key: cord-0000004-2b73a28n
authors: Fagan, Karen A; McMurtry, Ivan F; Rodman, David M
title: Role of endothelin-1 in lung disease
date: 2001-02-22
journal: Respir Res
DOI: 10.1186/rr44
sha: 348055649b6b8cf2b9a376498df9bf41f7123605
doc_id: 4
cord_uid: 2b73a28n

Endothelin-1 (ET-1) is a 21 amino acid peptide with diverse biological activity that has been implicated in numerous diseases. ET-1 is a potent mitogen regulator of smooth muscle tone, and inflammatory mediator that may play a key role in diseases of the airways, pulmonary circulation, and inflammatory lung diseases, both acute and chronic. This review will focus on the biology of ET-1 and its role in lung disease.

from Xenopus laevis [16] . ETA receptors in normal lung are found in greatest abundance on vascular and airway smooth muscle, whereas ETB receptors are most often found on the endothelium. Clearance of ET-1 from the circulation is mediated by the ETB receptor primarily in the lung, but also in the kidney and liver [17] .

Activation of both ETA and ETB receptors on smooth muscle cells leads to vasoconstriction whereas ETB receptor activation leads to bronchoconstriction. Activation of ETB receptors located on endothelial cells leads to vasodilation by increasing nitric oxide (NO) production. The mitogenic and inflammatory modulator functions of ET-1 are primarily mediated by ETA receptor activity. Binding of the ligand to its receptor results in coupling of cell-specific G proteins that activate or inhibit adenylate cyclase, stimulate phosphatidyl-inositol-specific phosholipase, open voltage gated calcium and potassium channels, and so on. The varied effects of ET-1 receptor activation thus depend on the G protein and signal transduction pathways active in the cell of interest [18] . A growing number of receptor antagonists exist with variable selectivity for one or both receptor subtypes.

Regulation of ET-1 is at the level of transcription, with stimuli including shear stress, hypoxia, cytokines (IL-2, IL-1β, tumor necrosis factor α, IFN-β, etc), lipopolysaccharides, and many growth factors (transforming growth factor-β, platelet-derived growth factor, epidermal growth factor, etc) inducing transcription of ET-1 mRNA and secretion of protein [18] . ET-1 acting in an autocrine fashion may also increase ET-1 expression [19] . ET-1 expression is decreased by NO [20] . Some stimuli may additionally enhance preproET-1 mRNA stability, leading to increased and sustained ET-1 expression. The number of ETA and ETB receptors is also cell specific and regulated by a variety of growth factors [18] . Because ET-1 and receptor expression is influenced by many diverse physical and biochemical mechanisms, the role of ET-1 in pathologic states has been difficult to define, and these are addressed in subsequent parts of this article.

In the airway, ET-1 is localized primarily to the bronchial smooth muscle with low expression in the epithelium. Cellular subsets of the epithelium that secrete ET-1 include mucous cells, serous cells, and Clara cells [21] . ET binding sites are found on bronchial smooth muscle, alveolar septae, endothelial cells, and parasympathetic ganglia [22, 23] . ET-1 expression in the airways, as previously noted, is regulated by inflammatory mediators. Eosinophilic airway inflammation, as may be seen in severe asthma, is associated with increased ET-1 levels in the lung [24] . ET-1 secretion may also act in an autocrine or paracrine fashion, via the ETA receptor, leading to increased transepithelial potential difference and ciliary beat frequency, and to exerting mitogenic effects on airway epithelium and smooth muscle cells [25] [26] [27] [28] .

All three endothelins cause bronchoconstriction in intact airways, with ET-1 being the most potent. Denuded bronchi constrict equally to all three endothelins, suggesting considerable modulation of ET-1 effects by the epithelium [29] . The vast majority of ET-1 binding sites on bronchial smooth muscle are ETB receptors, and bronchoconstriction in human bronchi is not inhibited by ETA antagonists but augmented by ETB receptor agonists [30] [31] [32] . Since cultured airway epithelium secretes equal amounts of ET-1 and ET-3, which have equivalent affinity for the ETB receptor, bronchoconstriction could be mediated by both endothelins [33] .

While ET-1 stimulates release of multiple cytokines important in airway inflammation, it does not enhance secretion of histamine or leukotrienes. ET-1 does increase prostaglandin release [32] . Inhibition of cyclo-oxygenase, however, has no effect on bronchoconstriction suggesting that, despite the release of multiple mediators, ET-1 mediated bronchoconstriction is a direct effect of activation of the ETB receptor [32] . ETA mediated bronchoconstriction may also be important following ETB receptor desensitization or denudation of the airway epithelium, as may occur during airway inflammation and during the late, sustained airway response to inhaled antigens [31, 34, 35] . Interestingly, heterozygous ET-1 knockout mice, with a 50% reduction in ET-1 peptide, have airway hyperresponsiveness but not remodeling, suggesting the decrease in ET-1 modulates bronchoconstriction activity by a functional mechanism, possibly by decreasing basal NO production [36, 37] .

Asthma is also an inflammatory airway disease characterized by bronchoconstriction and hyperreactivity with influx of inflammatory cells, mucus production, edema, and airway thickening. ET-1 may have important roles in each of these processes. While ET-1 causes immediate bronchoconstriction [38] , it also increases bronchial reactivity to inhaled antigens [35] as well as influx of inflammatory cells [39, 40] , increased cytokine production [40] , airway edema [41] , and airway remodeling [28, 42, 43] . Airway inflammation also leads to increased ET-1 synthesis, possibly perpetuating the inflammation and bronchoconstriction [44] . ET-1 release from cultured peripheral mononuclear and bronchial epithelial cells from asthmatics is also increased [45, 46] . Inhibition of ETA or combined ETA and ETB receptors additionally leads to decreased airway inflammation in antigen-challenged animals, suggesting that the proinflammatory effects of ET-1 in the airway are mediated by ETA receptors [39, 47] .

