Airway vascular reactivity and vascularisation in human chronic airway disease

Pulmonary Pharmacology & Therapeutics 22 (2009) 417–425

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Airway vascular reactivity and vascularisation in human chronic airway disease Simon R. Bailey a, *, Sarah Boustany b, Janette K. Burgess b, Stuart J. Hirst c, Hari S. Sharma d, David E. Simcock e, Padmini R. Suravaram d, Markus Weckmann b a

Faculty of Veterinary Science, University of Melbourne, Parkville, Victoria, Australia CRC for Asthma and Airways and Discipline of Pharmacology, University of Sydney, NSW Australia c Department of Physiology, Monash University, Clayton, Melbourne, Victoria, Australia d Angiogenesis and Tissue Remodelling Group, Erasmus University Medical Centre, Rotterdam, The Netherlands e King’s College London, MRC & Asthma UK Centre in Allergic Mechanisms of Disease, Department of Asthma, Allergy and Respiratory Science, Guy’s Campus, London, UK b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 June 2008 Received in revised form 1 April 2009 Accepted 21 April 2009

Altered bronchial vascular reactivity and remodelling including angiogenesis are documented features of asthma and other chronic inflammatory airway diseases. Expansion of the bronchial vasculature under these conditions involves both functional (vasodilation, hyperperfusion, increased microvascular permeability, oedema formation, and inflammatory cell recruitment) and structural changes (tissue and vascular remodelling) in the airways. These changes in airway vascular reactivity and vascularisation have significant pathophysiological consequences, which are manifest in the clinical symptoms of airway disease. Airway vascular reactivity is regulated by a wide variety of neurotransmitters and inflammatory mediators. Similarly, multiple growth factors are implicated in airway angiogenesis, with vascular endothelial growth factor amongst the most important. Increasing attention is focused on the complex interplay between angiogenic growth factors, airway smooth muscle and the various collagen-derived fragments that exhibit anti-angiogenic properties. The balance of these dynamic influences in airway neovascularisation processes and their therapeutic implications is just beginning to be elucidated. In this review article, we provide an account of recent developments in the areas of vascular reactivity and airway angiogenesis in chronic airway diseases. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Angiogenesis Asthma Bronchial circulation Chronic obstructive airway disease Airway vasculature Extracellular matrix Lymphangioleiomyomatosis Smooth muscle Vascular reactivity

1. Introduction The tracheobronchial vasculature is the principal arterial supply to the airway wall, deriving its blood supply from the systemic circulation [1]. Bronchial arteries arise from the aorta and form a peribronchial plexus surrounding the airway wall. The bronchial and pulmonary circulations anastomose freely throughout the tracheobronchial tree. Although relatively small compared with pulmonary blood flow, the bronchial circulation is considered the primary source of oxygenation and tissue nutrients available to the airway wall. Other functions of the bronchial circulation include thermoregulation and humidification of inspired gas and the elimination of drugs. It is now appreciated that the pathophysiological consequences of altered bronchial blood flow can contribute to the clinical manifestations of chronic airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). These alterations may arise acutely from local vasodilatation and increases in

* Corresponding author. Tel.: þ61 3 8344 6315; fax: þ61 3 8344 7374. E-mail address: [email protected] (S.R. Bailey). 1094-5539/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.pupt.2009.04.007

microvascular leakage. Remodelling of the vasculature, including the formation of new blood vessels due to increased levels of angiogenic growth factors, can also in turn facilitate the delivery and increased adhesion and trafficking of inflammatory cells to sites of inflammation (Fig. 1). Asthma is a chronic inflammatory disease of the airways that gives rise to a heterogeneous syndrome characterised by airway hyperresponsiveness (AHR), leading to symptoms of variably reversible airflow obstruction including cough, chest tightness, wheeze and dyspnoea. The inflammatory process that is characteristic of asthma alters the normal tissue architecture of the airway wall [2]. Such changes in the composition, content, and organisation of the tissue constituents of the airway wall are collectively referred to as ‘airway remodelling’. The precise timing of the appearance of pathological hallmarks of asthma in susceptible individuals is uncertain, although it is likely that this process can begin early in life either in parallel with inflammation, or as a prerequisite for persistent inflammation [3]. Two of the most striking and consistent features of remodelled airways in asthma include accumulation of airway smooth muscle (ASM) [4] and increases in bronchial wall blood vessels [5].

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Airway vessels

Vasodilatation

Mediators

Permeability

Cell infiltration

Remodelling

Angiogenesis

Fig. 1. Diverse roles of bronchial vessels in chronic airway diseases. Adapted from [105].

