is now on the bidirectional relationship between ASM cells and the ECM. In addition to increased synthesis of ECM proteins, ASM cells can be involved in downregulation of matrix metalloproteinases (MMPs) and upregulation of tissue inhibitors of metalloproteinases (TIMPs), thus eventually contributing to the alteration in ECM. In turn, ECM proteins promote the survival, proliferation, cytokine synthesis, migration, and contraction of human airway smooth muscle cells. Thus, the intertwined relationship of ASM and ECM and their response to stimuli such as chronic inflammation in diseases such as asthma and COPD contribute to the remodeling seen in airways of patients with these diseases.     

Interaction of tgf-beta with immune cells in airway disease

Asthma, chronic obstructive pulmonary disorder (COPD), and cystic fibrosis (CF), chronic diseases of the airways, are characterized by symptoms such as inflammation of the lung tissue, mucus hypersecretion, constriction of the airways, and excessive fibrosis of airway tissue. Transforming growth factor (TGF)-beta, a cytokine that affects many different cell processes, has an important role in the lungs of patients with some of these chronic airway diseases, especially with respect to airway remodeling. Eosinophils can be activated by and are a major source of TGF-beta in asthma. The action of TGF-beta also shows associations with other cell types, such as T cells and neutrophils, which are involved in the pathogenesis of asthma. TGF-beta can perpetuate the pathogenesis of COPD and CF, as well, through its induction of inflammation via release from and action on different cells. The intracellular signaling induced by TGF-beta in various cell types has been elucidated and may point to mechanisms of action by TGF-beta on different structural or immune cells in these airway diseases. Some possible treatments, especially that prevent the deleterious airway changes induced by the action of either eosinophils or TGF-beta in asthma, have been investigated. TGF-beta-induced signaling pathways, especially those in different cell types in asthma, COPD, or CF, may provide potential therapeutic targets for the treatment of some of the most devastating airway diseases.     

支气管哮喘小鼠模型气道血管变化及其影响因子分析

目的明确支气管哮喘时血管网络及血管内皮细胞变化,以及血管内皮生长因子（VEGF）亚型及其受体在支气管哮喘小鼠模型气管及肺组织中的变化及作用。方法在建立小鼠支气管哮喘模型的基础上,应用免疫荧光染色和HE染色观察气道旁血管密度变化并计数不同血管单位长度上内皮细胞数量,应用逆转录多聚酶链反应（RT-PCR）方法检测气管和肺组织血管内皮生长因子（VEGF）亚型及其受体mRNA表达情况。结果1.随着疾病时间的延长,小鼠气道壁血管密度增加,血管内皮细胞数量逐渐增加,特别在慢性期小血管尤为明显;2.应用RT-PCR技术及琼脂糖凝胶电泳,小鼠气管及肺组织VEGF120,VEGF164,VEGF188和VEGF205各个亚型mRNA均增高。其中VEGF164在气管组织,VEGF188在周围肺组织中mRNA表达在慢性哮喘期明显增高,少见的亚型VEGF144只在周围肺组织中检测到,而在支气管组织中无表达;VEGFR1 mRNA水平没有明显变化,而VEGFR2在正常气管组织中未检测到,而在支气管哮喘气管组织明显表达,并且在支气管哮喘慢性期高于急性期。结论随着疾病的进展,小鼠支气管哮喘时气道存在血管密度及血管内皮细胞数量增加,VEGF在此过程中起着重要的作用,各亚型的作用存在差异。另外,VEGFR2在支气管哮喘中对于介导和增强VEGF信号和VEGF活性起着重要的作用,并且可能是导致VEGF诱导小鼠气道血管再生与重塑的重要介质。