Children with asthma have increased circulating levels of ET-1 [48] . Adult asthmatics have normal levels between attacks but, during acute attacks, have elevated serum ET-1 levels that correlate inversely with airflow measurements and decrease with treatment [49] . Bronchoalveolar lavage (BAL) ET-1 in asthmatics is similarly increased to concentrations that cause bronchoconstriction and inversely correlates with forced expiratory volume in 1 s (FEV 1 ) [29, 50, 51] . As in cultured epithelial cells, ET-1 and ET-3 are found in equal amounts in BAL fluid from asthmatics [33, 52] . There is also a relative increase in ETB versus ETA receptor expression in asthmatic patients, which may contribute to increased bronchoconstriction [53] . Not all asthmatics, however, have increased ET-1 as patients with nocturnal asthma have decreased BAL ET-1 levels [54] . Treatment of acute asthma exacerbations with steroids, beta-adrenergic agonists or phosphodiesterase inhibitors resulted in decreased BAL ET-1 [52, 55] . Immunostaining and in situ hybridization for ET-1 in biopsy specimens from asthmatics have shown an increase in ET-1 in the bronchial epithelium that correlates with asthma symptoms [46, 56] .

Cigarette smoking leads to increased circulating ET-1 [57] but patients with chronic obstructive pulmonary disease, in the absence of pulmonary hypertension and hypoxemia, do not have increased plasma ET-1 [58] [59] [60] . Increases in urinary ET-1 instead correlate with decreases in oxygenation, possibly through hypoxic release of ET-1 from the kidney [61, 62] . Smokers also have impaired ET-1 mediated vasodilation that correlates with bronchial hyperresponsiveness and may contribute to pulmonary hypertension [63, 64] .

ET-1 has been implicated in the pathogenesis of bronchiectasis by its ability to promote neutrophil chemotaxis, adherence, and activation [65] [66] [67] [68] [69] . Sputum ET-1 levels are increased in patients with cystic fibrosis [59] , and sputum ET-1 correlated with Pseduomonas infection in noncystic fibrosis related bronchiectasis [70] .

ET-1 has also been implicated in the pathogenesis of bronchiolitis obliterans (BO), which is characterized by injury to small conducting airways resulting in formation of proliferative, collagen rich tissue obliterating airway architecture. BO is the leading cause of late mortality from lung transplantation, and ET-1 is increased in lung allografts [71] . The pro-inflammatory and mitogenic properties of ET-1 in the airways has led to speculation that ET-1 may be involved in formation of the lesion [28] . This is further supported by the increase in BAL ET-1 in lung allografts [72, 73] . The in vivo gene transfer of ET-1 to the airway epithelium using the hemagglutinating virus of Japan in rats recently resulted in pathologic changes in the distal airways identical to those seen in human BO specimens [74] . These changes were not due to nonspecific effects of the hemagglutinating virus of Japan itself, but could be attributed to the presence of the ET-1 gene, which was localized to the airway epithelium, hyperplastic lesions, and alveolar cells.

Pulmonary hypertension is a rare and progressive disease characterized by increases in normally low pulmonary vascular tone, pulmonary vascular remodeling, and progressive right heart failure. ET-1 has been implicated as a mediator in the changes seen in pulmonary hypertension. In the pulmonary vasculature, ET-1 is found primarily in endothelial cells and to a lesser extent in the vascular smooth muscle cells. The endothelium secretes ET-1 primarily to the basolateral surface of the cell. ET-1 secretion may be increased by a variety of stimuli including cytokines, catecholamines, and physical forces such as shear stress, and decreased by NO, prostaglandins, and oxidant stress [20, [75] [76] [77] [78] . Hypoxia has been reported to increase, have no effect, or decrease ET-1 release from endothelial cells [79] [80] [81] [82] [83] .

Activation of the receptors for ET-1 in the pulmonary vasculature leads to both vasodilation and vasoconstriction, and depends on both cell type and receptor. In the whole lung, ETA receptors are the most abundant and are localized to the medial layer of the arteries, decreasing in intensity in the peripheral circulation [84, 85] . ETB receptors are also found in the media of the pulmonary vessels, increasing in intensity in the distal circulation, while intimal ETB receptors are localized in the larger elastic arteries [85] . This distribution of receptors has important implications in understanding ET-1 regulation of vascular tone. Vascular ET-1 receptors may be increased by several factors including angiotensin and hypoxia [80, [85] [86] [87] .

ET-1 can act as both a vasodilator and vasoconstrictor in the pulmonary circulation. Generation of NO or opening of ATP-sensitive potassium channels leading to hyperpolarization results in vasodilation mediated by ETB receptors on pulmonary endothelium [88, 89] . In hypertensive, chronically hypoxic lungs with increased ETB receptor expression, augmented vasodilation is due to increased ETB mediated NO release that is inhibited by hypoxic ventilation, while inhibition of NO synthesis leads to increased ET-1 mediated vasoconstriction [85, [90] [91] [92] . Both ETA and ETB receptors, conversely, acting on vascular smooth muscle, mediate ET-1 induced vasoconstriction. In the normal lung, ET-1 causes vasoconstriction primarily by activation of the ETA receptors in the large, conducting vessels of the lung [93, 94] . In the smaller, resistance vessels of the lung, ETB receptors in the media predominate and are responsible for the ET-1 induced vasoconstriction [93] . Interestingly, preconstriction of the pulmonary circulation resulted in a shift from primarily ETA mediated to ETB mediated vasoconstriction [94] .