COPD is a leading global cause of morbidity and mortality that is characterised by inexorable deterioration of small airway function with emphysema associated with cellular inflammation and lung tissue remodelling. The condition is characterised by chronic inflammation of the entire bronchial tree, is perpetuated by inhaled irritants in cigarette smoke, and results in airway narrowing and modest AHR. Airways become oedematous, having excessive mucus production and impaired epithelial ciliary function. Accordingly, patients develop a chronic productive cough, as well as wheeze and dyspnoea. The mechanisms of COPD are heterogeneous, involving fibrosis, cell proliferation or apoptosis as well as inflammation [6,7]. Vascular remodelling in COPD, evidenced by vascular intimal thickening and the emergence of smooth muscle cells within the intima of small pulmonary arterial branches, has been attributed to this chronic inflammatory process accompanied by airway fibrosis [7]. Lymphangioleiomyomatosis (LAM) is a rare multisystem disorder that occurs predominantly in women of reproductive age. The disease is characterised by cystic lung disease, leading to impaired respiratory function. Symptoms results from proliferation of neoplastic cells (LAM cells), which have a smooth muscle cell phenotype, express melanoma antigens, and have mutations in one of the tuberous sclerosis complex genes (TSC1 or TSC2) [8]. LAM frequently involves lymphatic structures, loss of alveolar structure and the development of cystic spaces [8]. Patients with LAM show altered patterns of extracellular matrix (ECM) in the lung [8] resulting in poor functional support to the airways. Several investigators have suggested that the disorganisation of the lung tissues seen in LAM underlies the impaired pulmonary function [9,10]. Consistent with extensive lymphatic involvement in LAM, elevated serum concentrations of the vascular endothelial growth factor (VEGF) family member, VEGF-D, a vascular and lymphangiogenic growth factor, have recently been associated with LAM [11]. Expansion of the bronchial vasculature may occur through either vasodilatation or neovascularization, or potentially both mechanisms (Fig. 1). Numerous mediators of the inflammatory response in asthma (histamine, bradykinin, prostaglandins, leukotrienes, nitric oxide) and sensory nerve stimulation have been shown to cause vasodilatation and to increase microvascular permeability [12,13], leading to interstitial oedema, airway thickening and mucus hypersecretion, luminal narrowing and AHR during bronchoconstriction. Animal studies have demonstrated that the bronchial microvascular volume can double during vasodilatation leading to airway narrowing [14]. This is supported by the observation that the rapid infusion of intravenous fluids in both healthy humans [15] and those with left ventricular heart failure [16] leads to airflow obstruction and AHR in response to methacholine challenge. It has also been suggested that vascular engorgement and oedema may account for the increased airway resistance observed following allergic bronchial provocation challenge [17] and for reduced forced expiratory flow rates seen in exercise-induced asthma [18]. Non-invasive human studies have demonstrated an increase in airway mucosal blood flow (AMBF) in stable asthmatic subjects when compared to healthy controls [19],

and animal models show further increases in blood flow after allergen challenge [20]. The consequences of increased AMBF in asthma are theoretically both beneficial and detrimental. It could enhance delivery of systemically administered medication to the airway and accelerate the clearance of locally produced spasmogens. However, increased AMBF could also enhance airway oedema, luminal narrowing and loss of airway distensability during inspiration (all typical of asthma) through microvascular hyperpermeability as well as promote increased clearance of inhaled antiasthma medications. A dominant feature of the bronchial mucosa in cases of fatal asthma is dilatation of the small blood vessels [21]. A study of airway vascularity (total vessel area as a percentage of submucosal area in specimen sections) in resected lung and post-mortem specimens found a vascularity of 3.3% in asthmatic individuals and 0.6% in control subjects [22]. In addition to an increase in size of submucosal blood vessels, the overall number of vascular structures is increased in those with asthma [5]. Blood vessels are therefore capable of acting through a range of mechanisms, involving both vasodilatation and vascular proliferative responses, to promote the inflammatory response and airway remodelling typical of airways disease (Fig. 1). 2. Vascular reactivity in airway disease The reactivity of bronchial blood vessels to vasoactive mediators, encompassing both changes in blood flow and in vascular permeability, is potentially an important aspect of the pathophysiology of obstructive airway disease and its treatment. 2.1. Airway blood flow The majority of blood flow to the conducting airways from the bronchial artery in man is distributed to the subepithelial mucosa [13,14]. Because asthma-associated airway inflammation is generally believed to involve this airway tissue compartment to a large extent, the measurement of subepithelial blood flow is of particular importance. Indeed, AMBF is increased in stable asthmatic subjects when compared to healthy controls [19], which may be due to a combination of increased tissue vascularity and vascular engorgement. The tracheobronchial vasculature is controlled by adrenergic, cholinergic, and peptidergic neuronal mechanisms. Sympathetic nerves release noradrenaline and neuropeptide Y (NPY), and parasympathetic nerves release acetylcholine and usually vasoactive intestinal polypeptide (VIP) [23]. Activation of pulmonary c-fibre receptors by irritants and inflammatory mediators causes a powerful vasodilatation, mainly via sympathetic motor nerves. Also local axon reflexes in response to irritants and inflammatory mediators release vasodilator neuropeptides such as substance P (SP), neurokinins, and calcitonin gene-related peptide (CGRP). Thus, the prevailing airway inflammation in conditions such as asthma may evoke mucosal vasodilation due to the direct action of mediators on vascular smooth muscle, neuropeptides released by