The overall effect of ET-1 on vascular tone depends on both the dose and on the pre-existing tone in the lung. ET-1 administration during acute hypoxic vasoconstriction will result in transient pulmonary vasodilation [89] . This effect is dose dependent, with lower doses leading to vasodilation while higher or repetitive doses cause vasoconstriction following an initial, brief vasodilation [89] . The role of ET-1 in the acute hypoxic vasoconstriction in the lung is not certain. ETA receptor antagonism attenuates hypoxic pulmonary vasoconstriction in several species [95] , and ET-1 may be implicated in the mechanism of acute hypoxic response by inhibition of K-ATP channels [96] .

Several lines of evidence have suggested the importance of ET-1 in chronic hypoxic pulmonary hypertension. ET-1 is increased in plasma and lungs of rats following exposure to hypoxia [80, 97] . Treatment with either ETA or combined ETA and ETB receptor antagonists additionally attenuates the development of hypoxic pulmonary hypertension [98, 99] . ET-1 has also been implicated in the vascular remodeling associated with chronic hypoxia through its mitogenic effects on vascular smooth muscle cells [98, 100] .

ET-1 has also been implicated in other animal models of pulmonary hypertension. ET-1 is increased in fawn hooded rats that develop severe pulmonary hypertension when raised under conditions of mild hypoxia and in monocrotaline treated rats [101, 102] . The increase in ET-1 in both of these forms of pulmonary hypertension may be contributing to increases in vascular tone as well as in vascular remodeling [103] [104] [105] [106] 114] . Interestingly, transgenic mice overexpressing the human preproET-1 gene, with modestly increased lung ET-1 levels (35-50%), do not develop pulmonary hypertension under normoxic conditions or an exaggerated response to chronic hypoxia [107] .

Human pulmonary hypertension is classified as primary, or unexplained, or secondary to other cardiopulmonary diseases or connective tissue diseases (ie scleroderma). Hallmarks of the disease include progressive increases in pulmonary vascular resistance and pulmonary vascular remodeling, with thickening of the medial layer small pulmonary arterioles and formation of the complex plexiform lesion [108] . Circulating ET-1 is increased in humans with pulmonary hypertension, either primary or due to other cardiopulmonary disease [109] . Levels are highest in patients with primary pulmonary hypertension. Since the lung is the major source for clearance of ET-1 from the circulation, increased arterio-venous ratios as seen in primary pulmonary hypertension suggest either decreased clearance or increased production in the lung [17, 109] . ET-1 is also increased in lungs of patients with pulmonary hypertension, with the greatest increase seen in the small resistance arteries and the plexiform lesions [110] , and may correlate with pulmonary vascular resistance [111] . Interestingly, treatment with continuous infusion of prostacyclin resulted in clinical improvement and a decrease in the arterio-venous ratio of ET-1 [112] , possibly by decreasing ET-1 synthesis from endothelial cells [76] . Studies using ET-1 receptor antagonists in the treatment of primary pulmonary hypertension are underway and may offer hope to patients with this disease by inhibiting this pluripotent peptide's effects on vascular tone and remodeling.

Several lines of evidence suggest the importance of ET-1 in lung allograft survival and rejection. The peptide has been implicated as an important factor in ischemia-reperfusion injury at the time of transplant as well as in acute and chronic rejection of the allograft.

Circulating ET-1 is increased in humans undergoing lung transplant immediately following perfusion of the allograft. Plasma ET-1 increased threefold within minutes, remained high for 12 hours following transplantation, and declined to near normal levels within 24 hours [113] . This increase in ET-1 correlated with the increase in pulmonary vascular resistance occurring about 6 hours post-transplantation, suggesting that the release of ET-1 in the circulation may have mediated this event. ET-1 in BAL fluid from recipients of lung allografts is similarly increased several fold and remains elevated up to 2 years post-transplant [72, 73] . In recipients of single lung transplants, ET-1 was increased 10-fold in BAL fluid from the transplanted lung compared with the native lung, suggesting that the increase in ET-1 was due to the graft and not the underlying disease requiring transplant [72] . ET-1 in BAL fluid did not, however, correlate with episodes of infection or rejection.

The cellular source of ET-1 in lung allografts is unknown. The expression of ET-1 in nontransplanted human lungs is low and found primarily in the vascular endothelium [114] . Transbronchial biopsy specimens obtained either for surveillance or for clinical suspicion of infection or rejection following transplantation revealed the presence of ET-1 in the airway epithelium and in alveolar macrophages [115] . ET-1 was occasionally seen in lymphocytes but not in the endothelium or pneumocytes. ET-1 localization was no different in surveillance specimens compared with infected or rejecting lungs, or changed over time from transplantation. This study suggests that the source of the increased BAL ET-1 in transplanted lungs is due to the increased number of alveolar inflammatory cells and de novo expression in the airway epithelium. The biologic importance of the ET-1 from inflammatory cells is supported by the observation that peripheral mononuclear cells from dogs with mild to moderate lung allograft rejection cause vasoconstriction in pulmonary arterial rings, which is attenuated by the ETA blocker BQ123 [116] .

Analysis of ET-1 binding activity in failed transplanted human lungs suggested that ET-1 binding activity was not different compared with normal lung in the lung parenchyma, bronchial smooth muscle, or perivascular infiltrates. ET-1 binding was, however, decreased in small muscular arteries (pulmonary arteries and bronchial arteries) in the failed transplants, suggesting a role for ET-1 in impaired vasoregulation of transplanted lungs [117] .

Ischemia-reperfusion injury is the leading cause of early post-operative graft failure and death. In its severest manifestation, increased pulmonary vascular resistance, hypoxia, and pulmonary edema lead to cor pulmonale and death [118] . ET-1 has been implicated as a mediator of these events. The increase in pulmonary vascular resistance observed in human recipients of lung allografts follows an increase in circulating ET-1 and falls with decreases in circulating ET-1 [113] . A similar pattern is seen in dogs subjected to allotransplantation [119] . Conscious dogs with left pulmonary allografts demonstrate an increase in both resting pulmonary perfusion pressure and acute pulmonary vasoconstrictor response to hypoxia [120] . Administration of ETA selective or combined ETA and ETB receptor blockers did not change the resting tone. ETB receptor mediated hypoxic pulmonary vasoconstriction appeared, however, to be increased in allograft recipients. In another study, administration of a mixed ETA and ETB receptor antagonist (SB209670) to dogs before reperfusion of the allograft resulted in a marked increase in oxygenation, decreases in pulmonary arterial pressures and improved survival compared with control animals [121] . In a model of ischemia reperfusion, inhibitors of ECE additionally attenuated the increase in circulating ET-1 and the severity of lung injury [122] . ET-1 receptor antagonists did not, however, completely eliminate the ischemia-reperfusion injury, suggesting that changes in other vasoactive mediators, such as an increase in thromboxane, a decrease in prostaglandins, or a decrease in NO, may also contribute to the increased pulmonary vascular resistance. Administration of NO donor (FK409) to both donor and recipient dogs before lung transplantation reduced pulmonary arterial pressure, lung edema, and inflammation, and improved survival. This suggests that reductions in NO following transplantation may be partly responsible for early graft failure [123] . Treatment with NO donor was also associated with a decrease in plasma ET-1 levels.

Acute rejection is manifested by diffuse infiltrates, hypoxia, and airflow limitation, and may lead to respiratory insufficiency and death. BAL ET-1 was increased in dogs during episodes of acute rejection that decreased with immunosuppressive treatment [124] . Acute episodes of rejection in humans, however, are not associated with further increases in BAL ET-1 [72] . Chronic rejection of allografts, manifested as BO, is the major cause of morbidity and mortality in long-term lung transplant survivors [71] . The etiology of BO following transplant is unclear but may be related to repeated episodes of acute rejection, chronic low-grade rejection, or organizing pneumonia [125] . As discussed earlier, a chronic increase in ET-1, as seen in lung allografts, may contribute to bronchospasm and proliferative bronchiolitis obliterans due to the bronchoconstrictor and smooth muscle mitogenic effects of ET-1 [28, 126] . This is further supported by the increase of BAL ET-1 in the transplanted lung, which is susceptible to BO, but not the native lung in recipients of single lung transplants [72] .

The mitogenic effects of ET-1 may play a role in the development of pulmonary malignancy as well as metastasis to the lung. Many human tumor cell lines, including prostate, breast, gastric, ovary, colon, etc, produce ET-1. The importance of the ET-1 may lie in its mitogenic effects on tumor growth and survival. This has been suggested by blockade of ETA receptors resulting in a decrease in mitogenic effects of ET-1 in a prostate cancer and colorectal cell lines [127, 128] . ET-1 receptors in tumor cells may also be altered with increases in the ETA receptor and downregulation of ETB receptors [129] . Other tumors may have an increase in ETB receptors, however, and blockade of ETB results in a decrease in tumor growth [130, 131] . Tumor cells may, as a result of this altered balance, lose the ability to respond to regulatory signals from their environment. ET-1 may additionally protect against Fas-ligand mediated apoptosis [132] .

ET-1 has been detected using immunohistochemistry and in situ hybridization in pulmonary adenocarcinomas and squamous cell tumors and, to a lesser extent, small cell and carcinoid tumors [133] . In situ hybridization also demonstrated a similar pattern of ET-1 mRNA expression in non-neuroendocrine tumors. ET-1 receptors have also been found in a variety of pulmonary tumor cell lines. ETA receptors were found in small cell tumors, adenocarcinomas and large cell tumors, while ETB receptors were expressed primarily in adenocarcinomas and small cell tumors [134] . ECE, which converts big ET-1 to ET-1, the committed step in ET-1 biosynthesis, was also found in human lung tumors but not in adjacent normal lung [135] . These findings, combined with the presence of ET-1 in lung tumors, suggest a possible autocrine loop that sustains and supports the growth of lung tumors. A recent study, however, suggested that, while ETA and ECE-1 were detectable in lung tumors, these genes were downregulated compared with normal bronchial epithelial cell lines [136] . It was proposed that the role of ET-1 in lung tumors is not that of an autocrine factor, but that of a paracrine growth factor to the stroma and vasculature surrounding the tumor allowing angiogenesis.