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axon reflexes from sensory nerve receptors, and reflex vasodilation due to stimulation of sensory nerves. This vasodilation may increase the thickness of the mucosa, both by vascular engorgement and by increased interstitial fluid volume [13] and act to facilitate inflammatory cell trafficking to sites of inflammation. It has been proposed that defects in bronchial vascular function may particularly contribute to exercise- and cold and/or dry airinduced bronchoconstriction and asthma [24,25]. In contrast to most other vascular smooth muscle beds in which temperature is maintained at 37  C, bronchial vascular temperature is expected to vary considerably, particularly during exercise. Cooling of the airway wall dramatically interferes with the adrenergic control of bronchial perfusion but has been found to have little effect on thromboxane-mediated vasoconstriction [26]. Whether differences in vascular reactivity in response to temperature changes may contribute to cold- or dry air-induced asthma is yet to be determined. Both ASM and vascular smooth muscle in the airways express a-adrenergic and b-adrenergic receptors, although the receptor densities differ between the two types of smooth muscle [27]. a-Adrenergic receptors predominate on vascular smooth muscle with contraction in airway vessels being mediated primarily by the a1-adrenergic receptor. However, vasodilatory b2-adrenergic receptors are also present [28,29]. There appear to be differences in the adrenergic responsiveness of the airways, and presumably ASM, between healthy and asthmatic subjects. For example, inhaled a1-adrenergic agonists cause airflow obstruction in patients with asthma but not in healthy subjects [30,31]. The a1-adrenergic responsiveness of airway blood flow has similarly been shown to be potentiated in asthmatics [32]. The possible mechanisms of asthma-associated vascular hyperresponsiveness include increased expression and function of a-adrenoceptors, altered signal transduction, impaired inactivation of a-adrenergic agonists by cellular uptake processes, or a combination thereof [33]. A putative endothelial contractile factor has also been proposed to be responsible for the increased vascular sensitivity to a1-adrenergic agonists [25], but this has not been investigated in asthma. Conversely, b2-adrenergic agonist-induced bronchodilation may be impaired in some patients with asthma. This latter phenomenon has been attributed to, but not explained by, a blunted b2-adrenergic dilator response of the airway circulation in patients with asthma [32]. In a further study measuring AMBF, inhaled salbutamol at the clinically recommended dosage was found to be an ineffective vasodilator in asthmatics, while having a potent vasodilator effect in the airway of normal subjects [18]. Conceivably, reduced vascular dilatation and vascular engorgement might increase airway compliance and be protective in asthma. It remains to be determined whether the actions of asthma treatments such as b2-adrenoceptor agonists to modify airway blood flow are clinically important compared with their known bronchodilator properties. 2.2. Vascular permeability As well as increased subepithelial blood flow, increased vascular permeability is also a feature of asthma [34]. Plasma extravasation occurs in response to the asthma-associated inflammatory insult, involving the release of inflammatory mediators, growth factors, neuropeptides, eosinophil granule proteins, cytokines, and proteases in the airway [35]. Histamine, platelet-activating factor, leukotriene D4 and bradykinin can increase microvascular permeability through the formation of intercellular gaps [36–38]. In addition to inflammatory exudative agents, vasodilation and microvascular congestion have been shown to increase microvascular permeability [36]. These changes do not reflect a change in

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vascular responsiveness to these mediators per se. However, blood vessels that have been newly generated in chronic airway inflammation have been shown to be particularly leaky, immature, and unstable [39], which may contribute to increased vascular permeability in inflammatory airways disease such as asthma. Another function of the submucosal vasculature is to act as conduits for inflammatory cell trafficking into the bronchial microenvironment. This area has received little attention, however, it has been highlighted that blocking inflammatory cell binding to adhesion glycoproteins and specific integrins (a4b1) abrogates the airway response to airway challenge [40]. In addition to the increased vascularity and functional differences in the airway vasculature of patients with asthma, some morphological differences have also been observed. These include oedematous walls and thickening of the subendothelial basement membrane in asthmatic capillaries and venules, atrophic myocytes and fibrosis in the arterioles [41]. Such changes may affect clearance of inflammatory mediators and airway constrictors. Indeed, bronchial flow is thought to be important in reversing agonist-induced bronchoconstriction and it has been suggested that an impaired bronchial circulation may contribute to the mechanism of AHR [42]. Among inflammatory mediators which may further impair airway blood flow in asthma by causing vasoconstriction are TNFa, which induces the release of endothelin-1 from the vascular endothelium [43], and isoprostanes, formed from the actions of oxygen-free radicals on arachidonic acid [44]. The vascular endothelium may also contribute to functional or inflammatory changes in the subepithelial vasculature in COPD. Cigarette smoking is associated with attenuated vascular relaxation responses in the systemic circulation, due to vascular endothelial dysfunction, probably resulting from exogenous oxidative stress caused by cigarette smoke and/or the endogenous production of oxygen-free radicals [45]. The airway vasculature is derived from the systemic circulation and so, unsurprisingly, bronchial vascular endothelial function has been found to be impaired in smokers and ex-smokers with COPD [45], as is also the case in the pulmonary circulation of such patients [46]. Whether or not this vascular abnormality contributes to the development of chronic bronchitis is a matter of speculation, although endothelial dysfunction may contribute to many inflammatory vascular changes. These changes could include the exacerbation of inflammatory cell recruitment, leukocyte migration and the formation of endothelial gaps leading to plasma leakage in postcapillary venules [47]. All of the above changes in airway vascular reactivity have implications for the response to treatment of asthma and other inflammatory airway diseases. For example, corticosteroids, such as budesonide and fluticasone, potentiate the vasoconstrictor effects of norepinephrine in the airway vasculature by inhibiting its nonneuronal uptake, and therefore these agents cause a greater vasoconstrictor effect (reducing airway blood flow) in asthmatic subjects [48,49]. Steroids also may acutely restore the impaired b2adrenoceptor-mediated endothelium-dependent vascular dilatation in the airways of mild asthmatics following albuterol administration [50]. Conversely, leukotriene receptor antagonists (montelukast) are vasoconstrictors in the airway [51]. In addition to acute vascular responses, blood vessels also respond to inflammation by remodelling and angiogenesis (Fig. 1). 3. Mechanisms of angiogenesis Angiogenesis refers to the formation of new blood vessels from the extant vasculature and is mediated by multiple growth factors with complementary and coordinated roles [52,53]. The process of angiogenesis proceeds along a sequence of highly regulated steps. Diseased or injured tissues produce and release angiogenic growth