Tumor angiogenesis is necessary for continued growth of the tumor beyond the limits of oxygen diffusion. The growth of vessels into the tumor is also important to metastatic potential of the tumor. ET-1 may play an important role in angiogenesis and tumor growth and survival Available online http://respiratory-research.com/content/2/2/090 commentary review reports primary research through induction of vascular endothelial growth factor expression and sprouting of new vessels into the tumor and surrounding tissue [137, 138] . ET-1 binding activity was found in blood vessels and vascular stroma surrounding lung tumors at the time of resection, most markedly surrounding squamous cell tumors [139] . ET-1 production may be further augmented by the hypoxic environment found within large solid tumors [140] . Since metastasis is dependent on neo-vascularization, ET-1 may also be an important mediator of this phenomenon. ET-1 receptor antagonists may have a useful role in the treatment of neoplastic disease by inhibiting growth as well as metastatic potential of human tumors.

Experimental lung injury of many different types results in increased circulating ET-1, BAL ET-1, and lung tissue ET-1 [18] . ET-1 levels in humans are also increased in sepsis, burns, disseminated intravascular coagulation, acute lung injury, and acute respiratory distress syndrome (ARDS) [141] [142] [143] [144] [145] [146] [147] . ET-1 increases also correlate with a poorer outcome with multiple organ failure, increased pulmonary arterial pressure, increased airway pressure and decreased PiO 2 /FiO 2 , while clinical improvement correlates with decreased ET-1 levels [144, 145, 147] . The arterio-venous ratio for ET-1 is increased in patients with ARDS but it is not clear whether this is due to increased secretion of ET-1 in the lungs or decreased clearance [142, 144] . In patients who succumbed to ARDS, there was also a marked increase in tissue ET-1 immunostaining in vascular endothelium, alveolar macrophages, smooth muscle, and airway epithelium compared with lungs of patients who died without ARDS. Interestingly, these same patients also had a decrease in immunostaining for both endothelial nitric oxide synthase and inducible nitric oxide synthase in the lung [148] . ARDS is also characterized by the presence of inflammatory cells in the lung. Since ET-1 may act as an immune modulator, an increase in ET-1 may contribute to lung injury by inducing expression of cytokines including tumor necrosis factor and IL-6 and IL-8 [149] . These cytokines may in turn stimulate the production of many inflammatory mediators, leading to lung injury. ET-1 additionally activated neutrophils, and increased neutrophil migration and trapping in the lung [65] [66] [67] [68] [69] .

Another hallmark of ARDS is disruption and dysfunction of the pulmonary vascular endothelium leading to accumulation of lung water. The role of endothelin in formation of pulmonary edema is uncertain. Infusion of ET-1 raises pulmonary vascular pressure, but it is uncertain whether ET-1 by itself increased pulmonary protein or fluid transport in the lung [150] [151] [152] . ET-1 may rather be acting synergistically with other mediators to lead to pulmonary edema [153, 154] .

Pulmonary fibrosis is the final outcome for a variety of injurious processes involving the lung parenchyma. The final common pathway in response to injury to the alveolar wall involves recruitment of inflammatory cells, release of inflammatory mediators, and resolution. The reparative phase occasionally becomes disordered, resulting in progressive fibrosis.

ET-1 in the lung may be important in the initial events in lung injury by activating neutrophils to aggregate and release elastase and oxygen radicals, increasing neutrophil adherence, activating mast cells, and inducing cytokine production from monocytes [65] [66] [67] [68] [69] 149, 155] . Among the many cytokines induced by ET-1 that are important in mediating pulmonary fibrosis are transforming growth factor-β and tumor necrosis factor α [156, 157] . ET-1 is also profibrotic by stimulating fibroblast replication, migration, contraction, and collagen synthesis and secretion while decreasing collagen degradation [158] [159] [160] [161] [162] . ET-1 additionally enhances the conversion of fibroblasts into contractile myelofibroblasts [43, 163] . ET-1 also increases fibronectin production by bronchial epithelial cells [164] . Finally, ET-1 has mitogenic effects on vascular and airway smooth muscle [126, 28] . ET-1 may thus play an important role in the initial injury and eventual fibrotic reparative process of many inflammatory events in the lung.

Several lines of evidence regarding the importance of ET-1 in pulmonary fibrosis are available. Plasma and BAL ET-1 levels are increased in idiopathic pulmonary fibrosis [50, 165] . Lung biopsies from patients with idiopathic pulmonary fibrosis have additionally increased ET-1 immunostaining in airway epithelial cells and type II pneumocytes, which correlates with disease activity [166] . Scleroderma is commonly associated with pulmonary hypertension and pulmonary fibrosis. Plasma and BAL ET-1 is increased in these patients [160, 167, 168] , but it is unclear whether the presence of either pulmonary hypertension or pulmonary fibrosis increases these levels further [167] . BAL fluid from patients with scleroderma increased proliferation of cultured lung fibroblasts, which was inhibited by ETA receptor antagonist. This suggests that the ET-1 in the airspace may be contributing significantly to the fibrotic response [160] . An increase in ET-1 binding has also been reported in lung tissue from patients with scleroderma associated pulmonary fibrosis [169] . Pulmonary inflammatory cells also appear to be primed for ET-1 production because cultured alveolar macrophages from patients with scleroderma and lung involvement secrete increased amounts of ET-1 in response to stimulation with lipopolysaccharide [170] . These observations collectively suggest that augmented ET-1 release may contribute to and perpetuate the inflammatory process. Bleomycin-induced pulmonary fibrosis in animals is associated with increased ET-1 expression in alveolar macrophages and epithelium [171] . The increase in ET-1 proceeds the development of pulmonary fibrosis. The use of ET-1 receptor antagonists has produced mixed results in limiting the development of bleomycin-induced fibrosis. A decrease in fibroblast replication and secretion of extracellular matrix proteins in vitro but not a decrease in lung collagen content in vivo has been shown using ETA or combined ETA and ETB receptor antagonists after bleomycin [172] . Another group did, however, observe a decrease in fibrotic area in lungs of rats following bleomycin that were treated with a mixed ETA and ETB receptor antagonist [173] .