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factors that diffuse into the nearby tissues and bind to cognate surface receptors on endothelial cells of nearby pre-existing blood vessels. Endothelial cell-derived mediators including matrix metalloproteineases (MMPs) disrupt the integrity of the sheath-like ECM (basement membrane) surrounding all existing blood vessels. As endothelial cells begin to proliferate, they migrate through the disrupted basement membrane of existing vessels towards the diseased or injured tissue. MMPs perform multiple roles in this environment including ECM degradation and release of growth factors from known ECM storage sites, and their activity is regulated by local tissue inhibitors of metalloproteinases (TIMPs). Finally, sprouting endothelial cells roll up to form a blood vessel tube that is stabilized by specialized muscle cells (smooth muscle cells, pericytes) that provide structural support. Heparin binding peptide growth factors (VEGFs, FGFs) and nonheparin binding peptide growth factors (TGFbs, IGFs) are known angiogenic inducers (Table 1). Of these, the VEGF family is the most potent and well-studied group of direct angiogenic factors. Moreover, cytokines (IL-6, oncostatin-M) and growth factors (IGFs, TGFb), often referred to as indirect angiogenic factors, take part in angiogenesis via production of VEGF-like molecules. Thus, the switch to the angiogenic phenotype involves a change in the local equilibrium between these positive regulators of the growth of microvessels [52,53] and other negative regulators such as endostatin (Table 2, see later sections).

Table 1 Major growth factors in airway vascular remodelling. Growth factor

Source

Target

Function

VEGF

Epithelium ASM Endothelial cells VSM Macrophages ECM

Endothelial cell

Proliferation Migration Proliferation Proliferation Recruitment Recruitment

FGF-1

ECM Fibroblast

Epithelial cells Fibroblast Macrophages Fibroblast ASM VSM Epithelium

Proliferation Collagen production Proliferation Collagen production

FGF-2

ECM Endothelial cell ASM VSM Macrophages

Endothelial cell Fibroblast ASM VSM Epithelium

Proliferation Proliferation Proliferation Proliferation Proliferation

IGF-1

ECM Fibroblast

Fibroblast ASM VSM

Proliferation and Differentiation Collagen synthesis

IGF-2

ECM Fibroblast

Fibroblast

Proliferation Differentiation Collagen synthesis

TGFb

ECM Platelets Macrophages Fibroblast ASM

Fibroblast

ECM production Recruitment ECM production ECM production Differentiation ECM production Apoptosis Differentiation ECM production Chemotaxis

ASM VSM Endothelial cell

Epithelium Neutrophil T-lymphocytes Monocyte/macrophage

Abbreviations: airway smooth muscle (ASM); extracellular matrix (ECM); fibroblast growth factor (FGF); insulin-like growth factors (IGF); transforming growth factor beta (TGFb), vascular endothelial growth factor (VEGF).

4. Positive regulators of angiogenesis 4.1. Vascular endothelial growth factor system VEGF is a critical molecule in vascular remodelling in many tissues. The VEGF family comprises seven members (VEGF-A to VEGF-F and placenta growth factor PlGF), which bind to cognate cell surface tyrosine kinase-linked receptors (VEGFR-1/Flt-1, VEGFR-2/KDR/Flk-1 and VEGFR-3/Flt-4) [52,54], that are principally expressed by endothelial and epithelial cells. VEGF promotes an array of responses in the endothelium including hyperpermeability, endothelial cell proliferation and migration, followed by angiogenesis with new vessel tube formation in vivo [52,54,55]. VEGF is widely expressed in cells of the lung. This includes bronchiolar, submucosal glandular and alveolar type I and II epithelial cells, alveolar macrophages, neutrophils, eosinophils, dendritic cells, and stromal cells including airway and vascular smooth muscle and myofibroblasts [56–59]. The expression of VEGF can be induced under a variety of pathophysiological conditions, including pulmonary hypoxia and pulmonary hypertension with increased sheer stress [59] and its release is constitutively elevated in ASM cells from asthmatics [60]. Additionally, levels of VEGF correlate directly with increases in total vascular area of the airway [61], disease activity [62] and are inversely correlated with airway calibre and AHR [57,63]. These studies suggest a close relationship between VEGF, airway vascular remodelling, mucosal inflammation and clinical scores of asthma severity [62,64–67]. Hypoxia and pulmonary hypertension are pathological features often seen in advanced COPD patients and in exacerbations of chronic severe asthma, and increased VEGF expression under influence of hypoxia-inducible transcription factors (HIFs) may contribute to increased and abnormal proliferation of endothelial and vascular smooth muscle cells in pulmonary vessels leading to vascular remodelling [68–70]. In patients with COPD, higher pulmonary VEGF expression was found in bronchial and alveolar epithelial and vascular smooth muscle as well as alveolar macrophages, whereas higher VEGFR-1 and VEGFR-2 expression was found in the endothelium when compared with patients without COPD [58]. Over expression of VEGF in mice caused pulmonary emphysema, aberrant structure of capillary endothelium, and widespread pulmonary inflammation [71]. Transgenic over expression of PlGF caused apoptosis of pneumocytes, emphysema and reduced VEGF expression and numbers of endothelial cells [71,72]. Although not conclusive, results of these animal studies further support a role of VEGFs and their receptors in tissue and vascular remodelling in chronic airway diseases. Other members of the VEGF superfamily, such as VEGF-D and placenta growth factor (PlGF), and other angiogenic factors including the angiopoietins (Ang) may be important in vascular remodelling processes in asthma and LAM [52,53,73]. Recent data suggest that increased PlGF may modulate the availability/production of VEGF in emphysematous patients [71]. The relative balance between vascular growth factors that promote (VEGF and Ang-2) or inhibit (Ang-1 and Ang-4) leakage also may be important in determining capillary integrity [52,54]. 4.2. Fibroblast growth factor system Fibroblast growth factors (FGFs) have also been implicated in vascular remodelling in patients with chronic lung disease [7,74]. FGFs are mitogenic factors involved in development, tissue homeostasis and repair processes, while FGF-1 and FGF-2 are also angiogenic factors. The FGF family comprises 23 members in man and their functional receptors are designated FGFR1–FGFR5.