While ET-1 seems to correlate with pulmonary fibrosis, it remains uncertain whether the increase in ET-1 is a cause or consequence of the lung disease. Pulmonary fibrosis was recently reported in mice that constitutively overexpress human ET-1 [107] . These mice were known to develop progressive nephrosclerosis in the absence of systemic hypertension [174] . The transgene was localized throughout the lung, with the strongest expression in the bronchial wall. In the lung, the mice developed age-dependent accumulation of collagen and accumulation of CD4+ lymphocytes in the perivascular space. This observation suggests that an increase in lung ET-1 alone may play a causative role in the development of pulmonary fibrosis [107, 175] .

Since its discovery 12 years ago, much evidence has accumulated regarding the biologic activity and potential role of ET-1 in a variety of diseases of the respiratory track. As compelling as much of this evidence is, the causal relationship between ET-1 activity and disease is not complete. The increasing use of ECE and endothelin receptor antagonists in experimental and human respiratory disorders will help to clarify the role of this pluripotent peptide in health and disease.

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Sustained coronary vasoconstriction provoked by a peptidergic substance released from endothelial cells in culture

Endothelial cells in culture produce a vasoconstrictor substance

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Chromosomal assignments of the human endothelin family genes: the endothelin-1 gene (EDN-1) to 6p23-p24, the endothelin-2 gene (EDN-2) to 1p34, and the endothelin-3 gene (EDN-3) to 20q13.2-q13.3

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Conversion of bug endothelin-1 to 21-residue endothelin-1 is necessary for expression of full vasoconstrictor activity: structure-activity relationships of big endothelin-1

Biosynthesis, distribution and metabolism of endothelins in the pulmonary system

Immunocytochemical localization of endothelin in cultured bovine endothelial cells

Identification of endothelin-1 and big endothelin-1 in secretory vesicles isolated from bovine aortic endothelial cells

Evidence for vesicles that transport endothelin-1 in bovine aortic endothelial cells

Polar secretion of endothelin-1 by cultured endothelial cells

The induction of cell differentiation and polarity of tracheal epithelium cultured on amniotic membrane

Cloning of a cDNA encoding a non-isopeptide selective subtype of the endothelin receptor

Cloning and expression of a cDNA encoding an endothelin receptor

Functional evidence for different endothelin receptors in the lung

Human pulmonary circulation is an important site for both clearance and production of endothelin-1

Endothelins and the lung

Endothelin receptor subtype B mediates auto-induction of endothelin-1 in rat mesangial cells

Nitric oxide regulates the expression of vasoconstrictors and growth factors by vascular endothelium under both normoxia and hypoxia

Localization of endothelinlike immunoreactivity in airway epithelium of rats and mice

Autoradiographic localization of endothelin-1 binding sites in the cardiovascular and respiratory system

Endothelin receptor distribution and function in the airways

Endothelin-1 production is associated with eosinophilic rather than neutrophilic airway inflammation

Endothein-1 stimulates chloride secretion across canine tracheal epithelium

Endothelin stimulates cililary beat frequency and chloride secretion in canine cultured tracheal epithelium

Endothelin-1 stimulates proliferation of normal airway epithelial cells

Endothelin-1 promotes mitogenesis in airway smooth muscle cells

Effect of epithelium removal and enkephalin inhibition of the bronchoconstrictor response of three endothelins and the human isolated bronchus

Endothelin receptor subtypes in human and guinea pig pulmonary tissues

Endothelin-1 receptor density, distribution and function in human isolated asthmatic airway

Endothelin-induced contraction and mediator release in human bronchus

Formation of endothelin by cultured airway epithelial cells

Damage of the airway epithelium and bronchial reactivity in patients with asthma

Endothelin-1 contributes to antigen-induced airways hyperresponsiveness

Disruption of ET-1 gene enhances pulmonary responses to methacholine via a functional mechanism in knockout mice

Airway hyperresponsiveness in mutant mice deficient in endothelin-1

Endothelin-1-induced bronchoconstriction in asthma

Endothelin production and effects of endothelin antagonism during experimental airway inflammation

Sputum cellular responses to inhaled endothelin-1 in asthma

Endothelin-1 enhances vascular permeability in conscious rats: role of thromboxane A2

Potential role of the endothelins in airway remodeling in asthma

Endothelin-1 induces bronchial myofibroblast differentiation

Increased synthesis and release of endothelin-1 during the initial phase of airway inflammation

Increased expression of endothelin in bronchial epithelial cells of asthamtic patients and effect of corticosteroids

Constitutive expression of endothelin in bronchial and epithelial cells of patients with symptomatic and asymptomatic asthma and modulation by histamine and interleukin-1

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Decreased production of endothlin-1 in asthmatic children after immunotherapy

Circulating endothelin-1 levels in patients with bronchial asthma

Increased endothelin-like immunoreactive material on bronchoalveolar lavage fluid from patients with bronchial asthma and patients with interstitial lung disease

Endothelin in bronchoalveolar lavage fluid and its relation to airflow obstruction in asthma

Levels of endothelin in the bronchoalveolar lavage fluid of patients with symptomatic asthma and reversible airflow obstruction

Altered ration of ETa and ETb receptor mRNA in bronchial biopsies from patients with asthma and chronic airway obstruction