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Table 2 Collagen-derived angiogenic inhibitors. Collagen-derived angiogenic inhibitor

Collagen type

Arresten Canstatin Tumstatin Restin Endostatin

Collagen Collagen Collagen Collagen Collagen

IV a1 chain IV a2 chain IV a3 chain XV XVIII

Distinct FGF ligands bind with varying affinities to cognate FGF receptors. Alternative splicing and regulated protein trafficking further modulate the intracellular events and resultant response initiated by FGF ligand–receptor interaction [7,75]. In COPD, enhanced expression of FGF-2 has been shown in the airways, and levels of FGF-1, FGF-2 and FGFR1 are increased in vascular and in epithelial compartments [58,68]. FGF-1 induces tissue remodelling by increasing collagenase expression and downregulation of collagen I expression in lung fibroblasts [68]. In the normal pulmonary vasculature, FGF-1, FGF-2 and FGFR-1 are constitutively expressed in the media (vascular smooth muscle cells) of pulmonary vessels and FGF-2 is also found in endothelial cells. Singh and colleagues demonstrated that increased expression of FGF-2 in vascular smooth muscle and endothelium precedes arterial enlargement in response to increased arterial blood flow in vivo [76]. Furthermore, Bryant and colleagues found that FGF-2 was protective against a decrease in vessel luminal area and wall thickening in response to altered blood flow and that this inhibitory effect was prevented by anti-FGF-2 neutralizing antibodies [77]. Together, these reports suggest that FGFs have a role in airway and vascular remodelling in the development of COPD, although evidence for FGF-2 being elevated in asthma is conflicting [66,77,78].

4.3. Insulin-like growth factors Endocrinologic disturbances and the levels of circulating of IGF1 have been linked with the disease processes, such as airway and vascular remodelling in patients with COPD [79]. Recent antibody array studies indicate release of IGF-1 is constitutively increased in ASM cells from asthmatics [60]. IGF-1 and IGF-2 are single chain polypeptides, which play a pivotal role in regulating cell proliferation, differentiation and apoptosis. The mitogenic effects of IGFs are primarily mediated through the type I IGF receptor (IGFR-1), which is a tetrameric tyrosine kinase receptor comprising two achains and two b-chains joined by disulfide linkages [80]. Insulin receptor and IGFR-1 have 60% homology between them thus enabling the IGFs and insulin to cross-ligate with other IGF receptors, albeit weakly.

4.4. Transforming growth factor-b The TGFb superfamily is important in cell development and differentiation and proliferative regeneration, although its actions are concentration- and cell-type-specific [81]. In epithelial and endothelial cells, TGFb1 is usually associated with terminal differentiation, growth inhibition and even apoptosis, but during wound healing it can be involved in tissue regeneration processes [81]. In myofibroblasts, smooth muscle cells and other cells of mesenchymal origin, TGFb1 induces proliferation and synthesis of ECM proteins, including collagens, elastin, proteoglycans and fibronectin [81]. TGFb1 is also known to increase the expression and release of VEGF in many of these cell types including ASM cells [60,82].

Mechanism of action Inhibits endothelial cell proliferation, migration, tube formation and neovascularisation Inhibits endothelial cell migration, proliferation. Induces endothelial cell apoptosis Induces apoptosis of proliferating endothelial cells Inhibits endothelial cell migration Inhibits endothelial cell migration, proliferation. Induces endothelial cell apoptosis. Causes G1 arrest of endothelial cells

5. Negative regulators of angiogenesis Within a given microenvironment, the angiogenic response is determined by a net balance between pro- and anti-angiogenic regulators released from activated endothelial cells, monocytes, smooth muscle cells and platelets among other cells [52,53]. Angiogenic promoters, such as VEGF, FGF-2 and TGFb (Table 1) are counter-balanced by multiple angiogenic inhibitors including selected members of the interleukin and interferon families, and collagen-derived inhibitors such as endostatin, arresten and tumstatin. 5.1. The ECM as a source of angiogenic inhibitors in chronic lung diseases Alterations in airway ECM protein deposition are one of the most prominent features of tissue remodelling in asthma. Histological studies of the ECM in asthmatic airways show increases in collagen I, III and V [83–85]. In contrast, Bousquet and colleagues have described an abnormal superficial elastic fibre network in the asthmatic airways [86]. They reported not only a decrease in elastin, but also an abnormal elastolytic process. Of note, levels of collagen IV were shown to be decreased in asthmatic airways compared with non-asthmatics [87]. Distinct fragments derived from different types of collagen including collagen IV have been reported to exhibit anti-angiogenic properties (see Table 2). 5.2. Collagen IV-derived angiogenic inhibitors Collagen IV is one of the major components of all basal membranes. It is critical in endothelial cell proliferation [88] and in the regulation of cell adhesion and migration [89]. Six different collagen IV a chains, a1–a6, encoded by 3 separate genes, form collagen IV heterotrimers [90,91]. Each a-chain comprises 3 domains, a cysteine-rich N terminal 7S domain, a central triplehelical domain and a globular C-terminal NC1 domain. The NC1 domain is believed to be involved in the initiation and assembly of the a-chain heterotrimers [92–95]. At least three collagen IV a-chain NC1 domains, a1–a3, possess anti-angiogenic activity. Collagen IV a1 (arresten) inhibits VEGFinduced proliferation and migration of endothelial cells through binding of a1b1 integrin (major collagen IV receptor) [92]. As for collagen IV a2 (canstatin), inhibition of endothelial cell proliferation and migration is suggested to be mediated by a cell surface protein/receptor, the most likely candidates being the integrins, avb3 (major vitronectin receptor), a1b1 and a2b1 (major collagen I receptor) [96]. Lastly, collagen IV a3 (tumstatin) induces apoptosis of proliferating endothelial cells [94,97,98]. Its mechanism of action involves an interaction with avb3 integrin, which leads to the inhibition of focal adhesion kinase (FAK), phosphatidylinositol-3 kinase, protein kinase B (Akt) and mammalian target of rapamycin (mTOR) as well as the prevention of the dissociation of the eukaryotic translation initiation factor 4E/eukaryotic initiation factor 4E-binding protein 1 (eIF4E/4E-BP1) complex, resulting in

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the inhibition of cap-dependent protein translation in endothelial cells [99]. Whilst the levels of collagen IV are reported to be decreased in the asthmatic airway [87], it is unknown which of the a-chains, if any, are changed in asthma. 5.3. Other collagen-derived angiogenic inhibitors Endostatin is an internal 20 kDa fragment from the carboxyterminal non-collagenous (NC) domain 1, of collagen XVIII. Endostatin exerts its effects by inhibiting endothelial cell proliferation and migration, inducing apoptosis of proliferating endothelial cells and causing cell cycle arrest in G1 [100]. The mechanism of inhibition involves VEGF-induced endothelial nitric oxide synthase (eNOS) phosphorylation [101,102] which ultimately leads to activation of protein phosphatase 2A (PP2A) and subsequent dephosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and the retraction of newly-formed vessels [103]. The collagen XV NC1 domain (restin), which has 72% homology with endostatin, also exhibits anti-angiogenic properties by inhibiting migration of endothelial cells without suppressing proliferation. Collagen XV knock-out mice exhibits abnormalities which include degeneration of endothelial cells as well as the local collapse of capillaries [104]. 6. Bronchial vascular remodelling and angiogenesis in chronic airway disease Advances in tissue labelling methods and availability of vascular markers (collagen IV, PECAM-1/CD31, factor VIII antigen) have improved the visualisation of blood vessels in tissue specimens [105]. Direct evidence for hypervascularity and angiogenesis in COPD and asthma has come from post-mortem and endobronchial biopsy studies [5,106–108]. 6.1. Vascular remodelling in COPD During the progression of COPD, vascular intimal thickening and the emergence of smooth muscle cells within the intima of small pulmonary arterial branches is attributed to a chronic inflammatory process accompanied by fibrosis, that is analogous to arteriosclerosis in cardiovascular disease [7,68,109]. In patients with COPD, vascular medial thickness, assessed by video image analysis, is increased modestly in pulmonary vessels of various sizes but the ratio of the smooth muscle a-actin-labelled area to total vascular wall area is unchanged, indicating that the increase in wall thickness could be attributed to the deposition of ECM proteins and medial accumulation of inflammatory cells and fibroblasts [65]. Vascular remodelling in COPD could be a contributing event in the pathogenesis of pulmonary hypertension and the observed changes in the intimal fibrosis as well as medial thickening could narrow the vessel calibre and may eventually lead to more severe vascular obstruction in COPD patients. Furthermore, an inverse correlation with lung function (assessed by FEV1) in COPD showed that the degree of pulmonary vascular remodelling is related to the severity of obstructive lung function defect [65]. 6.2. Angiogenesis in asthma Angiogenesis can be visualised by the appearance of cystic outpouchings or angiogenic ‘sprouts’ from parent blood vessels. These are increased in number in the airway submucosa layers of patients with asthma [61]. Although the mechanisms triggering the angiogenic switch in asthma are unknown, recent studies show an imbalance between pro-angiogenic and protective anti-angiogenic factors in asthmatic airways [64] (Tables 1, 2). Exaggerated levels of the endothelial mitogen and permeability factor, VEGF, its receptors