Blood and bronchoalveolar lavage endothelin-1 levels in nocturnal asthma

Circulating endothelin-1 levels in patients with bronchial asthma

Endothelin immunoreactivity of airway epithelium in asthmatic patients

Elevated endothelin-1 levels after cigarette smoking

Plasma endothelin-1 levels in chronic obstructive pulmonary disease: relationship with naturitic peptide

Sputum endothelin-1 is increased in active fibrosis and chronic obstructive pulmonary disease

Local and peripheral plasma endothelin-1 in pulmonary hypertension secondary to chronic obstructive pulmonary disease

Increased 24 hour endothelin-1 urinary excretion in patients with chronic obstructive pulmonary disease

An endothelin-1 mediated autocrine growth loop involved in human renal tubular regeneration

Diminished vascular response to inhibition of endothelium-derived nitric oxide and enhanced vasoconstriction to exogenously administered endothelin-1 in clinically healthy smokers

Increased responsiveness of human pulmonary arteries in patients with positive bronchodilator responses

Endothelin-1 causes sequential trapping of platelets and neutrophils in pulmonary microcirculation in rats

Activated neutrophils by endothelin-1 caused tissue damage in human umbilical cord

Endothelin-1 enhances superoxide generation of human neutrophils stimulated by the chemotactic peptide N-formyl-methionyl-leucyl-phenalanine

Aggregation of human polymorphonuclear leukocytes by endothelium: role of platelet activating factor

Endothelin-1 causes accumulation of leukocytes in the pulmonary circulation

Endothelin-1 in stable bronchiectasis

Airway obstruction and bronchiolitis obliterans after lung transplantation

Increased levels of endothelin-1 in bronchoalveolar lavage fluid in patients with lung allografts

Endothelin in bronchoalveolar lavage fluid is increased in lung transplant patients

Experimental bronchiolitis obliterans induced by HVJ-liposome mediated endothelin-1 gene transfer

Hemodynamic shear stress stimulates endothelin production by cultured endothelial cells

Prostaglandin E2 and prostacyclin inhibit the production and secretion of endothelin from cultured endothelial cells

Hypoxia decreased endothelin-1 synthesis by rat lung endothelial cells

Oxidant stress regulates basal endothelin production by cultured rat pulmonary endothelial cells

Effects of vasoactive and inflammatory mediators on endothelin-1 expression in pulmonary endothelial cells

Enhanced endothelin-1 and endothelin receptor gene expression in chronic hypoxia

Endothelin 1: mitogenic activity on pulmonary artery smooth muscle cells and release from hypoxic endothelial cells

Hypoxia stimulates human prepro endothelin-1 promoter activity in transgenic mice

Effect of hypoxia on endothelin-1 production by vascular endothelial cells

Endothelin receptor subtypes in human vs rabbit pulmonary arteries

Localization and distribution of endothelin receptor subtypes in pulmonary vasculature of normal and hypoxia exposed rats

Angiotensin II up-regulates the expression of type A endothelin receptors in human vascular smooth muscle cells

Increased endothelin receptor gene expression in hypoxic rats

Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium derived relaxing factor

Endothelin-1 causes pulmonary vasodilation in rats

Chronic hypoxia augments endothelin-B receptor mediated vasodilation in isolated perfused rat lung

IF: Hypoxia inhibits ETB receptor mediated NO synthesis in hypoxic rat lungs

Endothelin-1 mediated nitro-L-arginine vasoconstriction of hypertensive rat lungs

Endothelin ETA-and ETBreceptor mediated vasoconstriction in rat pulmonary arteries and arterioles

ET-1 induced vasoconstriction shifts from ETA-to ETB-receptor mediated reaction after preconstriction

Endothelin-A receptor antagonist prevents acute hypoxiainduced pulmonary hypertension in the rat

Mechanism of hypoxic vasoconstriction involves ETa receptor-mediated inhibition of K-ATP channels

Expression of endothelin-1 in rats developing hypobaric-hypoxia induced pulmonary hypertension

ETA-receptor antagonist prevents and reverses chronic hypoxia-induced pulmonary hypertension in rat

BQ-123, and ETa-receptor antagonist, attenuates hypoxic pulmonary hypertension in rats

Vascular remodeling and ET-1 expression in rat strains with different responses to chronic hypoxia

Increased lung endothelin-1 production in rats with idiopathic pulmonary hypertension

Endothelin-1 is elevated in monocrotaline pulmonary hypertension

Nonspecific endothelin-receptor antagonist blunts monocrotaline-induced pulmonary hypertension in rats

Endothelin induced collagen remodeling in experimental pulmonary hypertension

Contribution of endogenous endothelin-1 to the progression of cardiopulmonary alterations in rats with monocrotaline-induced pulmonary hypertension

Overexpression of endothelin-1 and enhanced growth of pulmonary artery smooth muscle cells from fawn hooded rat

Theuring F: Pulmonary fibrosis and chronic lung inflammation in ET-1 transgenic mice

Monoclonal endothelial cell proliferation is present in primary but not secondary pulmonary hypertension

Increased plasma endothelin-1 in pulmonary hypertension: marker or mediator of disease

Expression of endothelin-1 in the lungs of patients with pulmonary hypertension

Endothelin-1 in the lungs of patients with pulmonary hypertension

Continuous infusion of epoprostenol improves the net balance between pulmonary endothelin-1 clearance and release in primary pulmonary hypertension

Plasma endothelin-1 levels in human lung transplant recipients

Distribution of endothelin-1 in transplanted human lungs. Transplantation

Distribution of endothelin-like immunoreactivity and mRNA in the developing and adult human lung

Mononuclear cells from dogs with acute lung allograft rejection cause contraction of pulmonary arteries