VEGFR-1/Flt-1 and VEGFR-2/KDR/Flk-1 [56,57,61,64,66], and other pro-angiogenic factors such as FGF-2 [57,78], angiogenin and MCP1 [66] have been detected in tissue and airway lining fluid from asthmatic individuals, which correlate directly with increased total airway vascular area [56,57,61,63,64,110], disease severity [62] and are inversely correlated to airway calibre and airflow obstruction [57,63,64]. Feltis and colleagues [61] have demonstrated that Ang1-positive vessels are increased in bronchial biopsies from individuals with asthma compared with those from healthy control subjects [57,61]. To date, endostatin is the only anti-angiogenic inhibitor that has been studied in asthma. Suzaki and colleagues reported that administration of endostatin/Fc to ovalbumin-sensitised mice inhibited airway hyperresponsiveness, pulmonary allergic inflammation, production of ovalbumin-specific IgE and markers of lung inflammation [111]. These investigators examined the expression of PECAM-1/CD31 (an endothelial cell marker) mRNA expression and found it was reduced in the endostatin treated mice. Other markers of angiogenesis were not examined [111]. Asai and colleagues showed levels of VEGF and endostatin to be increased in asthmatic compared with non-asthmatic sputum samples [64]. An imbalance was shown in the VEGF/endostatin ratio in asthmatics compared with non-asthmatics, and that the increase reflected increased levels of VEGF over those of endostatin [64]. Simcock and colleagues reported similar findings in the VEGF/endostatin ratio in bronchoalveolar lavage fluid from asthmatics and showed that bronchoalveolar lavage fluid from asthmatic but not healthy subjects induced VEGF-dependent vascular tubule formation in vitro [66]. Collectively, these studies suggest airway angiogenesis is an ongoing feature of stable asthma. 6.3. Airway smooth muscle and bronchial wall angiogenesis Appreciation of the role of ASM in respiratory disease progression has advanced significantly since the acknowledgment that ASM expresses an array of cytokines, adhesion molecules, ECM components and growth factors that can influence vascular reactivity and angiogenesis; in short ASM is a biological factory [60,112– 116]. Furthermore, bronchial blood vessels in man are anatomically juxtaposed between the bronchial epithelium and the increased ASM mass found in asthmatic airways [107,108], and the distance between the ASM and the epithelium reduces with increasing asthma severity [106], increasing the proximity of vessels to the ASM. Immunohistochemical studies of both animal and human airways show positive labelling in ASM for pro-angiogenic factors such as VEGF [68,117]. ASM cells in culture express VEGF splice variant mRNAs and VEGF protein, both constitutively and incrementally following stimulation by the proasthmatic inflammatory mediators bradykinin [118], TNF-a, IL-1b, TGFb and Th-2 cytokines IL-4, IL-5 and IL-13 [82,112,118,119]. Furthermore, ASM cells from asthmatics exhibit enhanced constitutive release of multiple proangiogenic factors including VEGF, angiogenin and angiopoietin (Ang)-1, with TGFb1 further increasing the production of VEGF from these ASM cells [60]. This enhanced expression or release of VEGF by smooth muscle cells in asthma has also been reported in diseases such as COPD [58,109], suggesting that these cells have an important role in promoting vascular remodelling and angiogenesis in chronic airway diseases [60,112,113]. Thus, the contribution that ASM cells make to the development and perpetuation of chronic airway disease may be greater than currently recognised. Recently, the interactions between ASM cells and endothelial cells have been studied in relation to angiogenesis in chronic respiratory disease. Several in vitro models have paved the way to decipher these cellular interactions including induction of

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primitive vascular tubules by ASM-derived VEGF [60,120]. Cell conditioned medium (CM) from a mechanical strain ASM model has been shown to be angiogenic by expressing hypoxia-inducible factor 1a, a transcription factor for VEGF expression, leading to induction of vascular tube formation in vitro [120]. CM from ASM from asthmatics, but not healthy controls, induced marked vascular tube formation that was abrogated by VEGF-depletion [60]. In addition, TGFb1 was shown to further increase the production of VEGF from ASM cells from asthmatics and to upregulate the capacity of these cells to induce the formation of vascular tubules [60]. Exogenous application of pro-angiogenic mediators to endobronchial biopsies from healthy volunteers increased tissue vascularity ex vivo to levels similar to those reported in bronchial biopsies from asthmatics [60]. The role played by ASM in regulating inhibitory effects on angiogenesis remains to be clarified, although an initial study indicates levels of multiple anti-angiogenic factors (endostatin, angiopoeitin-2) are not different between cells cultured from subjects with or without asthma, despite increased release of proangiogenic mediators including VEGF from asthmatic ASM [60]. Clearly, ASM has important synthetic functions that suggest it participates directly in the pathogenesis of airway wall vascular remodelling to perpetuate asthmatic airway inflammation and AHR. It is also possible that asthmatic ASM regulates angiogenesis to meet the metabolic requirements arising from its increased content in the airway wall in asthma [60]. In summary, angiogenesis is regulated by both pro- and antiangiogenic factors which under homeostatic conditions are in a state of equilibrium [52]. Levels of pro-angiogenic factors, such as VEGF, are reported to be increased in the airways of asthmatics [56,57,61,62,110]. However, levels of known angiogenic inhibitors have not been examined in detail in asthma, although, of these, endostatin appears unaffected in asthma [64,66]. Evidence to date, therefore, supports an imbalance between the pro-angiogenic factors and the anti-angiogenic factors, with the scale appearing to be tipped in favour of pro-angiogenic mediators and ASM being intimately involved in this outcome. 7. Conclusions A number of vascular changes (both functional and structural) observed in human chronic airway diseases have significant pathophysiological consequences, and thus participate in the clinical manifestations of airway disease. Current in vivo and in vitro data indicate that cross-talk between airway and vascular smooth muscle cells, endothelium, myofibroblasts and inflammatory cells via release of growth factors and cytokines, contributes to vascular remodelling. While ASM cells are emerging as key players in pathophysiological changes in the airway circulation, many different factors are involved in driving vascular changes including various components of the ECM. At present, our knowledge of the onset and progression of vascular remodelling and angiogenesis in chronic airway diseases is far from complete. With further understanding of the mechanisms involved in the functional and angiogenic changes in airway blood vessels in asthma and other chronic inflammatory human airway diseases, the potentially important therapeutic implications for targeting these alterations will become apparent. Acknowledgements Sarah Boustany was supported by National Health and Medical Research Council (NHMRC) of Australia and ALTANA Pharma AG. Janette Burgess is the recipient of a NHMRC Peter Doherty Fellowship (165722). Stuart Hirst acknowledges the support of

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NHMRC (566904) and Asthma UK (05/022, 05/027, 07/034). Hari Sharma was supported by the Netherlands Asthma Foundation (329773). David Simcock was supported by a King’s College London Studentship. The authors would also like to thank Astra-Zeneca for sponsoring and the travel grants to attend the Sixth International Young Investigators’ Symposium on Smooth Muscle in Sydney, Australia, November 2007. References [1] Bernard SL, Glenny RW, Polissar NL, Luchtel DL, Lakshminarayan S. Distribution of pulmonary and bronchial blood supply to airways measured by fluorescent microspheres. J Appl Physiol 1996;80:430–6. [2] Beckett PA, Howarth PH. Pharmacotherapy and airway remodelling in asthma? Thorax 2003;58:163–74. [3] Saglani S, Payne DN, Zhu J, Wang Z, Nicholson AG, Bush A, et al. Early detection of airway wall remodeling and eosinophilic inflammation in preschool wheezers. Am J Respir Crit Care Med 2007;176:858–64. [4] Carroll N, Elliot J, Morton A, James A. The structure of large and small airways in nonfatal and fatal asthma. Am Rev Respir Dis 1993;147:405–10. [5] Li X, Wilson JW. Increased vascularity of the bronchial mucosa in mild asthma. Am J Respir Crit Care Med 1997;156:229–33. [6] Chung KF, Adcock IM. Multifaceted mechanisms in COPD: inflammation, immunity, and tissue repair and destruction. Eur Respir J 2008;31:1334–56. [7] de Boer WI, Alagappan VK, Sharma HS. Molecular mechanisms in chronic obstructive pulmonary disease: potential targets for therapy. Cell Biochem Biophys 2007;47:131–48. [8] Merrilees MJ, Hankin EJ, Black JL, Beaumont B. Matrix proteoglycans and remodelling of interstitial lung tissue in lymphangioleiomyomatosis. J Pathol 2004;203:653–60. [9] Peyrol S, Gindre D, Cordier JF, Loire R, Grimaud JA. Characterization of the smooth muscle cell infiltrate and associated connective matrix of lymphangiomyomatosis. Immunohistochemical and ultrastructural study of two cases. J Pathol 1992;168:387–95. [10] Sobonya RE, Quan SF, Fleishman JS. Pulmonary lymphangioleiomyomatosis: quantitative analysis of lesions producing airflow limitation. Hum Pathol 1985;16:1122–8. [11] Glasgow CG, Taveira-Dasilva AM, Darling TN, Moss J. Lymphatic involvement in lymphangioleiomyomatosis. Ann N Y Acad Sci 2008;1131:206–14. [12] Laitinen LA, Laitinen A, Widdicombe J. Effects of inflammatory and other mediators on airway vascular beds. Am Rev Respir Dis 1987;135:S67–70. [13] Widdicombe JG. Asthma. Tracheobronchial vasculature. Br Med Bull 1992;48:108–19. [14] Mariassy AT, Gazeroglu H, Wanner A. Morphometry of the subepithelial circulation in sheep airways. Effect of vascular congestion. Am Rev Respir Dis 1991;143:162–6. [15] Rolla G, Scappaticci E, Baldi S, Bucca C. Methacholine inhalation challenge after rapid saline infusion in healthy subjects. Respiration 1986;50:18–22. [16] Cabanes LR, Weber SN, Matran R, Regnard J, Richard MO, Degeorges ME, et al. Bronchial hyperresponsiveness to methacholine in patients with impaired left ventricular function. N Engl J Med 1989;320:1317–22. [17] Hogg JC, Pare PD, Moreno R. The effect of submucosal edema on airways resistance. Am Rev Respir Dis 1987;135:S54–6. [18] McFadden ER, Jr. Hypothesis: exercise-induced asthma as a vascular phenomenon. Lancet 1990;335:880–3. [19] Kumar SD, Emery MJ, Atkins ND, Danta I, Wanner A. Airway mucosal blood flow in bronchial asthma. Am J Respir Crit Care Med 1998;158:153–6. [20] Long WM, Yerger LD, Abraham WM, Lobel C. Late-phase bronchial vascular responses in allergic sheep. J Appl Physiol 1990;69:584–90. [21] Dunhill MS. The pathology of asthma, with special changes in the bronchial mucosa. J Clin Pathol 1960;13:27–33. [22] Kuwano K, Bosken CH, Pare PD, Bai TR, Wiggs BR, Hogg JC. Small airways dimensions in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis 1993;148:1220–5. [23] Widdicombe JG. Neural control of airway vasculature and edema. Am Rev Respir Dis 1991;143:S18–21. [24] Kanazawa H, Nomura S, Asai K. Roles of angiopoietin-1 and angiopoietin-2 on airway microvascular permeability in asthmatic patients. Chest 2007;131: 1035–41. [25] Zschauer AO, Sielczak MW, Wanner A. Altered contractile sensitivity of isolated bronchial artery to phenylephrine in ovalbumin-sensitized rabbits. J Appl Physiol 1999;86:1721–7. [26] Janssen LJ, Lu-Chao H, Netherton S. Responsiveness of canine bronchial vasculature to excitatory stimuli and to cooling. Am J Physiol Lung Cell Mol Physiol 2001;280:L930–7. [27] Barnes PJ. Neural control of human airways in health and disease. Am Rev Respir Dis 1986;134:1289–314. [28] Carstairs JR, Nimmo AJ, Barnes PJ. Autoradiographic visualization of betaadrenoceptor subtypes in human lung. Am Rev Respir Dis 1985;132:541–7. [29] Onorato DJ, Demirozu MC, Breitenbucher A, Atkins ND, Chediak AD, Wanner A. Airway mucosal blood flow in humans. Response to adrenergic agonists. Am J Respir Crit Care Med 1994;149:1132–7.

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