Localization and characterization of endothelin-1 binding sites in the transplanted human lung

Effect of ischemia reperfusion or hypoxia reoxygenation on lung vascular permeability and resistance

Alterations in bronchoalveolar lavage and plasma endothelin-1 levels early after lung transplantation

Pulmonary vasoregulation by endothelium in conscious dogs after left lung transplantation

Efficacy of administering an endothelin-receptor antagonist (B209670) in ameliorating ischemia-reperfusion injury in lung allografts

Phosphoramidon abolishes the increased in endothelin-1 release induced by ischemia-hypoxia in isolated perfused guinea pig lungs

The effect of FK409 -a nitric oxide donor -on canine lung transplantation

Endothelin-1 in bronchoalveolar lavage during rejection of allotransplanted lungs

Risk factors for obliterative bronchiolitis in heart-lung transplant recipients

Endothelin, a potent vasoconstrictor is a vascular smooth muscle mitogen

Endothelin-1 and decreased endothelin B receptor expression in advanced prostate cancer

Stimulation of colorectal cancer cell line growth by ET-1 and its inhibition by ET(A) antagonists

Enhanced endothelin ETB receptor down-regulation in human tumor cells

Augmented expression of endothelin-1, endothelin-3, and the endothelin-B receptor in breast carcinoma

An endothelin receptor b antagonist inhibits growth and induces cell death in human melanoma cells in vitro and in vivo

Endothelin receptor blockade potentiates FasLinduced apoptosis in rat colon carcinoma cells

Detection of endothelin immunoreactivity and mRNA in pulmonary tumors

Endothelin receptor expression in lung cancer cell lines and bronchial epithelial cell lines

Evidence for an endothelin-1 autocrine loop in lung cancer

Studies on the expression of endothelin, its receptor subtypes and converting enzymes in lung cancer and human bronchial epithelium

Vasoactive peptides modulate vascular endothelin cell growth factor production and endothelial cell proliferation and invasion

Angiogenesis in cancer: the role of endothelin-1

Localization and characterization of endothelin-1 receptor binding in the blood vessels of human pulmonary tumors

The effect of oxygen and carbon dioxide on tumor endothelin-1 production

Released endothelin in oleic acid-induced respiratory distress syndrome

Endothelin-1 in adult respiratory distress syndrome

Endothelin production in sepsis and the adult respiratory distress syndrome

Endothelin-1 in acute lung injury and the adult respiratory distress syndrome

Role of endothelin in disseminated intravascular coagulation

Endothelin-1 and prostaglandin E2 levels increase in patients with lung injury

Circulating endothelin-1 concentrations in acute respiratory failure

Expression of endothelial nitric oxide synthase, inducible nitric oxide synthase and endothelin-1 in lungs of subjects who died with ARDS

Endothelins: polyfunctional peptides

Phosphoramidon blocks big-endothelin-1 but not endothelin-1 enhancement of vascular permeability in the rat

Endothelin-1-induced increase in microvascular permeability in isolated perfused rat lungs requires leukocytes and plasma

Endothelin-1 increases the pulmonary microvascular pressure and causes pulmonary edema in salt-solution but not blood perfused rat lungs

Vascular responses to endothelin-1 following inhibition of nitric oxide synthase in the conscious rat

Impact of endothelin-1 in endotoxininduced pulmonary vascular reactions

ET-1 released histamine from guinea pig pulmonary but not peritoneal mast cells

Macrophage production of transforming growth factor beta and fibroblast collagen synthesis in chronic pulmonary inflammation

Tumor necrosis factor/cachectin plays a key role in bleomycininduced pneumopathy and fibrosis

Endothelin 1 and endothelin 3 induced chemotaxis and replication of pulmonary fibroblasts

Effect of endothelin-1 on alpha smooth muscle actin expression and on alveolar fibroblast proliferation in interstitial lung diseases

Increased levels of endothelin-1 in bronchoalveolar lavage fluid from patients with systemic sclerosis contribute to fibroblast migration and activity in vitro

Endothelin produced by endothelial cells promotes collagen gel contraction by fibroblasts

Effects of endothelins on collagen turnover in cardiac fibroblasts. Cardiovsac Res

Paracrine interaction between fibroblast and endothelial cells in a serum-free coculture model: modulation of angiogenesis and collagen gel contraction

Endothelin-1 induces increased fibronectin expression in human bronchial epithelial cells

Endothelin-1 in idiopathic pulmonary fibrosis

Expresison of endothelin-1 in lungs of patients with cryptogenic fibrosing alveolitis

Plasma endothelin-1 levels, pulmonary hypertension, and lung fibroblasts with systemic sclerosis

Circulating endothelin-1 levels in systemic sclerosis subsets -a marker of fibrosis or vascular dysfunction

Increased levels of endothelin-1 and differential endothelin type A and B receptor expression in scleroderma-associated fibrotic lung diseases

Endothelin secretion by alveolar macrophages in systemic sclerosis

Increased endothelin-1 and its localization during the development of bleomycin-induced pulmonary fibrosis in the rat

Increased endothelin-1 in bleomycin-induced pulmonary fibrosis and the effect of endothelin receptor antagonists

Effect of endothelin receptor antagonists (BQ485, RO 47-0203) on collagen deposition during development of bleomycin-induced pulmonary fibrosis in rats

Endothelin-1 transgenic mice develop glomeulerosclerosis, interstitial fibrosis, and renal cysts but not hypertension

A cytokine reborn? Endothelin-1 in pulmonary fibrosis